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Characterization of an alphaherpesvirus resistant cell line Banfield, Bruce William 1994

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CHARACTERIZATION OF AN ALPHAHERPESVIRUS RESISTANT CELL LINE ty BRUCE WILLIAM BANFIELD B. Sc., the University of British Columbia, 1988  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology and Immunology)  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA February 1994 © Bruce William Banfield, 1994  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive  copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  A,rc,cv  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  1 /99V T  ‘‘‘‘Ai  _1Y  Abstract A mouse L cell mutant termed gro29 has been isolated (Tufaro et a!. 1987) and shown to be defective in the growth of two enveloped viruses, vesicular stom atitis virus (VSV) and herpes simplex virus type 1 (HSV-1). In this thesis it is demonstrated that the rate of transport and processing of HSV-1 glycoproteins was impeded in infected gro29 cells. In addition, HSV-1 virions failed to exit the cell and accumulated in cytoplasmic vacuoles. The phenotype of the uninfected gro29 cell was examined. It was determined that gro29 cells were resistant to the lectins ricin and modeccin, and showed a reduced ability to bind fluorescently conjugated ricin. In addition, gro29 cells failed to synthesize the glycosaminoglycan chondroitin sulfate. The lectin binding properties of gro29 cells and the glycosaminoglycan synthesis profile exhibited by these cells suggested that gro29 might have suffered a defect in the metabolism of N acetylgalactosamine (GalNAc). The metabolism of GalNAc was examined in gro29 cells and macromolecules directly involved in the synthesis and utilization of this molecule were found to be normal. The phenotype of gro29 suggested that HSV-1 requires a host cell component for efficient virus maturation and egress distinct from those components which facilitate the trafficking of viral membrane glycoproteins such as VSV G protein. The results presented in this thesis suggest that it may be possible to interfere with cell secretion or GalNAc metabolism such as to leave the cell viable yet impair the ability of cells to propagate herpes simplex virus.  Table of Contents Abstract  .  ii  Table of Contents  iii  List of Figures  vi  List of Abbreviations  viii  Acknowledgment  xi  Dedication  xii  Introduction  I  The herpesviruses  I  The medical significance of the herpes simplex viruses  2  Herpes simplex virus structure  3  Herpes simplex virus glycoproteins  7  The herpes simplex virus lifecycle  12  Study of the herpes simplex virus lifecycle  22  Isolation of gro mutants able to survive exposure to HSV-1  23  gro29 cells  23  Materials and Methods  26  Cells and viruses  26  Materials  26  Harvesting herpes simplex virus  27  Determination of virus titre  28  Pulse-chase labeling experiments  28  Immunoprecipitations  28  Endoglycosidase H digestions  29  Western blots  29  Fractionation of microsomes  30  Indirect immunofluorescence  31 III  Fluorescence microscopy using lectins  .31  Electron microscopy  32  Analysis of HSV-1 gD in BFA treated cells  32  Analysis of glycosaminoglycans  33  Enzyme and protein assays  34  Purification of Golgi membranes for nucleotide sugar translocation assays  34  Nucleotide sugar translocation assays  35  Glucose and sulfate uptake assays  36  Chapter 1- Analysis of HSV-1 infected gro29 cells  38 38  Results Production of infectious virus by gro29 cells  38  Pulse-chase analysis of HSV-1 glycoprotein processing  39  Analysis of gD transport in HSV-1 infected cells  46  Analysis of virus egress  49  Analysis of cell-associated virus  50  Immunofluorescence and electron microscopic analysis of infected cells  58  Brefeldin A treatment of HSV-1 infected cells  65 75  Discussion Chapter 2- Characteristics of uninfected gro29 cells Results  83 83  Lectin sensitivity of gro29  83  Fluorescence detection of lectin binding molecules  84  Analysis of glycosaminoglycan synthesis in gro29 cells  87  Investigation of Ga1NAc metabolism in gro29 cells  93  Rescue of chondroitin sulfate synthesis in gro29 cells  98  iv  Measurement of UDPGa1-4-epimerase activity in cell 101  extracts Measurement of nucleotide sugar translocation into Golgi  106  membranes  116  Discussion General Discussion  124  References  128  V  List of Figures Fig. 1: Herpesvirus structure  4  Fig. 2: Proposed models for the egress of alphaherpesviruses  19  Fig. 3. Titre of virus released from HSV-1 infected L and gro29 cells  41  Fig. 4. Processing of HSV-1 gD and gB in L and gro29 cells  44  Fig. 5. Western blot analysis of HSV-1 gD in membrane fractions of L and gro29 cells  48  Fig. 6. Comparison of HSV-1 particles released from L and gro29 cells  52  Fig. 7. Nucleocapsid assembly in L and gro29 cells  54  Fig. 8. Detection of intracellular virions  57  Fig. 9. Immunofluorescence analysis of HSV-1 gD in HSV-1 infected L and gro29 cells  60  Fig. 10. High resolution microscopy of HSV-1 infected L and gro29 cells  64  Fig. 11. Immunofluorescence localization of cytoplasmic virions  67  Fig. 12. Effects of BFA on the processing of HSV-1 gD during HSV-1 infection in L cells  70  Fig. 13. Effect of BFA on the processing of HSV-1 gD in gro29 cells  73  Fig. 14. Fluorescence analysis of lectin-binding molecules in L and gro29 cells  86  Fig. 15. Analysis of Glycosaminoglycan synthesis in L and gro29 cells  90  Fig. 16. Measurement of glucose uptake in L and gro29 cells  92  Fig. 17. Rate of sulfate uptake in L and gro29 cells  95  Fig. 18. Synthesis of UDPGaINAc  97  Fig. 19. Translocation of UDPGa1NAc into Golgi membranes  100  Fig. 20. Rescue of chondroitin sulfate synthesis in gro29 cells  103  Fig. 21. Measurement of UDPGa1-4-epimerase activity in L and gro29 cell extracts  105 vi  Fig. 22. Conversion of UDPGa1NAc to UDPG1cNAc L and gro29 cell extracts  108  Fig. 23. Rate of nucleotide sugar translocation into L and gro29 Golgi membranes  111  Fig. 24. Kinetics of nucleotide sugar translocation into L and gro29 cell Golgi membranes  113  vii  List of Abbreviations BFA  brefeldin A  BSA  bovine serum albumin  BrdU  5-bromo-deoxyuridine  CMP  cytidine monophosphate  CMPSA  cytidine monophosphate-sialic acid  E 3 Q  cytopathic effect  CPM  counts per minute  CS  chondroitin sulfate  d  day(s)  DEAE  diethylaminoethyl  DNA  deoxyribonucleic acid  EDTA  ethylenediaminetetraacetic acid  endo H  endoglycosidase H  ER  endoplasmic reticulum  FACS  fluorescence activated cell sorting  FBS  fetal bovine serum  FITC  fluoresciene isothiocyanate  Gal  galactose  GalA  glucuronic acid  Ga1NAc  N-acetylgalactosamine  Glc  glucosamine  G1cNAc  N-acetylglucosamine  G1cNSO3  N-sulfoglucosamine  h  hour(s)  HA  hyaluronic acid  HAT  hypoxanthine aminopterin thymidine  viii  HPI  hours post infection  HS  heparan sulfate  HSV-1  herpes simplex virus type 1  HSV-2  herpes simplex virus type 2  Kbp  kilobase pairs  KDa  kilodaltons  Man  mannose  mm  minute(s)  MOl  multiplicity of infection  PAGE  polyacrylamide gel electrophoresis  PBS  phosphate buffered saline  PFU  plaque forming unit(s)  PrV  pseudorabies virus  RN A  ribonucleic acid  RPM  revolutions per minute  SA  sialic acid  SDS  sodium dodecylsulfate  TCA  trichloroacetic acid  TGN  trans Golgi network  TLC  thin layer chromatography  TK  thymidine kinase  UDP  uridine diphosphate  UDPGa1  uridine diphosphate-galactose  UDPGa1NAc  uridine diphosphate-N-acetylgalactosamine  UDPG1c  uridine diphosphate-glucosamine  UDPG1cNAc  uridine diphosphate-N-acetylglucosamine  UL  unique long region of the herpes simplex virus genome ix  UMPK  uridine monophosphokinase  U  unique short region of the herpes simplex virus genome  VHS  virus host shutoff  VICV  virus-induced cytoplasmic vacuole  VS V  vesicular stomatitis virus  VZV  varicella-zoster virus  w/v  weight/volume  w/w  weight/weight  x  Acknowledgments I wish to acknowledge; F. Tufaro, S. L. Gruenheid, H. E. Meadows and K. Schubert for their contributions to data presented in this thesis, Michael Wies for assistance with microscopy and photography, the members of Frank Tufaro’s laboratory past and present for helpful discussions, Dr  J.  T. Beatty for reminding  me from time to time that doing science is really a lot of fun, the members of my advisory committee: Drs. Jefferies, McMaster and Spiegelman for sound advice through the years, Dr McMaster for invaluable assistance in the preparation of this thesis, and Renée Finnen for her patience and critical reading of this thesis. Most of all I wish to thank Dr Frank Tufaro for his enthusiasm and the opportunity to work under his tutelage.  xi  This thesis is dedicated to my parents, William and Gertrude, without whose constant support and encouragement this would not have been possible.  xii  1 Introduction The herpesviruses The Herpesviridae comprise a large family of viruses which share some common features (reviewed in Roizman and Batterson 1986). Herpesviruses are large enveloped viruses with a double stranded, linear, DNA genome enclosed in an icosahedral nucleocapsid composed of 162 capsomeres. A property shared by all herpesviruses is their ability to remain in a latent state in the host in which they multiply. In excess of 80 herpesviruses have been characterized, infecting organisms as diverse as frogs and man. The members of the virus family, Herpesviridae, have been classified into three subfamilies based on their biological properties; the alphaherpesvirinae, betaherpesvirinae, and gammaherpesvirinae (Roizman and Batterson 1986). The alphaherpesvirinae (eg. herpes simplex virus, pseudorabies virus, and varicella-zoster virus) are characterized by a variable host range, short reproductive cycle, rapid spread in cell culture, efficient destruction of infected cells, and the ability to establish latent infection in cells of the ganglia. It has recently been suggested that the alphaherpesvirinae be subclassified into the simplexviruses and the zosterviruses because of the differences in the requirements for entry of these viruses into cells (Spear 1993). The betaherpesvirinae (eg. cytomegalovirus), are distinguished by a restricted host range, long reproductive cycle, slow spread in culture, enlargement of infected cells (cytomegalia), and the ability to be maintained in a latent state in secretory glands, kidneys, lymphoreticular cells, and other tissues. Gammaherpesvirinae (eg. Epstein-Barr virus) are extremely restricted in their host range, infecting lymphocytes in the family or order to which the natural host belongs. Gammaherpesvirinae infection is often arrested in a prelytic or  2 lytic stage without production of infectious virus. Latency of these viruses has been found to occur in lymphoid tissues.  The medical significance of the herpes simplex viruses The use of the term herpes (from the Greek herpein, to creep) to describe  the typical “cold sores” produced by this agent dates back about 25 centuries (Beswick 1962). However, it was not until the late 19th century when it was determined that there was an infectious agent associated with the lesions. In 1920 Grüter published experimental results which showed that the virus could be passaged from an experimentally infected rabbit to the cornea of a blind man (Grüter 1920). It was not until 1967 when it was determined that two distinct serotypes of herpes simplex viruses exist, HSV-1 and HSV-2 (Dowdie et al. 1967). It is now known that there are significant molecular differences between the two viruses; HSV-1 and HSV-2 show roughly 50% homology at the nucleic acid level (McGeoch et al. 1991). The herpes simplex viruses (HSV-1 and HSV-2) are the causative agents of a number of medically important diseases. In addition, infections with HSV 1 and HSV-2 are among the commonest of human viral diseases (reviewed in Mindel 1989). HSV-1 and HSV-2 each have the ability to cause the all the clinical symptoms observed for these infections although one or the other tend to predominate in a specific condition. These viruses are responsible for highly contagious oral and genital lesions, which are the most common manifestations of infection; it has been estimated that half a million new cases are reported each year in the USA and, as of 1984, 98 million people were infected in that country alone. Usually HSV-1 is associated with oral lesions and HSV-2 with genital lesions although there are exceptions in some regions of the world. HSV-1  3 induced ocular infections are the leading cause of corneal blindness in the USA with 300,000 cases reported each year. HSV-1 is also the most common cause of fatal endemic encephalitis and the most prevalent viral infection of the central nervous system in the USA. Neonatal infection with HSV-2 is also a significant problem with estimates ranging from 2.6-50 cases per 100,000 live births in the USA, depending on the region. More recently, the role of herpes simplex infection in AIDS patients has been examined (reviewed in Quinn 1990). It has been suggested that herpes simplex virus infection may play a role in the transmission, clinical presentation and pathogenesis of HIV infection. Because of the significant medical impact of herpes simplex viruses it is of considerable importance to determine the function of viral and cellular molecules in the lifecycle of this pathogen, as this information may expose vulnerabilities which can be exploited in the treatment of disease.  Herpes simplex virus structure The herpes simplex virion consists of four structural elements: the core, capsid, tegument and the envelope (see Fig.1). It is estimated that between 30-40 structural proteins comprise the herpes simplex virion (Heine et al. 1974; Spear and Roizman 1972) which represents approximately half of the coding capacity of the viral genome. The core of the virus contains a linear, double stranded DNA genome of approximately l52Kbp which is wound into a tight toroidal structure in the virion (Furlong et al. 1972). In some electron micrographs of virions it appears that fibers connect the electron dense core to the inside of the capsid. The origin or function of these fibers is unknown. The entire genome of HSV-1 has been sequenced (McGeoch et al. 1988; McGeoch et al. 1985; McGeoch et al. 1986). HSV DNA consists of two covalently linked components termed long (L) and short  4 Fig. 1. Herpesvirus structure. A) The core containing 152 Kbp of linear double stranded DNA wound into a torrus. B) The icosahedral nucleocapsid. C) The tegument. D) The viral envelope. E) Viral glycoproteins embedded in the envelope. (schematic of the virion courtesy of Y. Leduc).  B  A  C  E  D  5 (S). The L region makes up about 82% of the genome and the S region about 18%. Each region consists of mostly unique sequence (UL and Us) flanked by inverted repeats. The repeated sequence flanking UL comprise 12% of the genome and the repeats flanking Us comprise 8.6% of the genome, thus a considerable portion of the viral genome is diploid. The genome encodes at least 77 genes; 58 of which are in the UL region (Barker and Roizman 1992; Liu and Roizman 1991b; McGeoch et al. 1988), 13 in the Us region (Georgopoulou et al. 1993; McGeoch et al. 1985), and 3 in the inverted repeats which flank the unique regions of the genome (Ackermann et al. 1986a; Chou and Roizman 1986). 5 region are Interestingly, all but one of the 13 genes which comprise the U nonessential for viral growth in culture (Longnecker et al. 1987; Longnecker 5 region and Roizman 1987; Weber et aL 1987). It has been suggested that the U contains genes which although not absolutely required for growth in culture evolved to allow HSV to survive in its human host, and that HSV may have arose by the insertion of Us sequences into a larger DNA component containing genes essential for viral replication (Longnecker and Roizman 1987). The capsid is an icosahedron consisting of 162 capsomeres. The capsomeres are made up of several different polypeptides and are in the form of a hexagonal prism with a hollow tube running the length of the long axis (Wildy et al. 1960). The capsid structure has 20 triangular faces and 12 apices. Each apex consists of a single capsomere surrounded by five others (pentons). The non-apical capsomers are surrounded by six others (hexons). The capsid therefore has 150 hexons and 12 pentons for a total of 162 capsomeres. Seven viral polypeptides have been identified as capsid components (Cohen et al. 1980; Gibson and Roizman 1972; Heilman et al. 1979) and are the product of six viral genes (Davison et al. 1992; Liu and Roizman 1991a; McNabb and Courtney 1992;  6 Pertuiset et al. 1989; Preston et al. 1983; Preston et al. 1992; Rixon et al. 1990a). These proteins are VP5, VP19C, VP21, VP22a, VP23, VP24 and VP26. The tegument surrounds the capsid and appears as an amorphous fibrous structure in electron micrographs. The tegument contains five major proteins, VPI /2, VP13, VP14, VPI6 and VP22 (Honess and Roizman 1973; Spear and Roizman 1972). It has been estimated that approximately 15 viral polypeptides are found in this structure (Heine et al. 1974; Spear and Roizman 1972). Little is known about the nature of the tegument, however, it has been determined that two viral transcription factors, VPI6 (also known as: VMW65 or czTiF) and ICP4, are located in this structure (Honess and Roizman 1973; Spear and Roizman 1972; Yao and Courtney 1989). In addition, a viral host shutoff function, VHS, which destabilizes both viral and cellular mRNAs is thought to be associated with the tegument (Kwong and Frenkel 1987; Smibert and Smiley 1990). Surrounding the tegument is a phospholipid bilayer envelope. Embedded in this envelope are viral glycoproteins. There are at least 11 HSV glycoproteins: gB, gC, gD, gE, gG, gH, gI, gj, gK, gL and gM (Ackermann et al. 1986b; Baines and Roizman 1993; Baucke and Spear 1979; Buckmaster et al. 1984; Heine et al. 1972; Hutchinson et al. 1992a; Hutchinson et al. 1992b; Longnecker et al. 1987; Para et al. 1983; Spear 1976; Spear 1985), 8 of which have been shown to be present in the virion envelope (all but gj, gK and gL) (reviewed in Spear 1993). It is likely that all of the proteins resident in the viral envelope have not yet been identified because a number of viral open reading frames are predicted to encode polypeptides which have membrane spanning domains (McGeoch et al. 1991) and as of yet have not been characterized. Furthermore, viral genes encoding proteins which have multiple membrane spanning domains (serpentine membrane proteins) may also reside in the  7 viral envelope (MacLean et al. 1991). Baines and Roizman have recently described a viral glycoprotein, gM, which belongs to the class of serpentine membrane proteins and is a resident of the viral envelope (Baines and Roizman 1993). Interestingly a number of the genes encoding viral glycoproteins are clustered in the unique short region of the viral genome suggesting that these genes may have been inherited together by a precursor of the herpesviruses. These genes encode glycoproteins gE, gG, gI, and gD. The U6 gene, which encodes gD, is the only essential viral gene found in the U region of the viral genome.  