@prefix vivo: . @prefix edm: . @prefix ns0: . @prefix dcterms: . @prefix skos: . vivo:departmentOrSchool "Science, Faculty of"@en, "Microbiology and Immunology, Department of"@en ; edm:dataProvider "DSpace"@en ; ns0:degreeCampus "UBCV"@en ; dcterms:creator "Cheung, Peter"@en ; dcterms:issued "2010-11-04T22:13:11Z"@en, "1991"@en ; vivo:relatedDegree "Master of Science - MSc"@en ; ns0:degreeGrantor "University of British Columbia"@en ; dcterms:description """Herpes Simplex Virus (HSV) requires the host cell secretory apparatus for the maturation and egress of newly synthesized viral particles. Not only do viral glycoproteins rely on the host ER and Golgi compartments for their proper processing, it is believed that enveloped particles are transported through these same organelles for their export out of the cells. Brefeldin A (BFA) is a compound that induces retrograde movement of material from the Golgi apparatus to the ER and causes the disassembly of the Golgi complex. In this study, the effects of BFA on the propagation of HSV-1 in infected cells were examined. Release of viral particles from infected cells was inhibited by as little as 1 µg/ml BFA. Further analysis revealed that BFA did not affect the normal assembly of viral nucleocapsids, but did block the movement of newly-enveloped particles from the nucleus into the cytoplasm. Naked nucleocapsids were found in the cytoplasm of infected cells treated with BFA, however, these particles were neither infectious, nor were they released from the cells. Although BFA altered the distribution of viral glycoproteins in infected cells, this alteration was reversed within 2 hours after the removal of BFA. In contrast, the BFA-induced blockage to viral release was not fully reversed after BFA was removed and cells were allowed to recover in fresh medium for 3 hours. These findings indicate that the BFA-induced retrograde movement of material 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, exposure of infected cells to BFA during the exponential release phase of the viral life cycle can cause irreversible damage to the egressing particles. This suggests that productive growth of HSV-1 in infected cells relies on a series of events that, once disrupted by agents such as BFA, cannot be easily reconstituted."""@en ; edm:aggregatedCHO "https://circle.library.ubc.ca/rest/handle/2429/29798?expand=metadata"@en ; skos:note "BREFELDIN A ARRESTS THE MATURATION AND EGRESS OF HERPES SIMPLEX VIRUS PARTICLES DURING INFECTION By PETER CHEUNG B.Sc, The University of British Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Microbiology) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA January 1991 © Peter Cheung, 1991 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. Department The University of British Columbia Vancouver, Canada Date KA-re, tq«f / DE-6 (2/88) i i ABSTRACT Herpes Simplex Virus (HSV) requires the host cell secretory apparatus for the maturation and egress of newly synthesized viral particles. Not only do viral glycoproteins rely on the host ER and Golgi compartments for their proper processing, it is believed that enveloped particles are transported through these same organelles for their export out of the cells. Brefeldin A (BFA) is a compound that induces retrograde movement of material from the Golgi apparatus to the ER and causes the disassembly of the Golgi complex. In this study, the effects of BFA on the propagation of HSV-1 in infected cells were examined. Release of viral particles from infected cells was inhibited by as little as 1 pg/ml BFA. Further analysis revealed that BFA did not affect the normal assembly of viral nucleocapsids, but did block the movement of newly-enveloped particles from the nucleus into the cytoplasm. Naked nucleocapsids were found in the cytoplasm of infected cells treated with BFA, however, these particles were neither infectious, nor were they released from the cells. Although BFA altered the distribution of viral glycoproteins in infected cells, this alteration was reversed within 2 hours after the removal of BFA. In contrast, the BFA-induced blockage to viral release was not fully reversed after BFA was removed and cells were allowed to recover in fresh medium for 3 hours. These findings indicate that the BFA-induced retrograde movement of material 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, exposure of infected cells to B F A during the exponential release phase of the viral life cycle can cause irreversible damage to the egressing particles. This suggests that productive growth of HSV-1 in infected cells relies on a series of events that, once disrupted by agents such as B F A , cannot be easily reconstituted. iv ABBREVIATIONS USED: BFA Brefeldin A CPE cytopathic effects D M E M Dulbecco modified Eagles medium endo H Endoglycosidase H ER endoplasmic reticulum GalNAc ^-acetylgalactosamine Glc glucose GlcNAc iV-acetylglucosamine h.p.i. hours post infection HSV -1 herpes simplex virus type 1 M a n Mannose MOI multiplicity of infection PBS phosphate buffered saline PFU plaque forming units RSB reticulocyte standard buffer STEM scanning transmission electron microscope TCA trichloroacetic acid TABLE OF CONTENTS: ABSTRACT ii ABBREVIATIONS iv T A B L E OF CONTENTS v LIST OF FIGURES v i ACKNOWLEDGMENT vi i INTRODUCTION . 