Herpes simplex virus glycoproteins The study of the structure and function of the viral glycoproteins specified by herpes simplex virus is one of the most concentrated efforts in the field of herpes simplex virology. These molecules have been shown to be important in several stages of the viral life cycle including attachment, entry and egress and also in the host immune response to this pathogen. The glycoproteins gB, gD, gH and gL are essential for viral propagation in tissue culture (reviewed in Spear 1993). These glycoproteins are thought to have roles in the stable attachment of the virus with the cell and in the fusion of the viral envelope with the cell membrane. Glycoprotein gB of HSV-1 is one of the most well characterized of the HSV glycoproteins and is found in the virus envelope and in the plasma membrane of infected cells as an oligomer (Claesson-Welsh and Spear 1986; Spear 1976). gB is modified by the addition of N- and 0-linked carbohydrates and is essential for the growth of the virus in culture (Cai et al. 1987; Johnson  8 and Spear 1983; Spear 1976). The gene for this glycoprotein is highly conserved; in fact, 13 homologues from related herpesviruses have been cloned and sequenced and these genes show a great deal of homology. Consistent with these data is the observation that a gB homolog has been found to functionally substitute in a HSV strain with a deleted HSV gB gene (Misra and Blewett 1991). gB has been suggested to play a role in the fusion of the viral envelope with the plasma membrane of the cell (Cai et al. 1988a; Highlander et al. 1988). This activity was inferred from the observation that many viral mutants which form polykaryons on monolayers of cells (termed syn mutants) mapped to the gene encoding gB (Bzik et al. 1984b; Cai et al. 1988a; Cai et al. 1988b). Furthermore, antibodies against gB inhibit fusion of the virus envelope with the cell, but not the attachment of the virus to the cell, suggesting that the role of this molecule is in membrane fusion (Highlander et al. 1988). Recently, regions of the gB molecule which are important for fusogenic activity have been identified (Baghian et al. 1993; Gage et al. 1993). It has also been shown in vitro that gB isolated from solubilized viruses can bind to heparin (Herold et al. 1991). Heparin is a glycosaminoglycan which is structurally similar to heparan sulfate, a molecule found on the surface of cells and implicated in the initial attachment of the virus to the cell (Shieh et al. 1992; WuDunn and Spear 1989). In addition, viruses lacking gB in their envelopes have a slightly decreased ability to bind to cells (Herold et al. 1991). These data may suggest that gB can act in the initial attachment of the virus to the cell. Glycoprotein gC is another well characterized viral glycoprotein which possesses both N- and 0-linked carbohydrate modifications (Dall’Olio et al. 1985; Johnson and Spear 1983; Olofsson et al. 1981; Spear 1976) and can bind to the third component of complement, C3b (Friedman et al. 1984b). Although not  9 essential for viral propagation in culture this molecule appears to have a major role in the attachment of virus to cells through heparan sulfate glycosaminoglycans (Herold et al. 1991; Shieh et al. 1992). Addition of heparan sulfate to the extracellular medium of cells during HSV infection reduces the ability of the virus to infect cells (WuDunn and Spear 1989). Herold and others have show that mutant viruses lacking gC in their envelopes bind to cells with 10-fold less affinity, and virions devoid of gC do not respond as greatly to inhibition by heparin as do wild type virions (Herold et al. 1991). Glycoprotein gD is an essential viral glycoprotein (Ligas and Johnson 1988) which has both N- and 0-linked carbohydrate moieties attached (Johnson and Spear 1983; Spear 1976). It appears that gD binds to a specific receptor on the cell surface and is involved in an intermediate attachment step between the initial attachment of the virus with heparan sulfate at the cell surface and the fusion of the viral envelope with the plasma membrane of the cell. The evidence for this is as follows; 1) antibodies against gD inhibit penetration of the virus into the cell but not attachment (Highlander et al. 1987), 2) mutant viruses devoid of gD are able to attach to cells but are unable to penetrate into the cell (Ligas and Johnson 1988), 3) viruses lacking gD are unable to inhibit the penetration of wild type virus (Johnson and Ligas 1988), 4) a soluble truncated form of gD is able to inhibit penetration of virus into the cell, suggesting the presence of a cell surface receptor for gD (Johnson et al. 1990), and 5) expression of gD in the plasma membrane of cells inhibits the penetration of virus, also suggesting the presence of a cell surface receptor for gD (Campadelli-Fiume et al. 1990). In addition, studies with gp5O, the gD homologue of PrV, suggest that this glycoprotein enables the virus to attach to the cell in a manner which is not competable by exogenous heparan sulfate (Karger and Mettenleiter 1993). It has also been suggested that gD may function in the fusion of the virus envelope  10 with the cell membrane, as it has been shown that cell lines expressing gD can fuse (Campadelli-Fiume et al. 1988b). gD has been shown to be a predominant target of the host humoral and cellular immune responses (Cohen et al. 1978; Martin et al. 1987; Torseth et al. 1987; Zarling et al. 1986). Furthermore, immunization with gD can protect animals from a lethal challenge of HSV (Chan 1983; Long et al. 1984), and monoclonal antibodies reactive with gD exhibit potent neutralizing activity against the virus (Para et al. 1985). Much is also known about the structure of gD. In particular, the mapping of epitopes on gD reactive with a panel of monoclonal antibodies (Cohen et al. 1986; Eisenberg et al. 1982; Eisenberg et al. 1985; Highlander et al. 1987; Showalter et al. 1981) has allowed the study of disulfide bond formation and N-glycosylation on the structure and function of gD (Cohen et al. 1988; Isola et al. 1989; Long et al. 1990; Muggeridge et al. 1988; Muggeridge et al. 1990; Sodora et al. 1989; Sodora et al. 1991a; Sodora et al. 1991b; Wilcox et al. 1988). gE has been shown to be modified by N- and 0-linked oligosaccharides, fatty acylation and sulphation (Baucke and Spear 1979; Hope et al. 1982; Johnson and Spear 1983; Para et al. 1983). gE has been identified as being an immunoglobulin G Fc binding protein (Baucke and Spear 1979). gE forms a complex with another viral glycoprotein, gI, and it is this complex which acts as the functional Fc receptor (Johnson and Feenstra 1987; Johnson et al. 1988). The role of this function in viral infection is unclear, but it has been suggested that it may aid in the viruses evasion of the host immune system. It has also been proposed that the Fc binding activity might act in the binding of the virus to the cell through cell surface molecules structurally related to immunoglobulins (Johnson and Feenstra 1987) (such as cell surface adhesion molecules, and molecules of the major histocompatability complex). If this interaction exists it  11 not essential for the replication of the virus in culture, as deletion of the gene encoding gE has little effect on the replication of the virus (Longnecker and Roizman 1987). Relatively little is known about the function of the other viral glycoproteins. As mentioned above gI forms a complex with gE to form an Fc receptor. gH is an essential viral glycoprotein thought to be involved in the fusion of the virus membrane with the cell (Fuller et al. 1989). gH has been shown to form a heterodimer with a recently described glycoprotein, gL, and this interaction is essential for the normal folding and cell surface expression of gH (Hutchinson et al. 1992a). In addition, it has been suggested that gH has a role in viral egress (Desai et al. 1988). gL has also been shown to be essential for the growth of the virus in culture (Roop et al. 1993) and is likely involved in the fusion of the viral envelope with the membrane of the cell. gK is thought to have a role in the fusion of viral and cellular membranes based on the observations that mutations in the gene for gK can cause increased fusion of infected cells (DebRoy et al. 1985; Pogue-Guile and Spear 1987), although it has not yet been determined if gK is present in the viral envelope. gM, the most recently identified glycoprotein, is the only viral glycoprotein described that is a member of the class of serpentine membrane proteins (Baines and Roizman 1993). A number of studies have determined that complete glycosylation of viral glycoproteins is not essential for virion infectivity (Campadelli-Fiume et al. 1982; Serafini-Cessi et al. 1983). These studies indicated that HSV infection of mutant cell lines defective in glycosyltransferases involved in the biosynthesis of N-linked glycans produced infectious progeny virus. The precise role of glycosylation on the viral glycoproteins remains unclear.  12 The herpes simplex virus lifecycle Attachment, binding and penetration Binding and penetration of HSV into cells is mediated by the interaction of viral envelope glycoproteins with cell surface molecules. The first step in the herpes simplex virus lifecycle is the adsorption of the virus to the cell surface (reviewed in Spear 1993). This process is mediated by the interaction of the viral glycoprotein gC with components of the cells extracellular matrix, in particular heparan sulfate proteoglycans (Herold et al. 1991; WuDunn and Spear 1989). It should be noted that this is an oversimplified view of these processes, as other studies have implicated the role of molecules independent of the gC/heparan sulfate interaction which mediate viral infection in some cell types. In particular, Sears and others were able to demonstrate that in MDCK cells, a polarized epithelial cell line, at least two cell surface receptors exist; one on the apical surface of the cell, and one on the basal surface of the cell. The apical receptor was dependent on gC in the virus envelope for its function whereas the basal receptor functioned independently of gC and was as equally efficient as the receptor on the apical surface (Sears et al. 1991). These data indicated that although the gC/heparan sulfate interaction appeared to be the predominant interaction in the absorption of the virus to the non-polarized cell lines tested in culture, it was clear that the virus has other means of interaction with the cell independent of this route. In another study (Gruenheid et al. 1992), mouse L cell lines selected for their ability to survive herpes simplex virus infection were isolated and found to be defective in the biosynthesis of heparan sulfate glycosaminoglycans. HSV plaques could still form on monolayers of these mouse L cells although the numbers were reduced by approximately 10 fold; similar to the reduction in plaque formation observed with virus lacking gC in the envelope (Herold et al. 1991). Two important conclusions can be drawn  13 from this study; heparan sulfate is an important molecule for the interaction of the virus with the cell under “normal ’ circumstances because a mutation in the t biosynthesis of this molecule was identified using the virus as a selective agent, and the presence of this molecule is not essential for infection which suggests that other as yet unidentified interactions take place that facilitate infection. The next step in the viral entry pathway is thought to be a “stable” attachment of the virus to the cell surface through gD with a cell surface receptor (Johnson et al. 1990; Johnson and Ligas 1988). Unlike the gC/heparan sulfate interaction the gD/receptor reaction is essential for productive infection to occur because mutant virus lacking gD cannot infect cells. The gD/receptor binding has been termed “stable” in that wild type viruses can enter into an attachment with the cell which is not elutable with heparin whereas viruses lacking gD do not become resistant to the addition of exogenous soluble heparin as readily (Fuller and Lee 1992). The specific roles of gill, a gC homolog, and gp5O, a gD homolog, in the attachment of the closely related alphaherpesvirus, PrV, has been examined in great detail. Karger and Mettenleiter (Karger and Mettenleiter 1993) have demonstrated the biphasic attachment of PrV to cells from a heparin sensitive binding to a heparin insensitive binding. The heparin sensitive binding was shown to depend on the presence of gill and the heparin insensitive binding was shown to depend on the presence of gp5O in the viral envelope. It is likely that the same mechanisms of attachment exist for both PrV and HSV. HSV is thought to enter the cell primarily by fusion of the viral envelope with the plasma membrane of the cell. Although the virus has been found to enter the cell by endocytosis, it is not thought that this route leads to a productive infection insofar as drugs affecting endocytosis and endosomal acidification have no effect on the efficiency of infection (Campadelli-Fiume et  14 al. 1988a; Koyana and Uchida 1984; Wittels and Spear 1991). It is likely that HSV which enters by this route is degraded in the lysosome (Campadelli-Fiume et al. 1988a). Studies using monoclonal antibodies directed against viral glycoproteins have suggested that HSV glycoproteins gB, gD and gH might be involved in the fusion of the virus envelope with the cell (reviewed in Spear 1993), furthermore the deletion of the genes encoding these proteins from the viral genome results in defective viruses which are unable to penetrate cells (Cal et al. 1988a; Forrester et al. 1992; Ligas and Johnson 1988). In addition, identification of viral molecules involved in this process has come in part from the observation that viral mutants which stimulate the fusion of infected cells  (syn mutants) occur at relatively high frequency (Brown et al. 1973; Hoggen and Roizman 1959; Person et al. 1976; Read et al. 1980). A number of genes which encode HSV glycoproteins have been shown to harbor these mutations. In particular, gB, gD, gK and gL have been found to have syn mutations, which suggested that these molecules may be involved in fusion of the virus envelope with the cell membrane (reviewed in Spear 1993). Although the mechanism by which fusion of these two membranes takes place is unclear, a model has been proposed based on what is known about the molecules involved. The process of gD interacting with its cellular receptor it is thought to stimulate conformational changes in the viral molecules involved in fusion such that a functional “fusion complex” (likely consisting of gD, gB, gH, gL and possibly gK) can form. It is also possible that other components of the virion are involved in the fusion reaction. In particular, a temperature sensitive HSV-1 mutant with a defect in penetration has been isolated and shown to have a mutation in the UL25 gene, the product of which is thought to be a constituent of the tegument (Addison et al. 1984). It is clear that much work remains to be  15 done before a comprehensive understanding of the events involved in the absorption, binding and penetration of herpes simplex viruses exists.  Uncoating, gene expression and replication Following the fusion of the viral envelope with the cell membrane the nucleocapsid enters the cytoplasm and travels to a nuclear pore where the genome is released into the cell’s nucleus. It has been hypothesized that the nucleocapsid travels down cytoskeletal elements in a fashion similar to that described for adenoviruses (Dales and Chardonnet 1973; Roizman and Batterson 1986). Release of the viral DNA from the nucleocapsid through the nuclear pore and into the nucleus is known to require a viral function because a temperature sensitive mutant of HSV-1 has been isolated which can travel to the nuclear pore but is unable to release viral DNA into the nucleus at the nonpermissive temperature (Batterson et al. 1983). Upon release of the viral genome into the nucleus of the cell a temporal cascade of gene expression ensues (Roizman and Batterson 1986). The virus uses the host cell RNA polymerase II in association with cellular and virally encoded transcription factors to transcribe its genes. There are at least five classes of HSV-1 genes; a,  f2, Yi, and Y2• The a genes are the first to be  transcribed and reach a peak level of expression from 2-4 hours post-infection (HPI). Some of the a gene products are required for the synthesis of the other groups of genes. The j3i and the  132  genes reach peak expression levels from 5-7  HPI. These temporal classes of genes are distinguished from each other in that the 13i genes require the a gene product, a4 (ICP4), for their transcription whereas the 132 genes do not. The Ii and the 132 genes encode proteins involved in viral genome replication. The Yi and Y2 genes have a requirement for concurrent viral DNA synthesis for their maximal expression. In the presence  16 of inhibitors of viral DNA replication the y genes are expressed at a much lower level than that observed during normal infection, whereas  f  gene expression is  unaffected. Conversely, if viral DNA synthesis is blocked the Y2 genes are not expressed at all. The majority of y genes encode viral structural proteins. As mentioned previously HSV encodes a protein, VHS, that destabilizes both cellular and viral mRNAs. It has been suggested that a possible role of this molecule is to help regulate the temporal cascade of gene expression by helping to degrade messages soon after their synthesis. This action of VHS would help to modulate the transition between the translation of early genes and late gene transcripts. Viral DNA synthesis is thought to take place at least in part by a rolling circle mechanism. This means that the parental genome must circularize prior to DNA replication, a process which has been demonstrated in vitro (Wadsworth et al. 1975). The viral genome is replicated using a virally encoded DNA polymerase. The concatomeric replicated viral DNA is cut into genomes  and packaged into empty nucleocapsids, in a process requiring at least seven virally encoded proteins (Addison et al. 1984; Addison et al. 1990; Al-Kobaisi et al. 1991; Liu and Roizman 1991a; Sherman and Bachenheimer 1987; Sherman and Bachenheimer 1988). These proteins are the products of the UL6, UL25, 36 genes, and their role in the assembly of nucleocapsids has 32 and UL UL28, UL been inferred from the analysis of temperature sensitive mutations in these genes. In addition, the role of the two products of the UL26 gene in the encapsidation of viral DNA has been examined and these proteins have been found to be essential for viral replication (Gibson and Roizman 1972; Liu and Roizman 1991a; Preston et al. 1983; Weinheimer et al. 1993).  17 Egress of herpes simplex viruses An outline of the models for viral egress are presented in Fig.2. The exact mechanism of herpesvirus egress is unknown. Several views exist on the site and mechanism of viral envelopment. The early stages of viral egress are well accepted (reviewed in Roizman and Bafterson 1986). In the first step, nucleocapsids containing viral DNA associate with a modified region of the inner nuclear membrane and become enveloped resulting in enveloped particles in the perinuclear space (Darlington and Moss 1968; Morgan et al. 1959; Nii et al. 1969). It is thought that the modifications at the site of envelopment represent the accumulation of viral tegument proteins, and that upon budding of the nucleocapsid into the perinuclear space the virion acquires a tegument as well as an envelope derived from the inner nuclear membrane. The inner nuclear membrane of infected cells has been shown to contain precursor forms of viral envelope glycoproteins (Compton and Courtney 1984). This is not surprising because the membrane of the rough endoplasmic reticulum (the site of synthesis of viral glycoprotein) is continuous with the inner nuclear membrane. Therefore it is likely that the enveloped virus particles in the perinuclear space contain “immature” precursor forms of the viral glycoproteins. It is at this point where current models for viral egress diverge. In one model, the virions in the perinuclear space enter the endoplasmic reticulum, and are transported through the secretory apparatus en route out of the cell. The viral envelope glycoproteins acquired in an immature state from the inner nuclear membrane (Compton and Courtney 1984) are processed to their mature forms as the virion is transported through the successive cisternae of the Golgi apparatus. Finally, the mature virion is secreted from the cell. The evidence for this model comes from electron microscopic studies (Morgan et al. 1959), and from the biochemical analysis of HSV infection in the presence of the  18 Fig. 2. Proposed models for the egress of alphaherpesviruses. 1) Model proposed by Johnson and Spear. Briefly, virions bud into the perinuclear space and obtain a tegument and an envelope containing “immature” viral glycoprotein. The virions then move from the perinuclear space to the endoplasmic reticulum (ER) and are transported through the Golgi apparatus and the trans Golgi network (TGN) before being secreted from the cell. 2) Model proposed by Jones and Grose/ Whealy and others. Briefly, virions which have budded into the perinuclear space de-envelope at the outer nuclear membrane or the endoplasmic reticulum releasing naked nucleocapsids into the cytoplasm. The cytoplasmic nucleocapsids then bud into virus induced cytoplasmic vacuoles (VICV) (Jones and Grose) or a late Golgi component (TGN) (Whealy et al.) which contain “mature” viral glycoprotein, before being secreted from the cell. 3) Point at which the models converge.  19  us Penucle space  *  * ‘ Cytoplasm  Cis Golgi  D  1  Medial Golgi  (  Trans Golgi -  I  *  /  Plasma Membrane  20 carboxylic polyether ionophore monensin (Johnson and Spear 1982). Monesin treatment of cells has been shown to cause the accumulation of vacuoles derived from the Golgi apparatus and block the secretion of soluble proteins and the transport of membrane proteins from the Golgi to the plasma membrane of the cell (Johnson and Schlesinger 1980; Tartakoff and Vassalli 1977; Tartakoff and Vassalli 1978). Monensin is thought to act by disrupting ion gradients in the cell which appear to be critical for the budding of transport vesicles from the Golgi complex (Pressman 1976). Johnson and Spear (Johnson and Spear 1982) were able to show that HSV infected cells treated with monensin did not release infectious virus from the cell, although infectious virus was found inside the cell. In addition, viral glycoproteins were underprocessed and were found in association with intracellular enveloped virions, and virus particles inside the cell accumulated in vacuoles likely derived from the Golgi apparatus. Taken together these results suggested that HSV particles associated with membranes of Golgi apparatus while the viral envelope glycoproteins were in an “immature” form. The processing of glycoproteins to “mature” forms takes place in the Golgi apparatus. Therefore it is conceivable that the virus is transported through the successive cisternae of the Golgi apparatus and the glycoproteins resident in the viral envelope become fully glycosylated as this occurs. These results suggested that HSV may be released from cells in a fashion similar to that described for soluble, secreted proteins. In another model for alphaherpesvirus egress, the nucleocapsid which has budded into the perinuclear space de-envelopes at the outer nuclear membrane or the endoplasmic reticulum (effectively the same membrane) releasing the nucleocapsid into the cytoplasm (Whealy et al. 1991). These cytoplasmic particles then bud into cytoplasmic membranes derived from the Golgi apparatus (Jones and Grose 1988; Stackpole 1969; Whealy et al. 1991;  21 Whealy et al. 1990b) and thereby acquire an envelope. These membranes contain fully processed viral glycoproteins. The virus is then secreted from the cell. This model is supported by the study of two alphaherpesviruses, VZV and PrV (Jones and Grose 1988; Whealy et al. 1991). Jones and Grose showed by quantitative electron microscope autoradiography that the predominant site of VZV envelopment was at virally induced post-Golgi cytoplasmic vacuoles which contained viral glycoprotein. Whealy and others were able to show that in PrV infected cells treated with the drug brefeldin A (BFA) enveloped particles accumulated in the perinuclear space and unenveloped particles were predominant in the cytoplasm. BFA causes the redistribution of membranes from the Golgi apparatus to the endoplasmic reticulum (Lippincott-Schwartz et al. 1990; Lippincott-Schwartz et al. 1989). Furthermore, in PrV infected cells not treated with BFA, nucleocapsids were found in close proximity to membranes of the trans-Golgi in structures which were thought to represent envelopment intermediates. Whether these data supporting the two models of viral egress conflict with each other or not remains to be determined. It may be that both pathways of egress described above are active in alphaherpesvirus infection and that one or the other predominate for a particular virus. There is also evidence to suggest that the type of cell line used for the study may have an influence on the observations of virus-Golgi interactions (Campadelli-Fiume et al. 1993). In particular, Campadelli-Fiume and others observed that infection with HSV induced the redistribution of Golgi components in some cell types but not in others. Furthermore, the role of virally encoded molecules involved in egress has been shown to be cell specific (Baines et al. 1991). Baines and others were able to determine that the product of the HSV-1 UL O gene was necessary for 2 viral egress beyond the perinuclear space in Vero cells but was not required for  22 HSV-1 egress in 143 tlc cells. It is clear that further study is required to elucidate the mechanisms of herpesvirus egress. Study of the herpes simplex virus lifecyde Several approaches have been taken to determine the roles of viral  proteins and cellular functions in the lifecycle of herpes simplex viruses. The involvement of particular cellular structures in the lifecycle of HSV has been inferred from microscopic examination of infected cells. The engineering of viral mutants defective in one gene and the subsequent examination of the ability of the mutant virus to propagate in a host cell has furthered the understanding of the role of many viral proteins. In addition, the isolation of temperature sensitive mutants in essential viral genes has proved useful for examining the role of these gene products. The use of drugs to block specific cellular processes has been very helpful in determining some of the cellular requirements for productive HSV-1 infection, and learning about the mechanisms of viral egress. Infection of well characterized somatic cell mutants defective in known enzymatic functions have also aided in the discovery of some of the cellular requirements for viral infection. Although the methods described above have yielded much information about herpes simplex virus infection, there is another effective way to identify important virus-cell interactions. The approach is to use the virus as a selective agent to isolate somatic cell mutants resistant to herpes simplex virus infection. This procedure, outlined below, has been used successfully to isolate several mutant cell lines. This technique has identified virus-cell interactions which are required for viral infection but are not essential for the growth of cells in culture. It is thought that this approach will be useful for identifying targets for antiviral drugs which could inhibit viral infection effectively yet not grossly effect cell viability.  23 Isolation of gro mutants able to survive exposure to HSV-1 The observation that cells can mutate from a virus sensitive state to a virus resistant state is not a new one (Luria and Deibruck 1943). The rationale for attempting to isolate and characterize somatic cell mutants able to survive exposure to HSV came from the great success of earlier studies in which mutant  Esherichia coli (E. coli) surviving exposure to  phage were isolated (Friedman  et a!. 1984a). These mutant strains of E. coli (some of which were called gro mutants) showed defects in many aspects of the ?. life cycle including attachment of the phage to cells, transcription of viral genes, phage DNA replication and assembly of new phage particles. To isolate mutant cells able to survive exposure to HSV-1 mouse LMtk cells were mutagenized with ethyl methanesulfonate prior to exposure to HSV 1. Colonies of cells surviving exposure to HSV-1 were cloned and further analyzed for their ability to propagate HSV-1. These mutant cells were termed gro mutants (Tufaro et al. 1987). Mouse L cells were chosen as a parental cell line because they readily propagate HSV-l and grow well in culture. The majority of the gro mutants isolated shared the same phenotype. One such gro mutant, gro2C, has been characterized extensively (Gruenheid et al. 1992). These cells were defective in the synthesis of the glycosaminoglycan heparan sulfate, a molecule which has been implicated in the initial interaction of the virus with the cell (Shieh et al. 1992; WuDunn and Spear 1989). Another mutant, gro29, had a considerably different HSV-1 resistance phenotype; the analysis of this cell line is the subject of this thesis. gro29 cells In a previous study, a mouse L cell mutant termed gro29 was isolated (Tufaro et al. 1987) and shown to be defective in the growth of two enveloped viruses, vesicular stomatitis virus (VSV), which buds from the plasma  24 membrane, and herpes simplex virus type 1 (HSV-1), which buds from the inner lamellae of the nuclear membrane (Darlington and Moss 1968; Morgan et aL 1959; Nii et al. 1969). Initial characterization of this cell line showed that VSV-infected gro29 cells were defective in the transport and processing of newly-made G protein, the envelope glycoprotein of VSV. Despite this defect, the release of infectious VSV from gro29 cells was diminished only three-fold when compared with the normal parental L cells, suggesting that the secretory defect in this cell line is not critical for the maturation and egress of VSV from the plasma membrane. The effect of this lesion on the release of infectious HSV-1 is very different, however. Although gro29 cells are infected efficiently and the replication cycle proceeds to the late stages of viral gene expression, the spread of HSV-1 from cell to cell does not occur and under-processed glycoproteins accumulate inside the gro29 cells at late times of infection (Tufaro et al. 1987). To further the understanding of the virus-host interactions governing these processes, the nature of the block to virus production in the gro29 cell line was investigated. In this thesis it is demonstrated that the rate of transport and processing of HSV-1 glycoproteins from their site of synthesis in the ER to the cell surface was impeded in infected gro29 cells. This defect in protein transport reduced but did not eliminate the appearance of viral glycoproteins in the plasma membrane of the infected cells. By contrast, newly-assembled virions failed to exit the host cell. In infected gro29 cells, the intracellular virions contained predominantly immature forms of the envelope glycoproteins and accumulated in cytoplasmic vacuoles resembling those that accumulate in cells treated with the carboxylic ionophore monensin, which also blocks HSV-1 egress (Johnson and Spear 1982). If the observations of HSV-1 infection in gro29 cells represent intermediates of a “normal” infection then the data presented in  25 this thesis suggest that the egress of HSV-1 in L cells occurred in a fashion  similar to that described by Johnson and Spear (Johnson and Spear 1982). Furthermore, the phenotype of the gro29 mutant cell line was unique with regard to HSV-1 infection and the experiments suggested that HSV-1 required a host cell component for efficient virus maturation and egress that was distinct from those components which facilitated the trafficking of viral membrane glycoproteins such as VSV G protein. To further examine the nature of the mutation(s) in gro29 cells the phenotype of the uninfected cell was examined. It was determined that gro29 cells were resistant to the lectins ricin and modeccin, a unique lectin resistant phenotype described for only one other cell line (Laurie and Robbins 1991). These lectins bind to galactose (Gal) and N-acetylgalactosamine (Ga1NAc) containing glycoconjugates. Furthermore, gro29 cells showed a reduced ability ; 120 to bind fluorescently conjugated ricin, but not fluorescently conjugated RCA which binds Gal. In addition, gro29 cells failed to synthesize the glycosaminogycan chondroitin sulfate normally but were able to synthesize heparan sulfate glycosaminoglycans. A major difference between chondroitin sulfate and heparan sulfate is that chondroitin sulfate contains Ga1NAc whereas heparan sulfate does not. Based on the lectin binding properties of gro29 cells and the glycosaminoglycan synthesis profile exhibited by these cells it was suggested that gro29 might have suffered a defect in the metabolism of Ga1NAc.  26  Materials and Methods  Cells and viruses The clone 1D line of LMtk mouse fibroblasts was the parental cell line of the mutant gro29 cells. The mutant gro29 cell line was obtained from F. Tufaro and was selected for the ability to survive HSV-1 infection (Tufaro et al. 1987). Vero cells were obtained from S. McKnight. All cells were grown at 37°C in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% fetal 2 atmosphere. The virus used was the HSV-1 bovine serum (FBS) in a 5% CO KOS strain obtained from D. Coen. Stocks of HSV-1 used for infections were grown in Vero cells. Materials Asialofetuin, 5-Bromo-deoxyuridine (BrdU), Bovine serum albumin (BSA, fraction five), UDPG1c dehydrogenase, UDPGa1NAc, UDPG1cNAc, UDPGa1, UDPG1c, pronase, chondroitinase ABC lyase, heparitinase, hyaluronidase, shark cartilage chondroitin sulfate, PET cellulose and lectins (except for modeccin) were obtained from Sigma (St. Louis, MO). Modeccin was obtained from Inland Scientific (Austin, Texas). Scintilation cocktails (Ready Safe and Ready Prot.), all ultracentrifuge rotors and tubes, and HPLC columns were from Beckman (Palo Alto, California). Anti-BrdU monoclonal antibody, cytochrome C (from horse heart), NADPH, CMP-sialic acid, and endoglycosidase H were from Boehringer Mannheim (Laval, Quebec). DEAE Sephacel was from Pharmacia LKB (Piscataway, New Jersey). Zwittergent was from Calbiochem (La Jolla, California). All radioactive reagents were purchased from Dupont-NEN ]D-[6H 5 and 3 S1-sulfate 3 (Mississauga, Ontario) with the exception of [ glucosamine which were purchased from ICN (St. Laurent, Quebec). Brefeldin A  27 (BFA) was from BioCan (Mississauga, Ontario). All tissue culture products and all conjugated secondary antibodies were purchased from Gibco/BRL (Burlington, Ontario). Antibodies to HSV-1 glycoproteins were kind gifts from M. Zweig and R. Philpotts. Polyclonal antisera against p58 was a kind gift from  J.  Saraste. Harvesting herpes simplex virus Medium was removed from infected cell monolayers (lOml) and subjected to low-speed centrifugation (600 Xg, 10 mm, 4°C) to pellet cell debris. The supernatant was centrifuged for 2 h at 20,000 Xg to pellet the virions, and the resulting pellet was resuspended in 1 ml of 10 mM Tris, pH 7.8, 50 mM NaC1 on ice. This material was sedimented through a 10 ml 5-40% Dextran T10 gradi ent formed in 50 mM NaCl, 10 mM Tris pH 7.8 for 1 h at 22,000 RPM in a Beckman SW41 rotor. Gradients were fractionated from the bottom of the tube into 0.5 ml fractions. For determination of radioactivity in insoluble material, 10% of each fraction to be analyzed was added to 50 jig BSA (lmg/ml) followed by I ml of 10% cold trichloroacetic acid (TCA). Insoluble material was collected onto filters after 1 h on ice and radioactivity was determined by liquid scintillation spectroscopy. For determination of virus titres, fractions were diluted serially with medium and used to inoculate monolayers of Vero cells growing in 96-well dishes. Titres were calculated when generalized cytopathic effect (CPE) was noticed in control infected samples. For electrophoretic analyses, samples of fractions to be analyzed were centrifuged at 436,000 g for 20 mm  in a Beckman TLA 100.2 rotor. Pelleted material was solubilized in sodium  dodecylsulfate (SDS) sample buffer and subjected to SDS polyacrylamide gel electrophoresis (SDS PAGE) as described (Laemmli, 1970). Following electrophoresis, gels were fixed, dried and autoradiography was performed. In  28 some cases, protein in gels was transferred to nitrocellulose membranes for western blot analysis prior to autoradiography. Determination of virus titre  Medium was completely removed from infected cell monolayers at various times of sampling and replaced with fresh medium. Each sample of medium to be titred was centrifuged (600 Xg, 10 mi  4°C) to remove cell debris.  Serial ten-fold dilutions of each sample were made and used to inoculate confluent monolayers of Vero cells. After I h, the innoculum was removed and the monolayers were overlayed with fresh DMEM containing 4% FBS and 0.8% agar or Methocell. Duplicate wells for each sample were analyzed after three and five days. Pulse-chase labeling experiments Monolayers of L cells and gro29 cells growing in 60mm dishes were infected with HSV-1 (multiplicity of infection (MOl)  =  10). After I h the virus  was removed and replaced with complete medium. At 5 h post infection (HPI) cells were washed three times with methionine-free medium and labeled for 30 mm  with 100 .tCi/ml [ S]-methionine 3 5 (1117 Ci/mmol) in methionine-free  medium containing 5% dialyzed FBS. At the completion of labeling, cells were  either harvested immediately or washed three times and incubated in DMEM/10% FBS (which contains excess methionine) for various times. Cells were harvested by washing the monolayer with cold phosphate buffered saline (PBS) and incubated for 15 mm  with 1 ml cold lysis buffer (10 mM Tris-HC1 pH  7.4, 150 mM NaCl, 1% Nonidet P-40 (NP-40), 1% Na-deoxycholate). Immunoprecipitations  1/10 volume of 10% NP-40, 10% Na-deoxycholate, 1% SDS was added to aliquots of cell lysates to be precipitated. The appropriate volume of anti-HSV-1  29 gD or anti-HSV-1 gB monoclonal antibody was added to each aliquot and the sample incubated overnight on ice. A 10% suspension of Staphylococcus aureus cells was then added to each sample equal to 20 volumes of primary antibody. This mixture was rocked at 4°C for 2 h before the cells were pelleted and washed once each with 0.5 ml of the following solutions; wash buffer #1 (20 mM Tris HC1 pH 7.5, 150 mM NaC1, 1% NP-40), wash buffer #2 (20 mM Tris-HC1 pH 8.8, 150 mM NaC1, 1% NP-40, 0.2% SDS), and wash buffer #3 (20 mM Tris-HCL pH 6.8, 150 mM NaC1, 1% NP-40, 0.2% SDS). The final pellet was resuspended in SDS sample buffer, heated at 100°C for 5 mm and subjected to electrophoresis through a 10% SDS polyacrylamide gel. Gels were fixed, dried and exposed to Kodak XAR-5 film for autoradiography. Endoglycosidase H digestions The Staphylococcus aureus pellets were resuspended in 40 p.1 2X endo H buffer (1% SDS, 5% 13-mercaptoethanol, 2 mM NaN3, 100 mM Na-citrate, pH 5.5), heated at 100°C for 5 mm, centrifuged to pellet the cells and the supernatants transferred to a fresh tube. Each supernatant was divided into two tubes containing 20 p.1 distilled water (dH O). The material was then digested or 2 mock-digested for 18 h at 37°C by the addition of 1 mU of endoglycosidase H O. Samples were subjected to polyacrylamide gel 2 (endo H) or 1 p.1 dH electrophoresis and autoradiography as described above. Western blots Cellular extracts were subjected to SDS polyacrylamide gel electrophoresis and electroblotted to nitrocellulose membranes as described by Towbin and others (Towbin et al. 1979). Proteins were stained using monoclonal antibodies and an alkaline phosphatase-based detection kit from Gibco/BRL, Ontario, Canada.  30 Fractionation of microsomes This procedure was adapted from Saraste and others (Saraste et al. 1986). Briefly, monolayers of infected or uninfected cells were harvested by incubation with trypsin and the trypsin was quenched by the addition of cold DMEM containing 10% FBS. All further steps were carried out in the cold. Cells were pelleted by centrifugation for 10 mm, washed with DMEM containing 10% FBS, followed by a wash with homogenization buffer (0.25 M sucrose, 10 mM Tris, pH 7.4, 10 mM KC1, 1.5 mM MgC12). Cells were then pelleted, resuspended in 4 pellet volumes of homogenization buffer and homogenized in a Dounce homogenizer. Homogenization was monitored by phase contrast microscopy with the aim of maximizing cell breakage and minimizing nuclear disruption. Nuclei and cell debris were removed from the homogenate by centrifugation (600 Xg, 10 mm, 4CC). The pellet was then washed with homogenization buffer and the supernatants combined. The supernatant was centrifuged at 10,000 Xg for 20 mm  to remove mitochondria. The post mitochondrial supernatant was  then decanted and layered on top of 5 ml of 0.33 M sucrose which had been layered over Imi of 2 M sucrose. This was centrifuged at 25 K rpm (107, 000 Xg) in a SW4I rotor for 1 h. Approximately 600 p.1 of the turbid band at the 2 M /0.33 M interface was removed with a syringe. This material was suitable for further purification of Golgi membranes. This total microsome sample (600 p.1) was made 50% in sucrose with the addition of 2.4 ml of 2 M sucrose and was added to a SW41 tube. The following sucrose solutions (w/w) were layered overtop: 1 ml of 45%; 1.5 ml each of 40%, 35%, 30%, 25%, and 2 ml of 20%. The step gradients were then centrifuged at 170,000 Xg for 19 h at 4°C. Following centrifugation, 0.5 ml fractions were collected from the bottom of the tube. 150 p.1 of each fraction was diluted with 0.5 ml dH2O and centrifuged at 100 K rpm (436,000 Xg) for 10 mm in a TLA 100.2 rotor. The supernatants were discarded  31 and the pellets resuspended in 30 jil of SDS sample buffer, boiled for 5 mm, and subjected to polyacrylamide gel electrophoresis. Polypeptides were transferred to nitrocellulose by western blotting and specific polypeptides were detected using monoclonal antibodies as described above. The density of the fractions ranged from 1.08 to 1.24. The location of the intermediate compartment between the ER and the Golgi was determined by western blotting using an anti-p58 monoclonal antibody. p58 served as a useful marker for the location of this compartment. This fractionation procedure yielded a protein peak in fraction 5 and a peak of the ER marker NADPH cytochrome C reductase in fraction 4. Indirect immunofluorescence Cells were grown on glass coverslips for 3 days and infected with HSV-1 (MOI=5). At 13 HPI, cells were rinsed with PBS, fixed with 2% formaldehyde in PBS. Cells were permeabilized in PBS! 1% BSA (PBS-BSA) and 0.2 % Triton X 100 for 3 mm. Permeabilization was stopped by rinsing the cells in PBS-BSA. The cells were then incubated with a 1 / 100 dilution of anti-gD monoclonal antibody in PBS-BSA. Monolayers were washed extensively and incubated with 1/100 dilution of rhodamine-conjugated goat anti-mouse IgG in PBS-BSA for 30 mm. Coverslips were then washed and mounted in 50% glycerol, 100 mM Tris, pH. 7.8. Images were photographed using a Zeiss microscope with epifluores cence optics. Confocal images were captured using a Bio-Rad MRC-500 confocal fluorescence microscope. Fluorescence microscopy using lectins Cells were grown on glass coverslips in 35 mm plastic dishes for 3 days prior to fixation. For fixation, cells were rinsed with PBS, and incubated in 2% formaldehyde in PBS for 10 mm. Fixation was stopped by rinsing cells in PBS. Cells were permeabilized in PBS-BSA and 0.2 % Triton X-100 for 3 mm.  32 Permeabilization was stopped by rinsing the cells in PBS-BSA. Cells were then incubated for 30 mm  in 20 p.g/ml FITC-conjugated ricin, wheat germ agglutinin  120 in separate incubations. Monolayers were then rinsed in (WGA) and RCA PBS-BSA to remove unbound molecules. After rinsing with PBS-BSA, the coverslips were mounted in 50% glycerol (w/v) in PBS and photographed using a Zeiss Axiophot microscope with epifluorescence optics. Electron microscopy Cells were grown on Millicell HA inserts (Millipore Corporation) for 24 h prior to infection with HSV-1 (MOI5). At 18 HPI, cells were rinsed with PBS and monolayers were fixed in 2.5% glutaraldehyde in 0.1 M Na-cacodylate, pH 7.3 for 1 h on ice. Cells were then rinsed and post-fixed in 1% 0504 for 1 h. These samples were rinsed, dehydrated and embedded in plastic. Specimens were sectioned, stained and photographed using a Zeiss EM1OC transmission electron microscope. Analysis of HSV-1 gD in BFA treated cells This procedure was adapted from Saraste and others (Saraste et al. 1986). Briefly, monolayers of BFA-treated and untreated infected cells were harvested by trypsinization, and washed extensively with cold DMEM/10% FBS. All further steps were carried out in the cold. Microsomes were prepared and subfractionated as described above. Following centrifugation, 1 ml fractions were collected from the bottom of the tube. 0.3 ml of each fraction was diluted with O and centrifuged at 436,000 Xg for 10 mm in a TLA 100.2 rotor. The 2 0.5 ml dH supernatants were discarded and the pellets containing the membranes were suspended in 40 l endoglycosidase H buffer (50 mM sodium citrate (pH 5.5), 0.5% SDS, 0.25% 3-mercaptoethanol, 1 mM NaN3), heated at 100°C for 5 mm, and centrifuged. Each supernatant was divided into two tubes containing 20 p.1  33 O. The material was then digested or mock-digested for 18 h at 37°C by the 2 dH addition of 1 mU of endo H or 1 j.il of dH O. Samples were subjected to 2 polyacrylamide gel electrophoresis and HSV-1 gD was detected on Western immunoblots. Analysis of glycosaminoglycans Biochemical labeling of glycosaminoglycans was performed by a modification of procedures described previously by Bame and Esko (Bame and Esko 1989). Briefly, glycosaminoglycans were radiolabeled by incubating cells for 35 (carrier free) and 20 p.Ci/ml D-[6[ 3 days with 10 Ci/ml S]sulfate Hlgiucosamine (38.8 Ci/mmol) in DMEM-FBS modified to contain 10 mM Na3 sulfate and 1 mM glucose. The cells were washed three times with cold PBS and solubilized with 1 ml of 0.1 M NaOH at 25°C for 15 mm. Samples were removed for protein determination. Extracts were adjusted to pH 5.5 by the addition of concentrated acetic acid, and treated with 2 mg/mi pronase in 0.32 M NaCl-40 mM sodium acetate, pH 5.5, containing shark cartilage chondroitin sulfate (2 mg/mi) as carrier, at 40°C for 12 h. For some experiments portions of the radioactive material were treated for 12 h at 40°C with 10 mU of chondroitinase ABC lyase, 0.5 U of heparitinase, or hyaluronidase. The radioactive products were quantified by chromatography on DEAE-Sephacel by binding in 100 mM NaCl, followed by elution with 0.7 M NaCl, or by cetylpyridinium chloride precipitation (Wasteson et al. 1973). For I-IPLC analysis, the glycosaminoglycan samples were de-salted by precipitation with ethanol (Bame and Esko 1989). Following centrifugation, the ethanol precipitates were suspended in 300 j.tl 20 mM Tris, pH 7.4 and 50 il was resolved by anion exchange HPLC using a 15 x 75 mm TSK DEAE-3SW column. Proteoglycans were eluted from the column using a 77 ml linear 50 mM to 700 mM NaC1 gradient formed in 10 mM KH2PO4 (pH 6.0). All buffers contained  34 0.2% Zwittergent 3-12 to extend the life of the column, as reported previously (Bame and Esko 1989). The glycosaminoglycans in the peaks were identified by digestion of the sample with the relevant enzymes prior to chromatography. Enzyme and protein assays NADPH cytochrome C reductase, an ER associated membrane protein which mediates the reduction of cytochrome C, was measured by the following spectrophotometric assay. Cell fractions (50tl) on ice were added to a freshly made reaction cocktail (410 ii 0.1 M potassium phosphate buffer pH 7.5, 20 jil 20% Triton X-100, 30 p1 5 mg/ml cytochrome C, 12 il 50 mM KCN) in a spectrophotometer cuvette. To start the reaction 120 p1 of 0.5 mM NADPH was added, the solution mixed and the increase in absorbance at 550 nm measured over time in a Hitachi spectrophotometer (Omura and Takesue 1970). The increase in absorbance at 550 nm reflects the conversion of NADPH to NADP by the enzyme. Sialyltransferase, a trans Golgi and trans Golgi network resident enzyme, was measured as described previously by Brändli and others (Brändli et al. 1988). UDPGa1-4-epimerase activity, and the conversion of UDPGa1NAc to UDPG1cNAc were measured exactly as described by Kingsley and others (Kingsley et al. 1986). Separation of nucleotide sugars on PET cellulose was performed as described (Kingsley et al. 1986; Randerath and Randerath 1965). Protein concentrations were determined using a Bio-Rad protein assay reagent. Purification of Golgi membranes for nucleotide sugar translocation assays Golgi membranes were purified from L and gro29 cells according to the method of Balch and others (Balch et al. 1984). All solutions and equipment used were kept as cold as possible throughout the procedure. Briefly, 50 150 mm  35 dishes of L cells and 70 150 mm dishes of gro29 cells were grown to subconfluency, harvested by incubation with trypsin, collected by low speed centrifugation (600 Xg, 10 mm, 4°C), washed two times with cold medium containing 10% FBS and once in homogenization buffer (250 mM sucrose, 10 mM Tris-HC1 pH 7.4) prior to resuspension of the cells in five pellet volumes of homogenization buffer, to yield a 20% suspension. Cell suspensions were homogenized on ice using a Dounce homogenizer with a tight fitting pestle. Homogenization was monitored by phase contrast microscopy with the aim of maximizing cell breakage with minimal nuclear disruption. 