1 I. Ultrastructure of HSV-1 virions: 2 II. Overview of HSV-1 life cycle: 4 A. Viral attachment, penetration, and uncoating: 4 B. Viral gene expression: 6 C. Viral DNA replication, assembly and egress: 7 D. Function of viral glycoproteins: 11 III. Importance of host cell glycosylation machinery to HSV-1 infection: 1 4 A. Chemical inhibitors of glycosylation affect HSV-1 propagation: 14 B. Cell lines defective in glycoprotein processing also affect growth of HSV-1: 15 IV. Experimental Rationale: 17 MATERIALS AND METHODS 2 0 RESULTS 2 5 DISCUSSION 4 6 REFERENCES 6 1 vi LIST OF FIGURES: Figure 1. Schematic diagram of the ultrastructure of HSV-1 3 Figure 2. Schematic diagram of the egress pathway of HSV-1 9 Figure 3. Effects of BFA on release of PFU into growth medium 2 6 Figure 4. Effects of BFA on viral release 2 8 Figure 5. Effects of B F A on nucleocapsid assembly. 3 0 Figure 6. Electron microscopy of BFA-treated and untreated cells 3 4 Figure 7. Immunofluorescence analysis of HSV-1 gD in infected cells treated with BFA 3 8 Figure 8. Determination of the rate of viral release following removal of B F A 4 2 Figure 9. Determination of BFA-sensitivity during viral propagation 4 4 Figure 10. Schematic diagram of an intranuclear pocket of enveloped particles seen in BFA-treated cells 5 0 ACKNOWLEDGMENT: I wish to express my sincere gratitude for all the people who helped to make my graduate studies experience a productive and enjoyable one. I am greatly indebted to Dr. Frank Tufaro, my supervisor, for his advice and guidance through the ups and downs of my research career during the past two years. I also wish to acknowledge the invaluable comments and help provided by my committee members, Dr. Wilf Jefferies and Dr. Gerry Weeks. Special thanks to Michael Weis for the help in the electron microscopy and photography work, and to Howard Meadows for the everyday technical assistance. Finally, I wish to say thanks to the past and present members of the Tufaro lab with whom I shared many wonderful and sometimes not so wonderful moments. 1 INTRODUCTION Members of the family Herpesviridae are characterised as enveloped, icosahedral viruses containing double-stranded D N A genomes. Herpesviruses are widespread in nature and are found in most animal species (Roizman and Batterson 1986). Of the 80 different types, herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), cytomegalovirus (CMV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7) are the herpesviruses that infect humans. These viruses cause a variety of diseases such as cold sores, varicella (chickenpox), and infectious mononucleosis, and can pose serious threats to the health of infected individuals. In addition to the lytic phase of their replication cycle, these viruses can also establish latent infections in the hosts, and can be reactivated after a long period of dormancy. The two most recently identified human herpesviruses, HHV-6, and HHV-7, are often isolated from T-lymphocytes of patients with lymphoproliferative diseases or AIDS (Josephs, Salahuddin et al. 1986; Salahuddin, Ablashi et al. 1986; Lusso, Markham et al. 1988; Takahashi. K., Sonoda et al. 1989; Frenkel, Schirmer et al. 1990). They in turn cause secondary diseases that can be fatal in these immuno-compromised individuals. The medical significance of human herpesviruses makes them one of the most intensely studied group of viruses worldwide. This thesis focuses on the viral-host interactions that facilitate 2 the maturation and egress of HSV-1 in infected animal cells. In this regard, the salient features of the structure and life cycle of HSV-1 are presented below. I. ULTRASTRUCTURE OF HSV-1 VIRIONS: The virion of HSV-1, like all other herpesviruses, consists of four distinct morphological components (figure 1): 1) an electron-opaque core containing the viral DNA, 2) an icosahedral capsid enclosing the core, 3) an outer envelope surrounding the capsid, and 4) a tegument between the capsid and envelope (Roizman and Batterson 1986). The HSV-1 genome consists of linear, double-stranded D N A that is about 100 x 106 daltons in molecular weight. It contains two covalently linked components designated as L (long) and S (short), representing 82 and 18%, respectively, of the viral D N A . The capsid, which has a diameter of approximately 100 nm, is made up of 162 capsomeres. Pentameric capsomeres are found on the vertices of the icosahedral structure, while hexameric capsomeres comprise the faces. Electron microscopy on thin sections shows that the viral envelope has a typical trilaminar appearance, and appears to be derived from patches of altered cellular membranes. Numerous glycoproteins embedded in this lipid membrane are seen as spikes ranging from 8 to 24 nm in length (Stannard, Fuller et al. 1987). While the tegument has no distinctive features in thin section electron microscopy, it does contain viral proteins that are required for the initiation of viral gene expression early in the infection cycle of HSV-1. 4 Figure 1. Schematic diagram of the ultrastructure of HSV -1 . 