6 ml portions of crude cell extract were adjusted to 1.4 M sucrose by the addition of a equal EDTA 2 volume of cold 2.3 M sucrose, 10 mM Tris-HC1 pH 7.4, made 1 mM Na from a 100 mM stock solution and loaded into a prechilled SW28 centrifuge tube. The homogenate was gently overlayed with 14 ml of ice cold 1.2 M sucrose 10 mM Tris-HC1 pH 7.4 followed by 8 ml of 0.8 M sucrose 10mM Tris-HC1 pH 7.4. The tubes were then centrifuged at 90 000 Xg for 2.5 h in a Beckman SW28 rotor. After centrifugation the white turbid band at the 1.2 M /0.8 M sucrose interface was removed with syringe in a minimal volume; this fraction contained the (l) and stored at -70° 2 Golgi apparatus. Aliquots of this material were frozen in N C. Using sialyltransferase as a Golgi marker activity it was determined that these fractions were enriched approximately 10 fold in this activity over crude cell extract for both L and gro29 Golgi membrane fractions. Nucleotide sugar translocation assays The transport of nucleotide sugars was measured by a modification of a procedure described previously (Brändli et al. 1988; Deutscher and Hirschberg 1986). [‘ C]-acetate was used as a membrane impermeable standard in these 4 assays. Assays were performed in the presence of concentrations of non radioactive nucleotide sugars varying from 0 to 25 p.M. 350 p.1 of assay buffer (250  36 mM sucrose, 150 mM KC1, 1 mM MgC12,10 mM Tris pH 7.5) containing 0.1 p.Ci H]-CMPSA (25.1 Ci/mmol) and 0.1 3 each of [ H]-UDPGa1NAc (8.3 Ci/mmol)(or [ 3  j.tCi [14C1 acetate (1.9 mCi/mmol) in a Beckman TLA 45 centrifuge tube was preincubated for 10 mm at 30°C. The reaction was started by the addition of 50  ill of Golgi membranes (17-25 .tg protein). The reaction mixture was incubated for a further 10 mm  at 30°C. The reaction was terminated by the addition of 600  p.1 ice cold assay buffer and immediate placement on ice. The membranes were then pelleted by centrifugation in a TLA 45 rotor at 100 000 Xg for 20 mm at 4°C. The surface of the pellets were rinsed once with 1 ml of ice cold assay buffer and recentrifuged to concentrate the membranes. The supernatant was decanted and 500 p.1 of lysis buffer (2% SDS, 5 mM EDTA, 500 mM Tris pH 8.8) was added to the pellets and the solubilized material analyzed by liquid scintillation spectroscopy. The relative amounts of solutes nonspecifically associated with the pelleted Golgi membranes was corrected for by determining the amount of radiolabeled C]-acetate 14 associated with the pelleted material. [ The time course assays were performed in a similar fashion except that the concentration of non-radioactive nucleotide sugar used was 0.5 p.M and the time of incubation was varied from 1- 20 mm. Glucose and sulfate uptake assays 35 mm dishes of containing 5 X iO L and gro29 cells were incubated at H1-glucosamine/ ml of low sulfate/low 3 37°C with medium containing 20 p.Ci [ glucose DMEM containing 4% dialyzed FBS (dialyzed against PBS) for times ranging from 1-60 mm. At the end of the labeling period dishes were rinsed 3 X with PBS prior to lysis of the cells in 700 p.1 0.1 N NaOH. Samples of extract were analyzed for protein concentration and radioactivity. To determine the kinetics of glucose uptake in L and gro29 cells the same procedure described above was employed with the exception that the incubation time was 60 minutes (which  37 was determined to be in the linear range for glucosamine uptake over time) and the incubation medium contained varying concentrations of glucose from 0-300 i.tM. Because glucosamine and glucose use the same translocator it was assumed that the translocator had similar affinity for both glucose and glucosamine. Therefore these assays should reflect the relative ability of L and gro29 cells to translocate glucose. To determine the rate of sulfate uptake in L and gro29 cells the procedure 35 was [ described above was used with the exception that 10 p.Ci/ml 5]-sulfate included in the incubation medium.  38 Chapter 1- Analysis of HSV-1 infected gro29 cells.  In a previous study the mouse L cell mutant gro29 was isolated on the basis of the ability to survive exposure to HSV-1 (Tufaro et al. 1987). This cell line was shown to be defective in the propagation of both VSV and HSV-1. Initial characterization of this cell line revealed that gro29 cells were defective in the transport and processing of newly made G protein, the envelope glycoprotein of VSV. Despite this defect the release of infectious VSV from these cells was diminished only three-fold when compared to the parental L cells, suggesting that the secretory defect in these cells was not critical to the maturation and egress of VSV from the plasma membrane. However, the effect of this lesion on the release of infectious HSV-1 from gro29 cells was very different. Although gro29 cells were infected efficiently and the replication cycle proceeded to the late stages of viral gene expression, the spread of HSV-1 from cell to cell did not occur and underprocessed glycoproteins accumulated inside the gro29 cell late in infection. To characterize the nature of the block to HSV-1 infection in gro29 cells the following study was performed. Results Production of infectious virus by gro29 cells.  It has been shown previously by indirect immunofluorescence that plaques do not form on monolayers of gro29 cells infected with HSV-1, despite the fact that infection occurs normally and progresses to the late stages of viral gene expression (Tufaro et al. 1987). To quantify the block to viral propagation in gro29 cells, the titres of the extracellular virus produced by infected parental L cells and gro29 cells were determined. The medium on each monolayer was replaced every two HPI to minimize the superinfection of cells during the  39 experiment. The titres were determined for each time point and then added together to reflect the total number of PFLJ released from the cells during the 20 h of sampling (Fig. 3). These analyses revealed that the parental L cells released 200 PFU/cell while gro29 cells released 0.1 PFU/cell. There are several possibilities that could account for this deficiency. If viral assembly or egress was defective, the release of virions from gro29 cells would be impaired. If the assembly and egress of the virions was normal, it may be that the specific infec tivity of the virus was diminished due to a structural defect caused by a lesion in the gro29 cells. It has already been established that gro29 cells have suffered a defect in glycoprotein transport and processing that leads to the accumulation of immature forms of HSV-1 glycoproteins in HSV-1 infected cells (Tufaro et al. 1987). Normal synthesis and processing of viral glycoproteins is very important for the propagation of HSV-1 (reviewed in Spear 1985); because of this, experiments were carried out to identify the defects in glycoprotein processing and to characterize the phenomena that contribute to the low yield of infectious virus from the gro29 cell line.  Pulse-chase analysis of HSV-1 glycoprotein processing. HSV—1 specifies at least eleven membrane glycoproteins: gB, gC, gD, gE, gG, gH, gI, gj, gK, gL and gM (Ackermann et al. 1986b; Baines and Roizman 1993; Buckmaster et al. 1984; Heine et al. 1972; Hutchinson et al. 1992a; Hutchinson et al. 1992b; Longnecker et al. 1987; Spear 1976; Spear 1985). gD and gB were investigated in these experiments. Glycoprotein D contains three sites for N linked oligosaccharides (Cohen et al. 1983) and two 0-linked chains (Serafini Cessi and Campadelli-Fiume 1981), whereas gB has nine potential N-linked sites (Bzik et al. 1984a) and an uncharacterized number of 0-linked chains. The  40 Fig. 3. Titre of virus released from HSV-1 infected L and gro29 cells. Monolayers of L and gro29 cells were infected with HSV-1 (MOI=1O). At the indicated times post-infection, the medium was removed and the numbers of infectious particles in the medium were determined by plaque assay on Vero cells as described in Materials and Methods. The results represent the cumulative release of PFU by L and gro29 cells over time Published in Banfield and Tufaro 1990).  0  !dLI 0I.  0  eoI (‘I  uI. 7 I  0  .  ,,  C CD  tO1. (‘I  8’  i• Cl) CD 0.  601.  117  42 precursor forms of these proteins (pgB and pgD) contain high-mannose oligosaccharides sensitive to endoglycosidase H (endo H) which become resistant to endo H as the oligosaccharide moieties are processed to more complex forms (Cohen et al. 1983; Johnson and Spear 1982; Mafthews et al. 1983; Serafini-Cessi et al. 1988; Wenske et aL 1982). To investigate the synthesis and processing of these proteins, pulse-chase analyses of the two glycoproteins in HSV-1 infected cells were performed at 5 HPI (Fig. 4A). When HSV-1 infected parental L cells and gro29 mutant cells  were labeled with S]-methionine 35 for 10 mm, the accumulation of label into gB [ and gD were similar for both cell lines. The major bands representing the newly-synthesized gD in L and gro29 cells had the same relative mobilities in the gel, indicating that the co- and post-translational modifications, such as the addition of N-linked oligosaccharides, occurred normally in both cell lines. Similarly, the synthesis and processing of gB in the two cell lines were indistinguishable at the end of the labeling period. After a 90 mm chase in the parental L cells (Fig. 4A), both gB and gD migrated more slowly in the gels, suggesting that processing of the high mannose oligosaccharides to complex forms, as well as modification by 0-linked glycosylation had occurred. In contrast, most of the gB and gD polypeptides synthesized in gro29 cells did not increase in size during the chase. There was no reduction in the amount of labeled polypeptides in the cells during the chase period, indicating that the bulk of newly-made material persisted in forms that were incompletely or aberrantly processed. The increase in radioactivity observed in several chase samples (L chase, gB for example) may be due to more efficient immunoprecipitation of the more highly processed species. Several explanations could account for the failure to modify newly-made glycoproteins in this cell line. The underprocessed species in the mutant gro29  43  Fig. 4. Processing of HSV-1 gD and gB in L and gro29 cells. Monolayers of L and gro29 cells were infected with HSV-1 (MOI=10). At 5 HPI, cells 5 for 20 mm, rinsed and chased with unlabeled S1-methionine 3 were incubated with [ methionine for 90 mm. HSV-1 gD and gB were immunoprecipitated from total cell extracts using monodonal antibodies and subjected to SDS -PAGE. (A) Autoradiogram showing HSV-1 gD and gB at the end of the labeling period (P) or after a 90 mm chase (C) in L cells (L) and gro29 cells (g29) as indicated. The positions of immature forms of gB and gD (pgB, pgD) and mature forms (gB, gD) are indicated to the left of the Fig. (B, top panel) Autoradiogram showing pulse-chase analysis of HSV-1 gD. The positions of pgD and gD and the duration of chase (mm or h) are indicated. (B, bottom panel) Histogram showing the percent of HSV-1 gD (B, top panel) that was resistant to digestion by endo H. Samples from pulse-chase experiments were digested with endo H and subjected to SDS -PAGE. The relative amounts of endo H resistant gD was determined by densitometric scanning of autoradiograms (not shown). was taken as the amount of bars, gro29 cells. Published in Banfield and Tufaro 1990.  .  Solid bars, L cells; Gray  C  A  gB gD L 2 9 9 29 9 L  gB Lg29  gD L29  pgBO  pgD  B  gD p9Db-  40  0 L 29  ——  L  29 g  90 Lg29  3hr  29 9 L  5 hr L 29  —  C  0  t 0 C ‘U  0  40  90  Duration of chase (mm)  180  300  45  cells may represent glycoproteins that were retained in the ER or Golgi. Alternatively, the newly-synthesized polypep tides may have traversed the Golgi without being processed. This could arise if a component of the ER- or Golgi resident processing machinery was defective in these cells. The following experiments were performed to characterize further the glycoprotein flux through the Golgi complex. Pulse-chase analyses of gD were repeated using a longer chase, and the rates at which newly-made glycoproteins became endo Hresistant were determined (Fig. 4B). Because endo H cleaves high-mannose chains but not complex oligosaccharides, it serves as a useful probe for the processing of N-linked glycoproteins (Kornfeld and Kornfeld 1985). Glycoprotein D was chosen for further analysis because all mature forms of this protein are endo H-resistant, whereas at least some of the high mannose Nlinked oligosaccharides attached to gB remain in a sensitive form (Johnson and Spear 1982; Wenske et al. 1982). It can be seen in Fig. 4B that newly-made gD was processed rapidly in L cells; by 40 mm post-labeling, greater than 95% of the polypeptides were modified to a higher apparent molecular weight, and by 90 mm, processing was complete. In contrast, gD synthesized in gro29 cells failed to become fully processed even after a 5 h chase. By 40 mm, two or three discrete  bands were detected that were larger than pgD, indicating that some modifications had occurred, although a comparison of relative mobilities suggests that these were not the forms that accumulated in the parental L cells. As the chase proceeded (Fig. 4B, bottom), there was a rapid and complete disappearance of endo H-sensitive forms in the L cell samples, indicating that most of the mass of the newly-made protein was modified by the host cell processing machinery. In contrast, the gD synthesized in gro29 cells persisted as endo H-sensitive forms. Oligosaccharide processing was not blocked entirely, however, and 60% of the newly-synthesized gD acquired endo H-resistance  46 during the 5 h chase (Fig. 4B).  These results indicated that the intracellular  transport of HSV-1 glycoproteins was slowed but not abolished in infected gro29 cells. The large shift in molecular weight that accompanies the maturation of gD in L cells was not observed in the gro29 cell samples. This is consistent with the notion that these modifications occur relatively late in the secretory pathway, after the acquisition of endo H resistance.  Analysis of gD transport in HSV-1 infected cells. The data obtained from the pulse-chase analysis suggested that the transport of glycoproteins through the secretory pathway was abnormal. To investigate the accumulation of glycoproteins in the organelles of the secretory pathway during HSV-1 infection, total microsomes were prepared from infected cells at 13 HPI and were subjected to centrifugation on sucrose step gradients. Because the membranes of the ER and Golgi cisternae have different densities, fractions enriched for different organelles and for different Golgi cisternae can be isolated by this technique. Samples of each fraction were subjected to SDS-PAGE and blotted onto nitrocellulose membranes. These gradients included all membranes in the microsomal fraction which separated at densities from 1.24 (fraction 1) to 1.08 (fraction 15). Furthermore, the location of the intermediate compartment that exists between the ER and Golgi complex was determined using an antibody to the protein p58, which has been shown to be located in this compartment and in cis Golgi elements (Jantti and Kuismanen 1993; Saraste et al. 1987). In gradients similar to those shown, the highest concentration of protein was in fraction 5, and the peak of the ER enzyme NADPH cytochrome C reductase was in fraction 4 (data not shown). HSV-1 gD was analyzed using a monoclonal antibody and an alkaline phosphatase-based detection system. In the L cell samples (Fig. 5A), the majority  47  Fig. 5. Western blot analysis of HSV-1 gD in membrane fractions of L and gro29 cells. Parental L and gro29 cells were infected with HSV-1 (MOI=1O) and incubated for 13 h. Cell monolayers were harvested and the total microsome fraction was prepared as described (Materials and Methods). Total microsomes were subjected to centrifugation in sucrose step gradients to fractionate membrane components of different densities. Fractions were solubilized and subjected to SDS-PAGE followed by electroblotting onto a nitrocellulose membrane. The high density membranes characteristic of the ER are in the lower numbered fractions, and the low density membranes characteristic of the Golgi complex are in the higher numbered fractions. The highest concentration of protein was in fraction 4, and the peak of the ER enzyme NADPH cytochrome C reductase was shown to be in fraction 5 in gradients similar to those shown. The HSV-1 gD present in these fractions was detected using an anti-gD monoclonal antibody and an alkaline phosphatase staining procedure. Western blots showing gD in the membranes of (A) L cells and (B) gro29 cells are shown. The arrowhead to the left of the lanes denotes a background band that was also present in uninfected cell samples (data not shown). The position of pgD is indicated to the left of the figure and the sample containing predominantly mature gD (fraction 14) is identified above the lane. The concentration of p58, a protein that has been shown to reside in the intermediate compartment between the ER and Golgi complex, was determined on separate blots using an anti-p58 antibody (data not shown). p58 was enriched in fractions 4-6, with a peak in fraction 5. Published in Banfield and Tufaro 1990).  cL  EL  LL  6  L  I •__  4  66 4  c  9  £  V  cd) 8 (  06 d 4  Sb  49  of pgD was contained in four fractions (4-7) and decreased in concentration towards the top of the gradient (right). Mature forms of gD were detectable by fraction 4 and were the only forms detectable in fraction 14. The distribution of gD in gro29 cell fractions was strikingly different (Fig. 5B). Whereas there was a  peak of pgD in fractions 4 and 5, little if any mature gD fractionated in the gro29 cell gradient. It was evident that gro29 fractions contained less gD overall than did L cell fractions, although the differences were not quantified in these experiments. When these same blots were probed for the presence of the Golgi marker p58, a protein that is enriched in the intermediate compartment be  tween the ER and Golgi complex (Jantti and Kuismanen 1993; Saraste et al. 1987), p 58 was equally abundant in the gro29 cell and L cell fractions, suggesting that the underrepresentation of gD in the gro29 cell fractions was not an artifact of the isolation procedure. This suggested that gD was underrepresented in the cellular organelles contained in these gradients. Because it has been shown that the rate of synthesis of gD was normal in these cells (Fig. 4), it may be that the defect in gro29 cells caused gD to localize into abnormal cellular structures that were lost during this isolation procedure. Analysis of virus egress. Based on the results demonstrating an inhibition of the transport and processing of HSV-1 glycoproteins, and the under representation of gD in the secretory organelles, it was reasoned that the ability of HSV-1 particles to be transported out of the cell might also be impeded. This follows from a current model of HSV-1 egress in which newly-formed virions are thought to traverse the Golgi complex en route out of the cell. To investigate this, parental L cells and gro29 cells were infected with HSV-1 and labeled with [ S]-methionine 3 5 for 18 h. Extracellular virions were harvested from the medium by centrifugation  and subjected to fractionation on Dextran T1O gradients. The TCA-insoluble  50 radioactivity was measured (Fig. 6A) and the titre of infectious particles was determined for each fraction (Fig. 6B). It can be seen in Fig. 6A that L cells released a large amount of virus which peaked in fraction 17. In contrast, there was only a small peak of material detected in the gro29 cell medium (0.2% of L cell), indicating that virus production was impeded. An analysis of the PFU present in each fraction revealed that the peak of radioactivity (Fig. 6A, fractions 15, 16 and 17) corresponded to the peak of infectious virus (Fig. 6B). Fractions 15-17, comprising the peak in each gradient, were pooled and subjected to SDS PAGE (Fig. 6C). A limited number of radioactive bands were detected, consistent with these samples being highly enriched for virions. No radioactive polypep tides were detected in the gro29 samples during the exposure times used. These data support the conclusion that gro29 cells are unable to release virions after infection.  Analysis of cell-associated virus. One way to account for the paucity of extracellular virions is if HSV-1 particles were unable to assemble in the mutant gro29 host cells. To investigate this, nucleocapsids were isolated from total cells and fractionated on sucrose gradients. Fractions were analyzed for TCA-insoluble radioactivity to detect the peak of nucleocapsids (Fig. 7A) and were subjected to SDS-PAGE to detect nude ocapsid proteins (Fig. 7B,C). In Fig. 7A, a small peak of nucleocapsids was evident in fractions 21-28 for both L and gro29 cells. There were fewer nucleocapsids isolated from gro29 cells, although the reduction was minor when compared with the reduction in virion release. Polyacrylamide gel analysis of the gradient fractions (Fig. 7C) revealed that the patterns of newly made polypeptides in the nucleocapsid fractions isolated from parental L cells and gro29 cells were similar. The polypeptides denoted with asterisks were  51 Fig. 6. Comparison of HSV-1 particles released from L and gro29 cells. Parental L cells and gro29 cells were infected with HSV-1 (MOl =10). At two HPI, the 5 S]-methionine. 3 medium was removed and replaced with labeling medium containing [ At 18 HPI, virions released into the medium were harvested by centrifugation, resuspended in buffer and centrifuged through a 5-40% dextran T-10 gradient as described (Materials and Methods). The gradients were fractionated and samples were retained for further analysis. Samples from the top of the gradient are shown to the left. (A) Total TCA-insoluble radioactivity in each sample. CPM; CPM x io. (B) number of PFU in the fractions indicated. PFU; PFU x iO. (C) Autoradiogram showing radioactive polypeptides present in the three peak fractions (16,17, and 18) from each gradient. To prepare the samples, the fractions were pooled, pelleted by centrifugation, solubiized and subjected to SDS-PAGE. The polypeptides in the gel were electro blotted to nitrocellulose membranes and viral proteins were identified using an anti-gD monoclonal antibody and an alkaline phosphatase detection system (data not shown). The blot was then exposed to film for 3 days to detect radioactive polypeptides in the pellets (shown in panel C). No signal was detectable for the gro29 sample. The positions of gD, nucleocapsid proteins (asterisks), and ovalbumin (45Kd) and carbonic anhydrase (29Kd) markers are shown. Titres of fractions were determined by H. Meadows. Published in Banfield and Tufaro 1990.  c2.  16 14 0  —  0  12  10 8 6 4  Fraction number  c)  0  0.  F  B  400  200 100  2  4  6  8  10  12  14  16  17  I  29 g  18  Fraction number  C  D 9  45.  ..  29  —  19  20  53 Fig. 7. Nucleocapsid assembly in L and gro29 cells. Parental L cells and gro29 cells infected with HSV-1 (MOI=10) were labeled with 50 j.iCi/ml S1-methionin e from 2-18 HPI. Cell monolayers were harvested and protein 35 [ extracts were prepared with Nonidet P40-Sodium deoxycholic acid extraction buffer. The extracts were sonicated, 0.5 M Urea was added and insoluble material was pelleted from the sample by low-speed centrifugation. The resulting extracts were layered onto a 1040% sucrose gradient and centrifuged. The gradients were fractionated into 35 samples and analyzed. (A) Radioactivity in TCA-precipitable material for each fraction. The bar over fractions 22-29 represents the peak of nucleocapsids. Open circles; L cells, closed circles; gro29 cells. (B,C) Autoradiograms showing radiolabeled polypeptides present in every other fraction. L cells (B) and gro29 cells (C). Two nucleocapsid proteins that fractionate with nucleocapsids and whole virions are indicated (asterisk). Published in Banfield and Tufaro 1990.  (1/  A  10  5  15  20  25  30  Fraction number  --  4  A Fracton#  1  5  9  13  :I.-  4 17  21  —  29  33  ——  ——  1  a —  I..’  25  —  -  35  55 specific to infected cells and fractionated consistently with nucleocapsids and virions. By comparing these gels to those published previously (Rixon et al. 1990), it was determined that the upper and lower bands represent VP21 and VP22a. The material sedimenting in gro29 fraction 35 represents urea-insoluble material that was not efficiently removed in the step prior to centrifugation. Although the nucleocapsid fractionation procedure gave an accurate assessment of the relative amounts of nucleocapsids present in the cells, it was of some interest to determine the number of intracellular particles that were also infectious. Cells were lysed in the absence of detergent, nuclei were removed, and extracts were fractionated on Dextran T10 gradients. Total TCA insoluble radioactivity in each fraction was determined, as was the number of infectious particles in selected fractions (Fig. 8A,B). In addition, samples were subjected to electrophoresis in polyacrylamide gels to detect the polypeptides sedimenting in the gradient (Fig. 8C). In L cells (Fig. 8B), fraction 12 contained 60% of the infectious particles, and 85% of the infectious particles were in fractions 10-14. gD was detectable in these fractions, as were the two nucleocapsid proteins (asterisks). gro29 cell extracts fractionated differently, however. Whereas the peak of infectious particles was in the same fraction as in the L cell gradient (fraction 12), the peak of radioactivity was at fraction 14 in the gro29 cells gradient (Fig. 8A). Analysis of the polypeptides in these fractions (Fig. 8C) revealed that a low number of polypeptides sedimented in the gradient, as expected for fractions enriched in virions. An analysis of the polypeptides in the L and gro29 cells revealed several differences. There were alterations in the polypeptides in the gB/gC region of the gel, which were not analyzed further. The gro29 fractions were enriched in immature forms of gD (pgD), whereas L cell fractions contained mostly mature gD. The identities of these polypeptides were confirmed by immunoprecipitation and western blots (data not shown).  56 Fig. 8. Detection of intracellular virions. Parental L cells and gro29 cells were infected with HSV-1 (MOI=10) and incubated for 35 Cell monolayers were harvested [ 18 h in the presence of 50 i.tCi/ml S]-methionine. and whole cell extracts were prepared by gentle homogenization in the absence of detergents. Cell debris and nuclei were pelleted from the extracts, which were then centrifuged for I h at 22,000 RPM through a 5-40% Dextran T10 gradient. The gradients were fractionated and samples were retained for further analysis. (A) Radioactivity in TCA-precipitable material. The arrow denotes the peak of infectious particles. CPM= counts per mm. x iO Closed circles=L cells; Open circles=gro29 cells.. (B) The number of PFU in each fraction was determined by limiting dilution analysis and plotted as the percent of total infectious particles in the sample applied to the gradient. Solid=L cells. Striped=gro29 cells. (C) A sample of every other fraction was subjected to SDS polyacrylamide gel electrophoresis and the radioactive polypeptides were detected by fluorography. The L cell fractions are shown in the left lanes (fractions 218); the gro29 fractions are shown in the right lanes (fractions 2-18). The positions of gD and pgD, as determined by immunoprecipitation and western blotting (not shown), are indicated. The relative positions of two nucleocapsid proteins are indicated (asterisk). Titres of fractions determined by H. Meadows. Published in Banfield and Tufaro 1990.  c7  60  A  50  40 30 C.) 20 10 0  4  2  0  10  8  6  12  14  16  18  20  14  16  18  20  Fraction number  a. 0 0  2  4  12  10  8  6  Fraction number  C  2  *:j  6  10V14  18  119D  2  6  1o!14  18  58 Furthermore, most of the mass of radiolabeled gD was associated with the virion-enriched fractions, suggesting that a large portion of the intracellular gD was embedded in viral envelopes. It is clear from these results that newlyformed particles were present in the cytoplasm of infected cells, and it appeared that they were enveloped inasmuch as viral glycoproteins fractionated with the peak of infectivity. The apparent differences in the sedimentation observed between L and gro29 cells were not investigated further. Although this procedure for isolating intracellular viral particles results in contamination of the sedimenting material with cellular polypeptides, it is useful for determining the infectivity of the intracellular particles. To determine the number of PFU in each fraction, samples of each gradient fraction were diluted in growth medium and used to inoculate monolayers of Vero cells. L cell samples contained 270 PFU/cell, whereas gro29 cell samples contained 1.5 PFLT/cell. These data and the data regarding total intracellular particles (Fig. 8) suggest that the lesion in gro29 cells inhibits or destroys the infectivity of the particles that are formed inside the cells, resulting in a decreased specific infectivity for the intracellular virions.  Immunofluorescence and electron microscopic analysis of infected cells. Based on the observations described above, the intracellular location of virions and glycoproteins was investigated. Cells were grown on coverslips, infected with HSV-1 and prepared for indirect immunofluorescence at 13 HPI. An anti-gD monoclonal antibody was used to detect gD on the cell surface and inside the cells after permeabilization with detergent (Fig. 9). Diffuse cytoplasmic staining was evident in infected L cells (Fig. 9A), with many discrete patches of gD scattered throughout the cytoplasm. These patches may represent virions in cytoplasmic vacuoles that were in transit to the cell surface prior to  59 Fig. 9. Immunofluorescence analysis of HSV-1 gD in HSV-1 infected L and gro29 cells. Parental L and gro29 cells were grown on glass coverslips and infected with HSV-1 (MOI=5). At 13 HPI, monolayers were fixed and prepared for immunofluorescence as described (Materials and Methods). The distribution of HSV-1 gD in permeabilized cells (A,B) or on the cell surface (C,D) of L cells (A,C) and gro29 cells (B,D) was detected by indirect immunofluorescence using an anti-gD monoclonal antibody followed by a rhodamine-conjugated second antibody. Published in Banfield and Tufaro 1990.  61 egress. In some cells, gD was concentrated in a juxtanuclear region which may contain the Golgi complex. These observations are consistent with biochemical data demonstrating that gD traversed the Golgi en route out of the cell (Fig. 5), and with the accumulation of gD-containing intracellular particles in the cytoplasm (Fig. 8). The localization of gD in gro29 cells was very different from that of the parental L cells (Fig. 9B). In these cells, gD was concentrated in a juxtanuclear region and there was very little of the diffuse cytoplasmic staining visible in L cells. The majority of the cytoplasmic gD appeared to be in small round cytoplasmic vacuoles. There was also some reticular cytoplasmic staining evident, consistent with gD being present in the ER. This data suggested that the intracellular distribution of gD was perturbed in these cells. To determine whether there was a block to the appearance of viral gly coproteins on the surface of gro29 cells, cells were infected, fixed and prepared for immunofluorescence without permeabilizing the cells. Comparisons of the cell surface staining (Fig. 9C,D) indicated that gD was transported to the cell surface in both L cells and gro29 cells. To determine the relative abundance of gD on the surface of gro29 cells compared with L cells, samples of infected cells were analyzed by flow cytometry in a fluorescence-activated cell analyzer (F. Tufaro, data not shown). Monolayers of L and gro29 cells were infected with HSV-1 (MOI=1O) and harvested by rinsing in growth medium containing 10 mM EDTA. Mock infected cells were also harvested for use as controls for non specific binding. Cells were rinsed and then stained with an anti-gD monoclonal antibody followed by a fluorescein-conjugated second antibody. The analysis of io L cells revealed a single population of cells with an average fluorescence 28-fold higher than the fluorescence in the uninfected cell controls. By contrast, gro29 cells exhibited a bimodal distribution with 80% of the cells  62 eliciting an average fluorescence intensity equivalent to 9% of the L cell population. The fluorescence of the remaining 20% of the gro29 cells was indistinguishable from the fluorescence intensity of L cells. Based on these results it was concluded that the flux of individual viral glycoproteins from the ER to the cell surface was perturbed only slightly in the mutant cell line when compared with the flux of viral particles out of the cells. To determine the intracellular location of virions, infected L cells and gro29 cells were examined by electron microscopy. In infected L cells, large cytoplasmic vacuoles containing many virions were observed (Fig. bA). An enlargement of a single vacuole is shown in Fig. bC. These structures were not common in L cells and are shown to allow for comparison to the structures seen in gro29 cells. Infected gro29 cells contained numerous small vacuoles in the cytoplasm, many of which also contained enveloped viral particles (Fig. 1OB,D). Whereas the large vacuoles in L cells were filled with particles, the vacuoles in gro29 cells were irregular in shape and contained few virions, and free nucleocapsids were often located adjacent to these vacuoles (Fig. IOD). Fig. 1OE shows a confocal immunofluorescence micrograph of infected gro29 cells at 13 HPI stained with anti-gD antibody and a rhodamine conjugated second antibody. In this procedure, decoration of the nuclear membrane and endoplasmic reticulum was well resolved, and gD was clustered in discrete locations of the cell. It is likely that this intense staining derived from the virion-containing vacuoles visible in the electron micrographs (Fig. 1OF). These vacuoles may represent the intermediate compartment between the ER and Golgi as the description of this structure (Saraste and Kuismanen, 1984) is consistent with this hypothesis as is the presence of immature viral glycoprotein.  63  Fig. 10. High resolution microscopy of HSV-1 infected L and gro29 cells. Parental L and gro29 cells were grown on Millicell HA inserts (Millipore) for 24 h prior to infection with HSV-1 (MOI=5) or mock-infection. Following inoculation, fresh DMEM containing 10% FBS was added and the infection was allowed to proceed for a further 18 h. For electron microscopy, cell monolayers were rinsed, fixed in glutaraldehyde, embedded and sectioned. (A) HSV-1 infected L cells; (B) HSV-1 infected gro 29 cells; (C, D) Higher magnification image of region 1 indicated in A and B, respectively. (E) Confocal image of HSV-1-infected gro29 cells prepared as in Fig. 5; (F) HSV-1 infected gro29 as in B prepared from a different sample showing HSV-1  particles in a cytoplasmic location. Nu, nucleus; Cyt, cytoplasm; v,virus particle; NC, nucleocapsid. Electron microscopy performed by H. Meadows. Published in Banfield and Tufaro 1990.  65 To confirm that the structures observed in the infected gro29 cells contained both viral glycoprotein and virus nucleocapsids double fluorescence labeling experiments were performed. Fig. 11 shows infected gro29 cells pulse labeled for 30 mm from 6.5 HPI to 7 HPI with 5-bromo-deoxyuridine (BrdU) and stained with anti-BrdU antibodies and FITC-conjugated ricin at 13 HPI. In this double-label immunofluorescence experiment, gro29 cells showed a strong cytoplasmic BrdU signal (Fig. hA) that co-localized with the ricin-binding sites (Fig. 11B). These results indicated that the viral DNA in the gro29 cell cytoplasm was associated with viral glycoproteins, and was likely in or near Golgi or Golgi derived membranes. The presence of a strong BrdU signal in the nucleus of these cells suggests that the newly-synthesized viral DNA can take longer than 6 h to be transported out of this compartment.  Brefeldin A treatment of HSV-1 infected cells. It is well established that BFA induces alterations in glycoprotein processing and transport in mammalian cells (Doms et al. 1989; Gamou and Shimizu 1988; Kato et al. 1989; Kato et al. 1987; Lippincott-Schwartz et al. 1990; Misumi et al. 1986; Perkel et al. 1988; Perkel et al. 1989; Urbani and Simoni 1990). It is also known that changes in glycosylation can have a profound effect on the propagation of HSV (Campadelli-Fiume et al. 1982; Johnson and Spear 1982; Olofsson et al. 1988; Peake et al. 1982; Pizer et al. 1980; Serafini-Cessi et al. 1983). Because gro29 modifies viral glycoproteins abberently it was of some interest to examine the effect of BFA on HSV-1 infection to see if any conclusions could be made regarding the two transport blockages.  66 Fig. 11. Immunofluorescence localization of cytoplasmic virions. Monolayers of cells growing on glass coverslips were infected with HSV-1 at an MOI=1O. Infected cells were incubated in medium containing BrdU from 6.5-7 HPI to label viral DNA. At 7 HPI the BrdU was removed and the cells received fresh medium. 13 HPI infected monolayers were processed for indirect immunofluorescence microscopy as described in the materials and methods section. Intracellular glycoconjugates were detected using FITC conjugated ricin. BrdU labeled DNA was detected using a monoclonal antibody against BrdU followed by incubation with a rhodamine conjugated secondary antibody. The figure shows the same cells labeled with anti-BrdU antibody (A) and with ricin (B).  68 Brefeldin A alters the processing of HSV-1 glycoproteins in infected cells. To study the effects of BFA on viral glycoprotein processing in HSV-1 in fected L cells, cytoplasmic membranes were isolated and the HSV-1 gD contained in those membranes was incubated with endo H, and analyzed by immunoblotting. Monolayers of infected cells were treated with 3 j.ig/ml BFA from 2 HPI and harvested at 13 HPI. Total microsomes were isolated from the cells and centrifuged in discontinuous sucrose gradients to separate heavy from light membrane fractions. Samples of membranes were collected from the gradients and the proteins contained in the fractions were subjected to digestion with endo H. Following SDS-PAGE of the digested and mock-digested samples, the polypeptides were transferred to nitrocellulose and HSV-1 gD was detected using an anti-gD monoclonal antibody. Fig. 12 shows the results of this assay for two fractions obtained from BFA-treated and untreated cells. These fractions (A  and B), contained most of the mass of protein loaded on the gradient and also contained the highest proportion of endo H-sensitive forms of HSV-1 gD. These fractions are the equivalent of fractions 4 and 5 in Fig. 5. In untreated cells (Fig 12, NO BFA), most of the mass of pgD was sensitive to digestion by endoglycosidase H. There was also a substantial amount of material which was resistant to endo H digestion (gD), representing polypeptides that had traversed the medial Golgi cisternae en route to the plasma membrane. Fraction B, which included membranes of lower density than those in fraction A, also contained a mixture of endo H-resistant and sensitive forms of gD. In BFA-treated cells, by contrast, both fractions (A and B) contained partially-processed forms of this glycoprotein, as judged by relative mobilities. The major species of gD exhibited a lower relative mobility than the endo Hsensitive forms accumulating in untreated cells. A small amount of this material was endo H-sensitive, and the digested species had lower relative  69 Fig. 12. Effects of BFA on the processing of HSV-1 gD during HSV-1 infection in L cells. L cells were infected with HSV-1 (MOI=1O), incubated without BFA (NO BFA) or with 3 jig/mi BFA (+BFA), and were harvested at 13 HPI. Total microsomes were prepared and fractionated further into ER- and Golgi-enriched samples on sucrose gradients as described (Materials and Methods). Fractions were collected from the bottom of the tube, and samples of each fraction were subjected to digestion with endo H and were analyzed by SDS-PAGE and western immuno-blotting. HSV-1 gD was detected using a monoclonal antibody and an avidin-biotin alkaline phosphatase detection system. The two fractions shown contained most of the mass of the protein and had the highest activity of the ER marker NADPH cytochrome C reductase (data not shown). These fractions are the equivalent of fractions 4 and 5 shown in Fig. 5. These fractions also had the largest proportion of endo H sensitive forms. The positions of several molecular weight markers representing 7lKd and 44Kd proteins are illustrated. The immature gD (pgD) and mature forms (gD) are indicated. The positions of the major endo H-sensitive and -resistant forms are indicated by arrowheads. Published in Cheung et al. 1991.  70  NO BFA Sample EndoH  B  A —  +BFA  +  —  B  A +  —  +  —  +  71k =  7gD pgD 44k  all  71  mobilities than the endo H-sensitive species detectable in untreated cells. Although the oligosaccharide structures in the fractions from BFA-treated cells were not characterized further, the observation that gD was largely resistant to endo H digestion indicated that most of the N-linked oligosaccharides on the HSV-1 glycoproteins were processed to intermediate forms by 13 HPI. These results could be explained if BFA induced the redistribution of Golgi processing enzymes into the ER of HSV-1 infected cells. This would explain the detection of the intermediate forms of gD in these ER enriched fractions. This interpretation is consistent with the BFA-induced perturbations that have been shown to occur in uninfected cells as well (Doms et al. 1989; Lippincott Schwartz et al. 1989). These data also suggested that the lesion affecting viral glycoprotein processing in the mutant gro29 cell was dissimilar to the effects seen when L cells were treated with the drug BFA in that viral glycoprotein processing occurs to a greater extent in the BFA treated cells. It was thus of some interest to examine the effects of BFA on viral glycoprotein processing in the mutant gro29 cell as these results may indicate whether the transport block observed in the gro29 cells is before or after the BFA effect. Two dishes of gro29 cells were infected with HSV-1 at an MOI=1O. At 1 1-IPI BFA was added to one of the dishes at a concentration of 3j.tg/ml. Cells  were harvested at 18 HPI. Fig.13 shows an immunoblot detecting glycoprotein D from infected gro29 cell membrane fractions in the absence (-) or presence (+) of BFA. In the untreated gro29 sample the immature form of gD, pgD, predominated. By contrast, in the BFA treated samples the majority of the gD was in an intermediate form similar to that seen in the BFA treated L cell sample (Fig.12).  72  Fig.13. Effect of BFA on the processing of HSV-1 gD in gro29 cells. Two dishes of gro29 cells were infected with HSV-1 at an MOI=1O. The dishes were then incubated or mock incubated with 3 tg/ml BFA from I HPI to 18 HPI when cells were harvested, homogenized and a membrane fraction prepared by differential centrifugation. Samples of the membrane preparations were subjected to SDS PAGE on a 10% gel. The protein in the gel was transferred to nitrocellulose and HSV-1 gD detected using a monoclonal antibody and an alkaline phosphatase based detection system.  +  74 Because viral glycoproteins are processed to a greater extent in the BFA treated gro29 cells than in untreated gro29 cells, these results suggested that the block to transport in gro29 cells occurred prior to the transport block caused by BFA. Moreover, these results indicated that the enzymes required for the acquisition of more highly processed forms of gD were present and functional in the infected gro29 cell, and as such the lesion in these cells with respect to herpesvirus glycoprotein processing was not due to the lack of functional glycosylation enzymes during herpesvirus infection. These data also indicated that the regions of the secretory apparatus normally affected by BFA were susceptible to the effects of this drug in gro29 cells because of the increased modification of gD observed in BFA treated gro29 cells. It is likely that the failure to glycosylate HSV-1 glycoproteins in gro29 cells in the absence of BFA was likely due to an inability of these molecules to reach the cellular compartments which contain these activities.  75 Discussion These data describe the maturation and transport of HSV-1 glycoproteins and virions in the mutant mouse cell line gro29. It has been shown that the release of infectious virus from HSV-1 infected gro29 cells was diminished 2000-fold (Fig. 3) due to a specific block in viral egress (Fig. 6). Although the assembly and envelopment of nucleocapsids occurred with high efficiency in gro29 cells, the low specific infectivity of the intracellular virus indicates that the maturation of the newly-formed virions into infectious particles was impaired (Figs. 7,8). It has also been determined that the viral particles that accumulate intracellularly in gro29 cells contain HSV-1 glycoproteins in the viral envelope (Fig. 8, 11). Analysis of the processing of HSV-1 gB and gD during infection indicates that these, and probably all HSV-1 encoded glycoproteins, were synthesized normally in gro29 cells and were modified by the addition of Nlinked oligosaccharide moieties in the ER (Fig. 4A). The rate of oligosaccharide processing of the HSV-1 glycoproteins was lower in infected gro29 cells (Fig. 4B), as most of the newly-synthesized glycoproteins were slow to convert to endo H-resistant forms (Fig. 4B). Moreover, subcellular fractionation of gro29 cells revealed that newly-made gD was under-represented in the membranes of the Golgi complex at 13 HPI (Fig. 5). Despite this impediment, immunofluorescence experiments (Fig. 9D) and flow cytometry indicated that HSV-1 glycoproteins were transported to the cell surface during infection (Fig. 9D). It has been shown previously that gro29 cells are defective in the transport and processing of glycoproteins (Tufaro et al. 1987). This property  76  likely accounts for the slow processing of the HSV-1 glycoproteins observed in this study. What could account for the extreme block to virion egress in this cell line? Several models have been proposed for the maturation and egress of virions in HSV-infected cells (see Fig. 2). In one model, HSV-1 virions begin assembly in the nucleus, acquire an envelope as they bud into the perinuclear space, are carried to the ER and Golgi apparatus and then to the plasma membrane in vesicles similar to those which carry newly-made membrane glycoproteins and secreted proteins (Johnson and Spear 1982). Budding occurs at the nuclear membrane, where immature glycoproteins are more prevalent than the processed mature forms (Compton and Courtney 1984). Because virions released from infected cells contain mature glycoproteins, it is likely that the envelope glycoproteins are modified in the Golgi apparatus while resident in the virus membrane (Johnson and Spear 1982; Spear 1985). A second model proposes that cytoplasmic vacuoles and not the nuclear membrane are the final sites of virion envelopment (Nii et al. 1969; Rodriguez and Dubois-Dalcq 1978). This second mechanism is thought to operate in the envelopment of two other herpesviruses, varicella-zoster virus (Jones and Grose 1988) and pseudorabies virus (Whealy et al. 1991). Both models predict that perturbations in the secretory apparatus of infected cells could affect the maturation and transport of glycoproteins and virions. The observed block to virus egress without a commensurate block in glycoprotein transport is unique to this cell line, and suggests that virions and glycoproteins have different cellular requirements during the HSV-1 lifecycle. Somatic cell mutants defective in glycosylation enzymes can effect the transport and processing of HSV-1 glycoproteins, and in some instances the cells fail to produce normal amounts of infectious virus. Immature forms of several  77 HSV-1 glycoproteins including gB and gD accumulated when mutant BHK cells defective in N-acetylglucosaminyltransferase I activity were infected with HSV-1 (Campadelli-Fiume et al. 1982). The release of infectious HSV-1 particles from these cells was relatively normal, however, in contrast to the results for gro29. A different mutant cell line, which is defective in the glycosyltransferases that add terminal sugars to glycoproteins (Vischer and Hughes 1981), displayed altered HSV-1 glycoprotein forms upon infection with HSV-1 but was reduced only three—five fold in the release of viral particles (Serafini-Cessi et al. 1983). The fact that gro29 cells have a novel phenotype with regard to virion egress indicates that gro29 harbors a lesion distinct from those characterized previously. Many of the described observations suggest that the properties of gro29 cells are similar to those induced in HSV-1 infected cells treated with the ionophore monensin (Johnson and Spear 1982). Monensin has been shown to interfere with the processing of N-linked oligosaccharides (Wenske et al. 1982), the addition of 0-linked sugars to HSV-1 glycoproteins, the transport of HSV-1 glycoproteins from the Golgi apparatus to the plasma membrane, and the egress of virions (Johnson and Spear 1982). A number of criteria have shown that processing of N-linked oligosaccharides, the transport of glycoproteins and the egress of virions are impeded in gro29 cells. The addition of 0-linked sugars to the HSV-1 glycoproteins was not investigated directly in this study, although the absence of the larger forms of the glycoproteins indicated that these modifications do not occur efficiently in gro29 cells (Johnson and Spear 1983). The subcellular fractionation of the secretory organelles of gro29 cells (Fig. 5) suggested that the failure to add 0-linked sugars may arise from inefficient transport preventing the bulk of the newly-made glycoproteins from entering  78 the trans Golgi and the trans Golgi reticulum, because gD was found associated with “early” elements of the secretory pathway. One of the most striking phenomena observed in HSV-1-infected gro29 and monensin-treated cells was the accumulation of virions in cytoplasmic vacuoles (Fig. 10). These vacuoles may represent intermediates in the pathway of virion egress. The electron micrographs (Figs. lOB and D) suggested that progeny virions produced in gro29 cells may be fusing with the membranes of the vacuoles in which they were contained. It is possible that the maldistribution of gD in the membranes of gro29 cells may allow the cells to be super-infected from within, resulting in excessive losses of progeny virions while they are in the process of being transported out of the cells. Alternatively, it may be that virions which are defective in some way are targeted to these vacuolar structures. Support for this notion comes from the observation that the virions contained in the vacuoles in gro29 cells were altered in their morphology when compared with L cell virions (Figs. 1OC and D). It is not known whether vacuoles of this type are Golgi-derived, as suggested previously (Johnson and Spear 1982), and it is not clear why they are abundant in gro29 cells and monensin-treated cells. Several important distinctions between the phenotypes of monensin treated cells and gro29 cells have been identified. Treatment of HSV-1 infected cells with toxic concentrations of monensin does not block virion egress as effectively as observed for gro29 cells (Johnson and Spear 1982). Moreover, HSV-1 glycoproteins were not detectable on the cell surface of monensin treated cells when analyzed by cell surface iodination (Johnson and Spear 1982), whereas HSV-1 glycoproteins on the surface of infected gro29 cells was readily detected by immunofluorescence (Fig. 9D) and flow cytometry. The capacity of  79 gro29 cells to transport at least a portion of their glycoproteins to the cell surface likely accounts for their ability to grow in culture. These results suggest that the targets for monensin are distinct from those affected by the lesion in gro29 cells. It may be that the mutant phenotypes common to monensin-treated and gro29 cells arise in all cells deficient in secretion. What accounts for the difference in the trafficking of individual glycoproteins versus intact virions? Virus glycoproteins encoded by HSV-1 can exist as membrane-resident proteins anchored in cellular organelles or embedded in the virus envelope. Consequently, their cytoplasmic and transmembrane domains can reside in the virion or in the cytoplasm and organellar membranes of the infected cells. Although the molecular features that regulate the rate of virus transport through the cell have not been characterized, it is likely that the glycoproteins embedded in the viral envelope influence the trafficking of the viral particle. It may be the case that the interactions of the virion with the secretory organelles are impaired in the gro29 cells. The fact that the requirements for virion and glycoprotein transport are differentiated in gro29 cells suggest that they are differentiated in normal cells as well. It is worth noting that other L cell mutants have been isolated that are unable to support the propagation of animal viruses.  The mouse L cell line  CL3, a ricin-resistant derivative, was unable to support the growth of Sindbis virus (Gottlieb et al. 1975; Gottlieb et al. 1979). Sindbis is an enveloped RNA virus that encodes two glycoproteins. The cleavage of one of these, PE2 to E2, occurs prior to assembly and is required for virion formation. This cleavage was blocked in CL3 cells and may account for the failure of Sindbis virus to bud from the plasma membrane. When Sindbis virus-infected cells were treated  80 with monensin, virus assembly takes place and enveloped particles can be found in monensin-induced cytoplasmic vacuoles (Johnson and Schlesinger 1980). The observations that CL3 cells, gro29 cells and cells treated with monensin exhibit defects in the processing of glycoproteins and in the maturation and egress of diverse families of enveloped viruses argues strongly that common cellular components facilitate these events. To further the understanding of the virus-host interactions governing the processes of viral maturation and egress, the effect of the fungal metabolite Brefeldin A (BFA) on the propagation of HSV-1 in culture was investigated. BFA is a macrocyclic lactone that causes the rapid redistribution of Golgi components into the ER (Doms et al. 1989; Lippincott-Schwartz et al. 1989). It is thought to act by releasing coat proteins from Golgi derived vesicles and thereby stimulate the fusion of the Golgi membranes with an intermediate “recycling” compartment (reviewed in Pelham 1991). Incubation of cells with BFA results in the loss of a discernible Golgi structure and blocks transport of proteins into post-Golgi cellular compartments (Misumi et al. 1986; Oda et al. 1987). Other processes such as endocytosis, protein synthesis and lysosomal degradation appear unaffected at the low concentrations of BFA used in these studies (Lippencott-Schwartz et al. 1991; Misumi et al. 1986; Nuchtern et al. 1989; Strous et al. 1993). Removal of BFA results in the rapid flux of Golgi components out of the ER and reorganization of the Golgi (Doms et al. 1989; Lippincott-Schwartz et al. 1989). The results presented with respect to treatment of HSV-1 infected cells with BFA indicated that the block to secretion elicited by this drug is clearly different than the block to secretion observed in gro29 cells. These differences are indicated by the ability of BFA treated L cells to process HSV-1 glycoproteins  81 to a greater extent than in gro29 cells (Fig. 12). In infected L cells treated with BFA, gD became more substantially processed to the point of becoming resistant to cleavage by endo H (Fig. 12). In contrast to this, gD synthesized in infected gro29 cells remained in an endo H sensitive form and did not display the decrease in electrophoretic mobility observed in the gD synthesized in infected L cells. The effects of BFA on the egress of HSV-1 have been well characterized (Chatterjee and Sarkar 1992; Cheung et al. 1991). Cheung and others were able to determine that release of virions into the extracellular medium was blocked by as little as 0.3 jtg/ml BFA when present from 2 h post-infection. Characterization of infected cells revealed that BFA inhibited the formation of infectious viral particles without affecting the formation of nucleocapsids. Electron microscopic analyses of BFA-treated and untreated cells demonstrated that viral particles were enveloped at the inner nuclear membrane in BFA treated cells and accumulated aberrantly in this region. Viral particles that entered the cytoplasm of BFA-treated cells lacked an envelope, suggesting that BFA prevented the transport of infectious particles into the cell cytoplasm. By removal of BFA at 18 HPI, it was demonstrated that the BFA-induced block to viral propagation was not fully reversible, despite the observation that the secretion of human growth hormone synthesized in these same cells from an expression plasmid was restored within 15 mm  of BFA removal. These findings  indicated that the BFA-induced retrograde movement of molecules from the Golgi complex to the ER early in infection arrests the ability of the host cell to support the maturation and egress of enveloped viral particles. Furthermore, the effects of BFA on HSV propagation were not fully reversible, indicating that the maturation and egress of HSV-1 particles relies on a series of events which  82 cannot be easily reconstituted after the block to secretion is relieved. Taken together these results suggested that the impediment to secretion effected by BFA occurs earlier in the secretory pathway than the lesion in gro29 cells. The action of BFA and the mutation in gro29 cells were shown to be distinct, therefore it was of some interest to determine if there were any observable effects of BFA on viral glycoprotein processing in gro29 cells (Fig.13). It was shown that the viral glycoprotein gD became more fully processed in the presence of BFA likely due to the retrograde movement of Golgi resident proteins to the endoplasmic reticulum in these cells. These data allowed the formation of two conclusions regarding the nature of the mutation in gro29 cells. First, gro29 cells were susceptible to the effects of this drug suggesting that the molecular target for BFA is present and functional in gro29 cells. This is perhaps not surprising because of the fact that most cells are sensitive to at least some of the effects of BFA (reviewed in Peiham 1991), and therefore the molecular targets of this drug are likely essential for cellular function. Second, the fact that more extensively glycosylated gD was identified in gro29 cells indicated that the enzymes required for the further maturation of the glycoprotein found in infected gro29 cells were present in these cells, and furthermore were located in a BFA sensitive compartment, likely the Golgi apparatus.  83 Chapter 2- Characteristics of uninfected gro29 cells.  A detailed analysis of the uninfected gro29 cell phenotype was required in order to determine the primary defect in this mutant cell and how it relates to herpesvirus infection. The discovery of alterations in cellular functions in gro29 might lead to a better understanding of the requirements for HSV-1 egress in culture. Results Lectin sensitivity of gro29. It is well established that alterations in glycoprotein processing (Stanley 1984) or protein transport into or out of cells (Colbaugh et al. 1988; Colbaugh et al. 1989; Laurie and Robbins 1991; Robbins et at 1984; Robbins et al. 1983; Roff et al. 1986) can result in resistance to specific lectins. Because gro29 cells showed alterations in transport and processing of proteins, their sensitivity to a variety of lectins was tested. Cells were incubated at low density in increasing concentrations of lectin. The lectin concentration resulting in a 90% decrease in the number of colonies formed (D ) was determined (Michaelis et al. 1992). In 10 these assays, clones of gro29 cells were 32-fold and 16-fold resistant to the toxins ricin and modeccin compared with parental L cells. They were not significantly altered in resistance to the other lectins tested, including wheat germ agglutinin (WGA), L-PHA, concanavalin A (ConA), or RCA . The substantial cross 120 resistance to both ricin and modeccin is a rare phenotype that has been reported for only one other cell line (Laurie and Robbins 1991). To determine whether the lectin-resistance phenotype was dominant or recessive, somatic cell hybrids were formed between clones of gro29 (gro29neo), which were resistant to G418, and a parent cell line expressing thymidine kinase  84 (LTK) (Michaelis et al. 1992). Hybrid cells were selected in HAT medium containing 0.48 mg/ml G418. After several weeks, cells were tested for lectin sensitivity. Results from multiple fusion experiments indicated that the lectin resistance phenotype was recessive when gro29 was fused to parental cells, suggesting that gro29 had a loss-of-function mutation. The hybrid cells also regained the ability to propagate herpes simplex virus.  Fluorescence detection of lectin binding molecules. Could lack of lectin binding molecules in gro29 cells account for the resistance to these toxins? An example of this is the MDCK cell mutant, MDCKIIRCAr, which is resistant to the lectin RCA 120 (binds Gal) and is defective in the translocation of UDPGaI into the lumen of the Golgi (Brãndli et al. 1988). This cell line shows a large decrease in the binding of fluorescently tagged RCA 120 to cell surface molecules. Based on these observations, lectin binding molecules in L and gro29 cells were examined. L and gro29 cells growing in monolayers were labeled with different fluorescently labeled lectins to characterize their intracellular glycoconjugates. Cells were labeled immediately after fixation and permeabilization with fluorescein conjugated ricin, WGA, and RCA 120 in separate incubations. Figs. 14A and 14B shows the binding of ricin (Gal and Ga1NAc) to parental L and gro29 cells. In parental L cells (Fig. 14A), ricin bound to a juxtanuclear region resembling the Golgi complex and to small, punctate spots distributed throughout the cytoplasm. By contrast, the binding of ricin to the mutant cells was strongly reduced compared to the parental L cells (Fig. 14B). Longer exposures of gro29 cells revealed binding to small, punctate cytoplasmic spots similar in appearance to those in L cells. This contrasts markedly with the pattern of staining of WGA (sialic acid), which bound substantially to Golgi-like  85  Fig. 14. Fluorescence analysis of lectin-binding molecules in L and gro29 cells. Parental L and gro29 cells were grown on glass coverslips in DMEM/ 10% FBS for 3 days. Monolayers were then fixed with 2% formaldehyde and permeabiized with Triton X-100 as described (Materials and Methods). The distribution of ricin binding molecules (A,B) and WGA molecules (C,D) in L cells (A,C) and in gro29 cells (B,D) was determined using fluorescein-conjugated lectins. Published in Michaelis et al. 1992.  98  87 regions of both L and gro29 cells (Fig. 14C,D). A similar pattern to that of WGA was observed when cells were stained with RCA 120 although the intensity of staining was much lower (not shown). The observed decreased ricin binding by fluorescence microscopy suggested that the Golgi has fewer ricin binding molecules. As mentioned above this lectin binds to Gal and Ga1NAc. Gal usage in gro29 cells appeared to be normal from the analysis of the transport and processing of VSV G protein (Michaelis et al. 1992; Tufaro et al. 1987), and the fluorescence microscopy of 120 binding appeared normal in comparison to L cells. Taken together, RCA these data suggested that Ga1NAc addition to macromolecules was reduced in gro29 cells. This conclusion was also supported by the analysis of PrV egress in gro29 cells (Whealy et al. 1992); where it was shown that the PrV glycoprotein gp5O, which contains no N-linked oligosaccharides but has 0-linked oligosaccharide modifications (Petrovskis et al. 1986; Whealy et al. 1990b), failed to become 0glycosylated efficiently and completely. The fact that 0-linked carbohydrate moieties are linked to serine or threonine through a Ga1NAc residue (Roseman 1970), suggested that the lack of available GalNAc in the gro29 cell could result in the lack of this modification.  Analysis of glycosaminoglycan synthesis in gro29 cells. Due to the apparent block to the addition of Ga1NAc to macromolecules in gro29, it was hypothesized that the synthesis of the glycosaminoglycan chondroitin sulfate might be defective as this molecule contains GalNAc in its repeated disaccharide unit (Kjellen and Lindahi 1991). To test this hypothesis, monolayers of gro29 and parental L cells were incubated with [ S]-sulfate 3 5 and H1-glucosamine to label glycosaminoglycans. After three days of labeling, 3 [  88 glycosaminoglycan fractions from cell extracts were prepared and analyzed by anion exchange HPLC. Fractions eluting from the HPLC column were collected and counted by liquid scintillation spectroscopy. It can be seen in Fig.15 that the control L cell glycosaminoglycans resolved into two major sulfated peaks containing predominantly heparan sulfate (HS) and chondroitin sulfate (CS) and three major glucosamine labeled peaks containing hyaluronic acid (HA), heparan sulfate (HS) and chondroitin sulfate (CS), respectively. Analysis of labeled gro29 proteoglycans showed that there was no detectable chondroitin sulfate associated with the gro29 cells. In contrast to these results gro29 was capable of synthesizing the proteoglycan heparan sulfate although the amount was decreased when compared to L cell heparan sulfate synthesis. It may be that the decrease in heparan sulfate observed was due to the reduced amount of functional Golgi membranes found in gro29 cells (Michaelis et al. 1992). One hypothesis that might explain the results observed with respect to glycosaminoglycan synthesis is that uptake of either glucosamine or sulfate by gro29 cells does not occur efficiently, and this affects chondroitin sulfate synthesis to a greater extent than heparan sulfate synthesis. To ensure that gro29 cells were capable of efficient glucose and sulfate uptake, the uptake of glucose and sulfate from the medium was measured directly. To determine the rate of glucose uptake by L and gro29 cells dishes of cells were incubated in a fixed concentration of radiolabeled glucosamine and varying concentrations of unlabeled glucose for a period of 1 h. At the end of the labeling period cells were harvested, and transported glucosamine measured by liquid scintillation spectroscopy. Results were corrected for differences in protein concentration, and the number of pmoles of glucose transported was determined. As shown in Fig.16 the rates of glucose uptake of L and gro29 cells were similar, although less glucose appeared to have been taken up by the gro29  89 Fig. 15. Analysis of glycosaminoglycari synthesis in L and gro29 cells. Monolayers of L and gro29 cells were incubated in low glucose! low sulfate medium H1-glucosamine for three days to label 3 5 and [ S]-sulfate 3 containing [ glycosaminoglycans. After this incubation glycosaminoglycan chains were isolated from the cells and analyzed by anion exchange HPLC as described (Materials and Methods). Fractions eluting from the column were collected and radioactivity in the fractions determined by liquid scintillation spectroscopy. The results are presented in the figure as CPM/fraction, glucosamine and sulfate counts are plotted independently. HA=hyaluronic acid, HS=heparan sulfate, CS=chondroitin sulfate. L cell glucosamine closed boxes; L cell sulfate open diamonds; gro29 glucosamine open boxes; gro29 sulfate closed diamonds. Glycosaminoglycan analysis by S. Gruenheid, F. Tufaro, and K. Schubert.  E  C3  1000  2000  3000  4000  5000  6000  0  Lcellsulfote gro29glucosomine  —*—---— —D——  20  gro29sullete  Lce11g1ucosmine  ——I—-—  40 Frctlon Number  HA  HS  60  Cs  ProteoglUcen AnelUsis of L end gro29 Cells  80  91  Fig. 16. Measurement of glucose uptake in L and gro29 cells.  35 mm dishes of L and gro29 cells were rinsed 3 times with low sulfate/low glucose medium prior to the addition of low sulfate/low glucose medium containing 0,25,50, H]-glucosamine. Dishes were incubated for 60 mm ( 3 100 or 300 M glucose and 20 jiCi [ 60 mm was shown to be within the linear range of glucosamine uptake versus time in , when they were rinsed 3 times 2 separate experiments (data not shown)) at 37°C 5% C0 with ice cold PBS followed by collection of the cell lysate in 0.1 N NaOH. Protein concentrations of the cell lysates were determined and the radioactivity in the sample determined by liquid scintillation spectroscopy. The specific radioactivity of each sample was determined and the kinetics of glucose uptake versus glucose concentration in the incubation medium was plotted. L cell open boxes, gro29 cell closed diamonds.  92  Glucose Uptake in L and gro29 Cells 12 —0—-—  L cell gro29 cell  10 ci)  4-,  0. ci) C.)  -  E  4.  0.  2 0•  0  .  50  •  I  100  •  I  150  •  I  200  •  [Glucose] (jiM)  I  250  •  I  300  •  350  400  93 cells per .tg of protein. Nonetheless, efficient uptake of glucose was observed in gro29 cells. The uptake of [ S1-sulfate 3 5 by L and gro29 cells over time was measured (Fig. 17). No significant difference was observed in the uptake of sulfate between L and gro29 cells. These results suggested that the failure to synthesize chondroitin sulfate was not a result of the failure to transport glucose or sulfate into gro29 cells. These data indicated that chondroitin sulfate was not being made in gro29 cells. As mentioned above the fundamental difference between heparan sulfate and chondroitin sulfate glycosaminoglycans is that the chondroitin sulfate repeating disaccharide unit is glucuronic acid (GalA) linked to GaINAc whereas the primary structure of heparan sulfate consists of repeating units of GalA linked to G1cNAc or N-sulfoglucosamine (G1cNSO ) (for review see 3 Kjellen and Lindahl 1991). The results of these experiments suggested that there was a deficiency in the incorporation of GalNAc into chondroitin sulfate in gro29 cells. Investigation of Ga1NAc metabolism in gro29 cells. The observed lectin resistance, lectin binding, deficiency in 0-linked glycosylation and the failure to synthesize chondroitin sulfate strongly suggested a defect in the metabolism of Ga1NAc in gro29 cells. Before Ga1NAc can be incorporated into macromolecules of cells growing in culture a number of reactions must occur. First, glucose (Glu) from the medium must be transported into the cell (Fig. 18). The Glu is converted to UDP-Ga1NAc by numerous enzymatic reactions in the cell cytoplasm (116, 117). Many of the reactions converting Glu to UDP-GalNAc are common to the de novo biosynthesis of many cellular metabolites (DelGiacco and Maley 1964; Glaser 1959; Maley and Lardy 1956; Maley et al. 1968; McGarrahan and Maley 1962; Richmond 1965), and as such defects in this pathway would be predicted to be lethal or to have  94 Fig. 17. Rate of sulfate uptake in L and gro29 cells. 35 mm dishes of L and gro29 cells were rinsed 3 times with low sulfate/low glucose medium prior to the addition of low sulfate/low glucose medium containing CPM 5 sulfate. Dishes were incubated for 0,5, 10,30 and 60 mm at 37°C, 5% CO2 prior to S] 3 [ being rinsed 3 times with ice-cold PBS followed by collection of the cell lysate in 0.1 N NaOH. Protein concentrations of the samples were determined and the amount of radioactivity in the sample determined by liquid scintillation spectroscopy. The amount of radioactivity transported into the cells versus time is plotted. L cell open boxes, gro29 cell closed diamonds.  95  Sulfate Uptake in L and gro29 Cells 4000 —El--—  L cell gro29 cell  3000  .  2000  ‘4-  Cl)  E  0  20  40 Time (mm)  60  80  96 Fig. 18. Synthesis of UDPGa1NAc. The biosynthesis of UDPGa1NAc and UDPGa1 from glucose, galactose, and N acetylgalactosamine is outlined. Note in particular the position of the enzyme UDPGa14-epimerase in the biosynthetic pathway. P=phosphate.  97  UDPGa1-4-Epimerase  UDPGIc  GIc-i -P  UDPGaI Gal-i -P Gal  Glucos  ‘  GIc  a  Galactose  Glc-6-P  I  4-”4-Fru-6-P  +  I GIcNAc-6-P I  4-”4-UDPGIcNAc 4  CELL  CYTOPLASM GalNAc GaINAc-1 -P  I  UDPGaINAc  UDPGa1-4-Epimerase  PLASMA MEMBRANE  N-Acetyl Galactosamine  98 broad consequences affecting cellular metabolism. Upon synthesis the nucleotide sugar is translocated into the organelle in which the incorporation reaction is to take place (Fig. 19). gro29 cells appear to be unable to incorporate Ga1NAc into macromolecules, so any defect in the metabolism of this sugar should be specific to the metabolism of Ga1NAc and not grossly affect the metabolism of other sugars. The addition of sugars such as glucosamine (Glc), galactose (Gal), mannose (Man), N-acetylglucosamine (GlcNAc) and sialic acid (SA) appear relatively normal in gro29 cells (based on the analysis of HSV-1 and VSV glycoproteins (Banfield and Tufaro 1990; Michaelis et al. 1992; Tufaro et al. 1987) and the ability to synthesize heparan sulfate Fig.15), therefore biochemical pathways having these sugars as intermediates should not be affected in gro29 cells. Furthermore, the enzymes which transfer GalNAc from UDPGa1NAc to a macromolecular substrate tend to be specific to each acceptor substrate, so it is unlikely that all of these gene products are affected in gro29 cells. Based on these hypothesis experiments were carried out in an attempt to determine the nature of the defect to Ga1NAc metabolism observed in gro29 cells. Rescue of chondroitin sulfate synthesis in gro29 cells. A Chinese hamster ovary (CHO) cell line has been described with a reversible defect in 0-linked glycosylation (Kingsley et al. 1986). It was found that this mutant cell line, idiD, was defective in the enzyme UDP-Gal 4epimerase, and that addition of Ga1NAc to the medium restored the ability of these cells to synthesize 0-linked carbohydrate chains (mammalian cells can synthesize UDPGa1NAc directly from Ga1NAc via a salvage pathway (Maley et al. 1968)), thereby bypassing the epimerase function (see Fig. 18). It was therefore of some interest to determine if the addition of Ga1NAc to the medium of gro29  99 Fig. 19. Translocation of UDPGa1NAc into Golgi membranes. The translocation of UDPGaINAc into the lumen of a Golgi vesicle is outlined. 1) UDPGaINAc is translocated into the Golgi lumen by an antiport exchanging UDPGa1NAc for UMP. 2) Ga1NAc is transferred from UDPGa1NAc to a macromolecular acceptor through the action of a specific UDPGa1NAc transferase. 3) The UDP released from UDPGa1NAc by the action of the transferase is dephosphorylated by a phosphatase to form Pi and UMP. 4) A permease enables the Pi to exit the Golgi. 5) The UMP generated by the action of the phosphatase can now be used to exchange for another nucleotide sugar. Adapted from Hirschberg 1987. Pi= inorganic phosphate.  100  Cytosol  UDPGaINAc  5  UMP  Golgi Membrane  101 cells restored the ability of these cells to synthesize chondroitin sulfate as would be predicted if the epimerase function were defective in gro29. Cells were grown for two days in medium supplemented with 10 p.M Gal, 100 p.M Ga1NAc (Kingsley et al. 1986), prior to labeling and HPLC analysis of glycosaminoglycans as described above. Fig. 20 shows the results of sulfate incorporation into heparan sulfate and chondroitin sulfate by gro29 cells in the presence or absence of Gal and GalNAc. As can be seen in Fig. 20 the addition of Gal and Ga1NAc to the medium of gro29 cells restored the ability of gro29 cells to synthesize chondroitin sulfate. These data indicated that gro29 cells contained the enzymes involved in the synthesis of chondroitin sulfate and that the defect in these cells was likely due to the availability of GalNAc. Measurement of UDPGa1-4-epimerase activity in cell extracts. To determine if the defect in gro29 cells was the lack of UDPGa1 4epimerase activity, as suggested by the ability of exogenous Ga1NAc to restore chondroitin sulfate synthesis, this enzyme activity was measured. The enzyme converts UDPG1c to UDPGa1 reversibly, and UDPG1cNAc to UDPGa1NAc reversibly (Maley and Maley 1959; Filler et al. 1983)(see Fig. 18). The UDPGa1 4epimerase activity was measured in L and gro29 cell extracts by a two-step spectrophotometric assay which quantitates the conversion of UDPGa1 to UDPG1c over time (Kingsley et al. 1986). The results of this experiment are shown in Fig. 21. It is clear from these results that the UDPGa1 4-epimerase activity is normal in gro29 cells. To insure that the epimerase activity measured above was not specific for the conversion of UDPGa1 to UDPG1c, the conversion of radiolabeled UDPGaINAc to UDPG1cNAc in L and gro29 cell extracts was measured. The measurement of the accumulation of radiolabeled UDPG1cNAc over time was facilitated by separating the nucleotide sugars by thin layer chromatography on  102 Fig. 20. Rescue of chondroitin sulfate synthesis in gro29 cells. Monolayers of gro29 cells were incubated in medium with or without 10 ji.M Gal, 100 I.LM GaINAc for two days prior to incubating the cells in low sulfate medium with  ] 5 [3S  sulfate (with or without 10 jiM Gal and 100 jiM GalNAc) for three days to label glycosaminoglycans. After this labeling period a glycosaminoglycan fraction was prepared from the cells and analyzed by anion exchange HPLC and subsequent liquid scintillation spectroscopy of fractions eluted from the column. These data are presented here as CPM/fraction. HS=heparan sulfate; CS= chondroitin sulfate. Control= gro29 without Gal/Ga1NAc preincubation, closed boxes; gro29 cells +Gal/Ga1NAc incubation, closed diamonds. Glycosaminoglycan analysis performed by F. Tufaro and K. Schubert.  L)  E  400  0 20  Frection Number  40  60  Proteog1Jc8n An6lysis of gro29 Cells +1— Gl 6ncI GlNAc  80  104 Fig. 21. Measurement of UDPGa1-4-epimerase activity in L and gro29 cell extracts.  Samples of L and gro29 cell extracts were assayed for UDPGa1-4-epimerase activity as described (Kingsley et al. 1986). Control samples were boiled for 5 mm prior to performing the assay. UDPG1c production is directly proportional to absorbance at 340 nm and is measured over time. L cell open boxes, gro29 cell closed diamonds, L control closed boxes, gro29 control open diamonds.  105  UDPGaI-4-Epimerase  Assay  020 ——  L cell gro29 cell  —*‘----  0  Lboiled gro2led...•  0.10• U I  0 -D  <  0.05  0.00• 0  20  10 Time  (mm)  30  106 PET cellulose (Kingsley et al. 1986; Randerath and Randerath 1965). Fig. 22 shows the results from the experiment. As can be seen in the graph, there were no significant differences detectable between the epimerase activity from the extracts of L and gro29 cells. These results indicated that the UDPGa1-4-epimerase activity in gro29 cells was normal. The addition of exogenous Ga1NAc to the medium of gro29 cells was able to stimulate chondroitin sulfate synthesis perhaps by raising the concentration of intracellular GaTNAc to a level which could overcome the observed defect. The addition of GalNAc to the medium of gro29 cells did not restore the ability of gro29 cells to bind fluorescently labeled ricin nor did it restore the ability of gro29 to propagate HSV-1, as might be expected if the UDPGa1-4-epimerase were defective. Measurement of nucleotide sugar translocation into Golgi membranes. It is well established that lectin resistant cell lines can be defective in the transport of specific nucleotide sugars into the Golgi apparatus. For example, the CHO cell mutant Lec2 is resistant to the lectin WGA (binds sialic acid), and was found to be defective in the translocation of CMP-sialic acid into the lumen of the Golgi (Deutscher et al. 1984). Also the MDCK mutant, MDCKIIRCAr, is resistant to the lectin RCA , which binds Gal, and is defective in the 120 translocation of UDP-galactose into the lumen of the Golgi apparatus (Brändli et al. 1988). Because of the lectin resistant phenotype, and the failure to incorporate GalNAc into macromolecules in gro29 cells, the translocation of UDP-Ga1NAc into Golgi membranes isolated from L and gro29 cells was measured (see Fig. 19). Considering that gro29 cells are capable of incorporating sialic acid into macromolecules (Tufaro et al. 1987, also see Fig.14D), the measurement of CMP-sialic acid translocation into Golgi membranes was used as a control.  107 Fig. 22. Conversion of UDPGa1NAc to UDPG1cNAc L and gro29 cell extracts.  Samples of L and gro29 cell extracts (containing 240 pg of protein) were incubated at H1-UDPGa1NAc. At 0,5,7, 10 and 12 mm samples of the 3 37°C in the presence of [ reactions were spotted on a PEI cellulose TLC plate which was pre-loaded with 50 nmole each UDPGa1NAc and UDPG1cNAc. Nucleotide sugars were resolved as described (Kingsley et al. 1986; Randerath and Randerath 1965), the UDPG1cNAc excised from the TLC plate and radioactivity determined by liquid scintillation H]-UDPG1cNAc is 3 H]-UDPGa]NAc converted to [ 3 spectroscopy. The amount of [ plotted versus time of incubation. L cell open boxes, gro29 cell closed diamonds.  108  UDPGaINAc  to  UDPGIcNAc  Epimerase  Assay  0.8—0--——  L cell extract gro29 cell extract  0.6t3  zC-, CD  D  0.4-  0  £ a. 0.2-  0.0  0  .  I  I  I  10  5 Time  (mm)  15  109 The translocation assays were performed as described in the materials and methods section, C]-acetate 14 was used as a membrane impermeable standard [ throughout these experiments. To ensure that the membrane preparations would be stable over the entire assay period, the rate of nucleotide sugar uptake was first determined (Fig. 23). Golgi membranes from L and gro29 cells were incubated at 30°C in buffer containing a fixed specific activity of [ H1-CMP sialic 3 acid or [ H]-UDPGa1NAc, for various lengths of time. As can be seen from these 3 data, the rate of uptake of CMP sialic acid (Fig.23A) and UDPGa1NAc (Fig. 23B) appear linear over the sampling period although the rate of CMP sialic acid transport is reduced in the gro29 cell membranes. From these data it was determined that the kinetics of nucleotide sugar translocation would be measured using an incubation period of 10 mm  when the rate of translocation  was linear. Fig. 24 shows the kinetics of nucleotide sugar translocation in L and gro29 membrane fractions. L and gro29 Golgi membranes were incubated in varying concentrations of nucleotide sugar for 10 mm  at 30°C before determination of  the amount of nucleotide sugar translocated into the lumen. The amount of CMP-sialic acid translocated into the Golgi versus the CMP-sialic acid concentration in the reaction was considerably lower in gro29 cells as might have been predicted from the membrane stability assays (Fig. 23A), however it is clear from Fig. 14D that sialic acid is incorporated into macromolecules in gro29 cells and furthermore the glycoprotein of VSV is capable of becoming oversialated in gro29 in relation to VSV infected L cells (Michaelis et al. 1992). It has been shown that the majority of the CMP-sialic acid translocated into Golgi membranes from CHO cells during these assays is incorporated into indigenous macromolecular acceptors during the assay (Deutscher et al. 1984; Sommers and Hirschberg 1982), thus it is possible that the decrease in CMP-sialic acid  110  Fig. 23. Rate of nucleotide sugar translocation into L and gro29 Golgi membranes. Preparation of Golgi membranes and procedures for nucleotide sugar translocation assays are described (Materials and Methods). Golgi derived membranes from L and gro29 cells were incubated in the appropriate buffer for various times at 3O’C before  termination of the reactions. All reactions were performed in duplicate. (A) Time course of CMPSA translocation into Golgi membranes. (B) Time course of UDPGa1NAc translocation into Golgi membranes. L cell membranes open boxes, gro29 cell membranes closed diamonds.  111  Time Course of CMP Sialic Acid Tra nslocation into Goig i Membranes 0.04—D-—-—  C  15  2  L cell membranes  0.03-  0)  0.02C-)  0.01-  E 0.00  _  0  10  Time  (mm)  20  30  Time Course of UDPGaINAc Trarislocation into Golgi Membranes C  0.04 —D-———  0  B  L cell membranes gro29 membranes  0.03 0) C.)  0.02 0  D  0.01  E  0.  0.00  0  5  10 Time (mm)  15  20  112 Fig. 24. Kinetics of nucleotide sugar translocation into L and gro29 cell Golgi membranes. Preparation of Golgi membranes and procedures for nucleotide sugar translocation assays are described (Materials and Methods). L and gro29 membranes were incubated in various concentrations of the appropriate nucleotide sugar for 10 mm at 30°C before termination of the reactions. All reactions were performed in duplicate. (A) Kinetics of CMPSA translocation into Golgi membranes. (B) Kinetics of UDPGa1NAc translocation into Golgi membranes. L cell membranes open boxes, gro29 cell membranes closed diamonds.  113  CMP  : E  0  Sialic  into  Translocation  Golgi  Membranes  180160  —D———  gro29 cell membranes L cell membranes  140120100-  O 3 H . 2 4.lb.l. [CMPSA]  UDPGaINAc E  Acid  Translocation  (pM)  into  Golgi  Membranes  800700  -  600-  —D-——-  B  gro29 cell membranes L cell membranes  I  •  [UDPGaINAc]  (pM)  30  114 translocation is due to the reduced number of macromolecular acceptors present in the gro29 Golgi compartment because of the reduced Golgi capacity of these cells (Michaelis et al. 1992). Fig 24B shows the rate of translocation of UDP Ga1NAc versus UDPGa1NAc concentration for L and gro29 cells. The ability of gro29 membranes to translocate UDPGa1NAc is indistinguishable from L cells up to a substrate concentration of 5 p.M UDPGa1NAc. At a substrate concentration of 25 p.M the L cell UDPGa1NAc translocator appeared to approach Vmax whereas the gro29 translocator did not. These data suggested that the gro29 UDPGa1NAc translocator may be a “better” pump than the L cell pump. It is not known if the differences observed between the L and gro29 UDPGa1NAc translocator are physiologically relevant. A formal possibility which may explain these results is that the UDPGa1NAc pump is functional in gro29 cells but it is not located in the appropriate intracellular compartment. Unfortunately no reagents exist to detect the protein or proteins which comprise the pump, so it is not yet possible to determine if this is the case. A growing body of evidence suggests that the concentration and relative ratio of nucleotide sugar concentrations can have profound effects on protein transport and glycosylation in vitro (Davidson and Balch 1993). Moreover, other molecules such as uridine monophosphokinase have been shown to influence the translocation of nucleotide sugars into the lumen of the Golgi in vitro (Hiebsch and Wattenburg 1992). It is conceivable that gro29 cells have a defect in modulating the availability of nucleotide sugars such that under “normal” circumstances (i.e. uninfected cells) the cells are capable of sustaining a basal level of protein transport and glycosylation to permit cell survival but upon infection with HSV-1 the tremendous increase in glycoprotein synthesis  115 saturates the Golgi capacity of these cells quickly and irreversibly, resulting in the inability to produce progeny virus.  116 Discussion The most striking phenotype observed in uninfected gro29 cells was the substantial cross-resistance to the toxic lectins ricin and modeccin (Michaelis et al. 1992). Ricin and modeccin are potent cytotoxins comprising two disulfide linked peptides, the A chain and B chain. The B chain binds to galactose and/or N-acetylgalactosamine-containing receptors on the cell surface, and the lectin enters the cell by receptor-mediated endocytosis. Following intracellular vesicular transport, ricin and modeccin are translocated to the cytosol, where they catalytically inactivate the 60 S ribosomal subunit. However, there is evidence indicating that modeccin and ricin recognize different receptors on the cell surface, have different requirements for endosomal acidification, and have different ribosomal targets (Stanley et al. 1990). Based on these differences, it is possible that cells resistant to both toxins are defective in transporting the toxin into the cytoplasm from the cell surface. Alternatively, the cell-surface receptors for modeccin and ricin may share a requirement for a specific oligosaccharide modification, the loss of which may disrupt the affinity of the two lectins for their respective receptors. The possibility that gro29 cells are altered in a carbohydrate modification is supported by results showing that gro29 cells were lacking most of the ricin binding molecules normally present in the parental L cells (Fig. 14). Although modeccin, ricin and RCA 120 bind to galactose-containing structures, it is probable that sugar residues adjacent to those in glycoprotein oligosaccharides influence the extent of binding (Olsnes et al. 1978). It may be the case that ricin and modeccin recognize predominantly GalNAc containing structures in L-cells, whereas RCA 120 recognizes distinct Gal structures, because there was no apparent block to the addition of Gal to N-linked oligosaccharides in gro29 cells  117 (Michaelis et al. 1992; Tufaro et al. 1987; Whealy et al. 1992). The reduced labeling of gro29 cells with fluoresceinated ricin may result from a failure to add GalNAc to newly synthesized molecules. In support of this possibility was the observation that gro29 cells were severely impaired in their ability to synthesize chondroitin sulfate chains (Fig.15), which are composed of alternating disaccharides of glucuronic acid and Ga1NAc. By contrast, the synthesis of heparan sulfate chains, composed of hexuronic acid and N-acetylglucosamine, was reduced yet apparently normal (Fig. 15). Furthermore, it has been determined that gro29 cells were deficient in their ability to 0-glycosylate viral glycoproteins (Banfield and Tufaro 1990; Whealy et al. 1992), this is interesting because the first sugar added to serine or threonine in an 0-linked glycan chain is Ga1NAc, further suggesting that a defect in the metabolism of Ga1NAc may be responsible for the lesion observed in gro29 cells. One way to explain the observed cross-resistance to modeccin and ricin, the inability to synthesize chondroitin sulfate and the inability to 0-glycosylate viral glycoproteins is a reduced UDPGa1-4-epimerase activity in gro29 cells. This enzyme converts UDPG1c and UDPG1cNAc to UDPGa1 and UDPGa1NAc respectively. A defect in this enzyme would alter the UDPGa1NAc availability in cells. A cell line has been characterized which displays a defect in this enzyme (Kingsley et al. 1986)  .  This cell line, termed idiD, shows a defect in the  formation of 0-linked oligosaccharide moieties on glycoproteins. The addition of Gal and GalNAc to the culture medium of these cells restores their ability to O-glycosylate proteins. To determine if the epimerase was defective in gro29 cells the effect of adding exogenous Gal and GalNAc on the synthesis of chondroitin sulfate was determined (Fig. 20). Addition of these sugars to the medium of gro29 cells restored the ability of gro29 cells to synthesize  118 chondroitin sulfate, although it also modified the elution profile of heparan sulfate from an anion exchange HPLC column. Nevertheless, these data suggested that the epimerase might have been defective. This enzyme activity from L and gro29 cell extracts was measured and found to be normal (Figs. 21 and 22). These data suggested that the ability of additional Gal and GaINAc to restore the synthesis of chondroitin sulfate was not due to the lack of a functional epimerase, however, it also indicated that the levels of these sugars inside gro29 cells can have an effect on glycosaminoglycan synthesis. Moreover, the ability of gro29 cells to synthesize chondroitin sulfate upon addition of exogenous Gal and GalNAc indicated that these cells harbored the enzymatic machinery required to synthesize this complex glycosaminoglycan chain. Another possibility which might explain the apparent Ga1NAc deficiency in gro29 cells is if the UDPGa1NAc translocator were missing or defective in these cells. The UDPGa1NAc translocator is an antiport which translocates UDPGa1NAc from its site of synthesis in the cytoplasm into the lumen of the Golgi apparatus where incorporation of GalNAc into macromolecules takes place (see Fig. 19). A defect in this translocator would result in an underrepresentation of GalNAc in complex molecules synthesized by enzymes of the Golgi complex. There is precedent for such a lesion. Cell lines with defects in sugar nucleotide transporters have been isolated, and show resistance to a variety of lectins. In particular, the MDCKII cell mutant MDCKRCAr shows a defect in UDPGa1 transporter activity (Brändli et al. 1988). Interestingly, these cells are resistant to RCA , but not to ricin. Moreover, in the CHO mutant 120 Lec8, which shows 100-fold resistance to killing by WGA compared with the parental CHO cells (Stanley 1981), UDPGa1 transport is reduced to 3-5% of normal, whereas the transport of UDPGa1NAc is unaffected (Deutscher and  119 Hirschberg 1986). These data also indicate that distinct activities exist for the translocation of UDPGa1 and UDPGa1NAc. The transport of UDPGa1NAc into Golgi membranes isolated from L and gro29 cells was measured (Figs. 23B and 24B). The transport of UDPGa1NAc into gro29 cell Golgi was normal up to a nucleotide sugar concentration of at least 5 p.M. At higher concentrations of UDPGa1NAc it appeared that the UDPGa1NAc translocator was more efficient in gro29 cells than in L cells. Why this might be the case or whether or not this observation is physiologically relevant is not clear. The activity of this molecule may be up-regulated in an attempt to compensate for the decrease in availability of Ga1NAc in gro29 cells. There is no evidence to support this notion. The transport of CMP sialic acid into Golgi membranes was measured as a control because it appeared that the metabolism of sialic acid was relatively normal in gro29 cells (Fig. 14)(Michaelis et al. 1992; Tufaro et al. 1987). Interestingly, the rate and kinetics of transport of this nucleotide sugar were significantly reduced in gro29 cell membranes as compared to the control L cell membranes (Figs. 23A and 24A). This result may be due to the observation that most of the CMP sialic acid transported into Golgi membranes from normal cells is incorporated into indigenous macromolecules very rapidly in these assays (Deutscher and Hirschberg 1986). The difference in CMP sialic acid translocation observed between L and gro29 cells may be due to fewer macromolecular acceptors in the gro29 Golgi, because this assay measures both the soluble and incorporated nucleotide sugars translocated into the lumen of the Golgi membranes. Another possibility to explain this discrepancy is that a sialyltransferase activity is reduced in the gro29 cell membranes, which would result in the slower incorporation of sialic acid into indigenous macromolecules  120 and in turn be reflected in the results of the assay. These questions could be addressed by determining the relative amounts of acid insoluble radioactivity (incorporated sialic acid) versus the acid soluble radioactivity (CMP-sialic acid) in the Golgi membranes after the assay. Another explanation for the apparent defect in Ga1NAc metabolism in gro29 cells is that the UDPGa1NAc translocator is not located in the appropriate cellular compartment. This could result in the lack of available UDPGa1NAc for the enzymes which perform the macromolecular synthesis reactions requiring this substrate. The Golgi apparatus is a highly ordered, compartmentalized structure and, as such, any perturbations in its organization may have drastic effects on the biochemical capabilities of this organelle. Unfortunately, the molecule(s) which comprise the UDPGa1NAc translocator have yet to be identified and therefore no reagents are available to test this hypothesis directly. A further possibility to explain the decrease in GalNAc incorporation into macromolecules in gro29 cells is a lack of transferase enzymes required for these functions. Many enzymes would likely be required to form the various Ga1NAc linkages found in higher eukaryotic cells. It is clear that the enzymes responsible for the incorporation of GalNAc into chondroitin sulfate are present in gro29 cells, but perhaps another enzyme deficiency exists which has a general effect on GalNAc metabolism. The enzyme Ga1NAc:polypeptide N acetylgalactosaminyltransferase which transfers GalNAc from UDPGa1NAc to serine or threonine on 0-linked glycoproteins has been characterized in some detail (Elhammer and Kornfeld 1986; Elhammer et al. 1993) and the gene encoding this activity has recently been cloned (Homa et al. 1993). However, many questions regarding the substrate specificity and the precise intracellular location of this enzyme remain unclear. It would be of some interest to  121 investigate the nature of this molecule in gro29 cells. In particular it will be interesting to study the expression and intracellular location of this enzyme in relation to L cells as this may provide further clues as to the origin of the defect in gro29 cells. It may be that this enzyme has weak activity, lower levels of expression, or it may be aberrantly localized in gro29 cells resulting in the observed phenotype. The possibility exists that over-expression of this enzyme in gro29 may be able rescue some of the defects in these cells. This work should now be possible due to the recent availability of reagents required for this study. Even though multiple transferases would likely have to be defective to result in the phenotype demonstrated by gro29, the paucity of information regarding these molecules suggests that the examination of UDPGa1NAc:polypeptide N acetylgalactosaminytransferase in gro29 cells may be fruitful. It has been observed that the level and ratio of the nucleotide sugars in the cytosol can have profound effects on the glycosylation of glycoproteins in vitro. Davidson and Balch (Davidson and Balch 1993) have shown that in semi permeable NRI< cells increasing concentrations of UDPG1cNAc can inhibit the sialylation of VSV G protein. In addition, it was determined that increasing the concentration of UDPGa1 in the assay blocked this inhibition and that the inclusion of UTP in their reactions stimulated the synthesis of more fully glycosylated forms of G protein. A conclusion to be drawn from this study is that the appropriate balance of individual nucleotide sugars is critical to the process of glycosylation in vitro. In another in vitro study, Hiebsch and Wattenberg (Hiebsch and Wattenburg 1992) discovered that the enzyme activity of uridine monophosphokinase (UMPK) stimulates the transport of nucleotide sugars into Golgi membranes. UMP is an inhibitor of the translocation of nucleotide sugars containing UDP into the Golgi apparatus (reviewed in Perez  122 and Hirschberg 1985). Therefore the effect of UMPK on the translocation of nucleotide sugars is likely due to the conversion of UMP to UDP by this enzyme thereby effectively decreasing the concentration of this inhibitor in the assay. Taken together, these data indicate that molecules not directly involved in protein glycosylation or secretion can have significant effects on these processes. gro29 cells may be missing such a molecule and this results in the inability of gro29 cells to efficiently incorporate Ga1NAc into macromolecules. It may be possible that the majority of the ricin-binding molecules detected in fluorescence experiments (Fig. 14) is irrelevant to the intoxication process, and that gro29 cells express functional ricin receptors on the cell surface. If this were the case, it may be that toxin uptake into the cytoplasm is defective in these cells. Previous studies have shown that following endocytosis, ricin molecules are found in Golgi elements and that ricin cytotoxicity is enhanced by low concentrations of nigericin or monensin, that disrupt Golgi structure and function, and by swainsonine and tunicamycin, that inhibit N-glycosylation (Ghosh and Wu 1988; Yoshida et al. 1990). Other studies have indicated that treatment of cells with BFA, that causes recycling of Golgi elements into the ER, increases the resistance of the cell to ricin and modeccin (Yoshida et al. 1991). Furthermore, hybridoma cells secreting anti-ricin antibody are relatively resistant to ricin (Youle and Colombatti 1987), suggesting that ricin interacts with the secretory pathway prior to entering the cytosol. Although it is not possible from these studies to make conclusions regarding the precise role of Golgi trafficking in the intoxication process, these results nevertheless implicate the Golgi complex, and provide evidence that ricin, and by analogy modeccin, associate with the Golgi complex prior to entry into the cytoplasm. The relative lack of functional Golgi membranes in gro29 cells may interfere with the lectin  123 intoxication pathway, thereby preventing the efficient entry of the toxins into the cytoplasm. Extensive cross-resistance to the two toxins is not common in mammalian cells, having been reported in only one variant of murine L cells, called LEFIC cells, which were selected originally for resistance to modeccin and Pseudomonas exotoxin (Laurie and Robbins 1991). LEFIC cells are 95- and 19fold resistant to modeccin and ricin, and show delays in the movement of indigenous proteins along the secretory pathway. Examination of the endocytic pathway in LEFIC cells has shown that uptake and acidification-dependent activities within early endosomes, and the delivery of endocytosed lysosomal hydrolases to lysosomes appears normal, but there is some evidence that transport from the Golgi to lysosomes may be impaired. Based on these results, it was suggested that toxin resistance may arise in LEFIC cells due to a failure to deliver a protein required for intoxication from the Golgi to the late endosome in a timely manner, ultimately leading to toxin degradation in lysosomes. Alternatively, a defect in a cellular compartment such as the trans Golgi network or the late colgi compartments may affect intoxication in these cells. Although any similarity between LEFIC cells and gro29 cells is still at the phenotypic level, it is interesting that both cell lines show significant deficiencies in protein secretion and lectin intoxication.  124 General Discussion  The analysis of PrV infection in gro29 cells has yielded much information about the defect in gro29 cells (Whealy et al. 1992). PrV encodes two glycoproteins which have useful properties for the study of glycosylation and secretion. One of these glycoproteins, gil, a HSV-1 gB homolog, is cleaved by a cellular protease in the trans Golgi or the trans Golgi reticulum after it has oligomerized and been transported out of the endoplasmic reticulum (Whealy et al. 1991; Whealy et al. 1990). The other glycoprotein is gp5O, a HSV-1 gD homolog, which is modified by the addition of 0-linked oligosaccharides but not by N-linked oligosaccharide moieties (Petrovskis et al. 1986; Whealy et al. 1991). The post translational modifications of these glycoproteins were monitored by pulse-chase experiments at various times after infection of gro29 cells with PrV. At 4 HPI gil was slow to become cleaved and the majority of the gil synthesized during the pulse remained intact. The 0-glycosylation of gp5O occurred slowly and incompletely in comparison to the PrV infection in the control parental L cells at 4 HPI. By contrast, at 6 HPI no detectable gil was cleaved after a 2 hour chase and much less 0-glycosylation of gp5O occurred during the chase than occurred at 4 HPI. These data suggested that a cellular component required for infection became limiting or saturated in gro29 cells as the infection proceeded. These results are consistent with the observation that during infection of gro29 cells with HSV-1 the virus was released from gro29 cells early in infection but this release slowed down drastically as the infection progressed (Fig. 3). These results may also explain why the uninfected gro29 cells can grow well in culture; there may be enough activity of the defective component to support normal cell growth but not enough to support viral infection.  125 A deficiency in the amount of Golgi or a modification of Golgi structure could also have profound effects on the lifecycle of herpes simplex viruses. Disruption of this organelle could hinder the maturation and egress of these viruses because they rely heavily on the Golgi apparatus to transport and process viral glycoproteins and virions (Fig. 2). The same lesion which affects the lectin intoxication pathways described above could also influence the release of herpes simplex viruses from the gro29 cell. Moreover, a deficiency of gro29 cells to incorporate GalNAc into macromolecules may be responsible for the inability of herpes viruses to traverse the secretory pathway; failure of gro29 cells to modify a molecule required for the interaction of the virus with the secretory pathway could result in the observed phenotype. At present, it is not known if the phenotype of gro29 cells resulted from a single genetic defect, and an effective selection procedure for revertants has not been developed. An alternative approach to this problem will be to determine whether gro29 cells represent a new complementation group by fusion to other toxin-resistant cell lines (Laurie and Robbins 1991; Stanley 1983b; Stanley 1987; Stanley et al. 1990). This should be possible because cell hybridization studies have shown that the gro29 phenotype is recessive (Michaelis et al. 1992). Unfortunately, the majority of the well-characterized toxin-resistant cell lines are derived from CHO cells; one of the few cell lines in culture which are non permissive to herpes simplex virus infection. Nonetheless, fusion of gro29 to these cell lines should be useful for determining whether the modeccin and ricin resistance phenotypes arose from a single genetic defect. It is also interesting to note that the toxin resistance phenotype of gro29 cells arose without prior exposure to lectin. The mutation(s) in gro29 cells may impinge on a cellular component common to the pathways of lectin intoxication and  126 herpes virus maturation and egress. The fact that the toxin resistance phenotype exhibited by gro29 is rare coupled with the block to herpesvirus propagation being unique to gro29 suggests that it is possible that gro29 cells harbor a defect in a component which facilitates both processes. How did the selection procedure employed to isolate cells deficient in the propagation of herpes simplex viruses generate gro29? Other mutant cell lines have been isolated by this procedure and none of them show the secretion defect observed in gro29 cells. All of the other cell lines isolated in this manner have been defective in the synthesis of heparan sulfate glycosaminoglycan chains (F. Tufaro, unpublished observation). One such cell line termed gro2C has been characterized in detail (Gruenheid et al. 1992). These cells showed approximately a 10 fold reduction in the ability to be infected by HSV-1. Regardless of the reduction in infectivity of gro2C these cells were readily killed by the virus. Re-selection of these cells by the same procedure to isolate mutant cell lines which were more resistant to HSV-1 than gro2C yielded cell lines that failed to synthesize chondroitin sulfate in addition to heparan sulfate (Banfield et al. manuscript in preparation). These results suggested that the ability of cells to survive infection in a mixed population of cells was linked to the biosynthesis of glycosaminoglycan chains. This was not surprising in that it had been determined (partially from the characterization of these cell lines) that heparan sulfate can act as a receptor for the binding of HSV-1 to cells, and furthermore, in the absence of heparan sulfate, chondroitin sulfate can act as a receptor for the virus (Gruenheid et al. 1992; Leduc et al., manuscript in preparation). The inability of gro29 cells to synthesize chondroitin sulfate and their reduced ability to make heparan sulfate may have been enough of a  127 selective advantage to isolate these cells from a mixed population consisting primarily of cells able to synthesize glycosaminoglycan chains normally. It may be possible to isolate the defective gene that confers the lectin resistance in gro29 cells by taking advantage of the fact that gro29 cells do not express ricin binding molecules abundantly. Pools of cDNA clones from an L cell expression library can be transfected into gro29 cells and a pool containing positive clones detected by examination of transfected cells with fluorescent conjugated ricin and epifluorescence microscopy. A positive pool of clones can then be diluted further until a single clone can confer the ability of gro29 cells to bind ricin. Once isolated, the ability of this cDNA to confer susceptibility to productive HSV-1-infection, lectin intoxication and to restore chondroitin sulfate synthesis in gro29 cells can be examined. Although this protocol may not isolate a gene capable of restoring all of the observed defects in these cells it will be interesting to see if any genetic link between these various phenotypes exist. 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