1. viral DNA core 2. icosahedral capsid 3 . viral envelope 4. glycoprotein spikes 5. tegument 4 II. OVERVIEW OF HSV-1 LIFE C Y C L E : The sequence of events leading to an infection starts by the attachment of the virus to the host cell and the penetration of an HSV-1 particle into the cell. Once the virus enters the cell, capsid uncoating occurs at the nucleus where viral gene transcription and D N A replication takes place. Viral particles are assembled in the nucleus, transported through the cytoplasm, and released from the infected cell. The length of the replication cycle of HSV-1 is relatively short compared to other herpesviruses such as C M V (Smith and DeHarven 1973). The entire process from the start of an infection to the first release of viral particles takes approximately 8 h for HSV-1 versus 4 days for C M V . The wide host cell range and the rapid multiplication cycle of HSV-1 makes it one of the best characterised herpesviruses. A. Viral attachment, penetration, and uncoating: To initiate an infection, HSV-1 particles attach to the cell surface through heparin-like cell-associated glycosaminoglycans (WuDunn and Spear 1989). After this primary binding process, stable attachment of virus to cell membrane likely occurs by interactions of viral components with specific receptors. Although such a receptor has not yet been found, ongoing research will likely identify this cell surface molecule in the near future. After the virus is adsorbed to the cell membrane, penetration of virus is mediated by the fusion of viral envelope with host cell plasma membrane. This model of viral entry is supported by the 5 observation that HSV-1 envelope components are detected in the cell surface membrane immediately after viral penetration, and in the absence of viral gene expression (Para, Baucke et al. 1980). In addition, agents that raise lysosomal pH, such as ammonium ions and chloroquin, do not block the infectivity of HSV-1, suggesting that viral entry does not occur by receptor-mediated endocytosis (Holland and Person 1977). Upon entry, the viral capsid is transported to the nuclear pores via the cellular cytoskeleton (Roizman and Batterson 1986). It is likely that the viral genome gains entry to the nucleus via the pores, although the mechanism of uncoating of the DNA has not been elucidated. One of the earliest events following uncoating is the shutoff of cellular protein synthesis (Read and Frenkel 1983; Fenwick 1984; Kwong and Frenkel 1987; Kwong, Kruper et al. 1988; Oroskar and Read 1989). This early inhibition of host protein synthesis is accomplished by a protein that is present in the virus particle. Frenkel and Kwong (Kwong and Frenkel 1987; Kwong, Kruper et al. 1988) found that a viral gene product, vhs, causes the disaggregation of cellular polyribosomes, and the degradation of host and viral mRNAs. This allows the newly-made viral mRNAs to out-compete host cell mRNAs for the available ribosomes. In addition to the virion-mediated shutoff of protein synthesis, a late secondary shutoff mechanism causes the degradation of cellular mRNA and reduces further the synthesis of host proteins (Fenwick 1984). In contrast to the early shutoff function, the delayed shutoff function is mediated by virally-encoded proteins synthesized in the infected cell. The combined effects of the early and delayed shutoff 6 processes inhibit completely the synthesis of host cell proteins, and favour the expression of viral genes. B. Viral gene expression: Al l herpesvirus genes are transcribed in the nucleus using host cell RNA pol II (Constanzo, Campadelli-Fiume et al. 1977), and viral mRNAs are translated in the cytoplasm by free or membrane-bound ribosomes. HSV-1 genes can be grouped into five families which are coordinately regulated (for review, see Knipe 1989). Expression of viral genes occurs in a cascade fashion and involves the sequential turning on and off of the transcription of different groups of genes. Early in the infection, the viral tegument protein, VP 16, induces the expression of immediate-early (IE) genes in the absence of prior viral protein synthesis. VP 16 is a major late viral phosphoprotein that is present in the tegument at approximately 400-600 molecules per particle (O'Hare and Goding 1988). At least three cellular proteins are believed to form a complex with VP 16 before binding to viral D N A (Kristie, LeBowitz et al. 1989). One of these proteins has been identified as the cellular transcription factor OTF-1, which recognises the T A A T G A R A T (where R is a purine) sequence present on all IE genes (Kristie, LeBowitz et al. 1989). Binding of this complex to the IE promoter region allows the catalytic domain of VP 16 to cis-activate the transcription of IE viral genes. Five infected cell polypeptides, ICPO, ICP4, ICP22, ICP27, and ICP47, are encoded by the IE genes, and they reach their peak rate of synthesis at about 2-4 hours post infection (h.p.i.). At least three of these proteins, ICPO, ICP4, and ICP27, are localized to the nucleus 7 and influence subsequent gene expression. ICP4 has also been shown to down-regulate IE gene expression by binding to viral D N A sequences and blocking transcription. Two groups of delayed-early (DE) genes are expressed under the induction of one or more IE gene products. These proteins, such as thymidine kinase, ICP8 (a D N A binding protein), and D N A polymerase, reach their peak of synthesis at about 5-7 h.p.i., and are involved principally in viral replication. Towards the end of the infection cycle, viral glycoproteins and structural proteins are made from two groups of late genes. The first group is transcribed prior to D N A replication, whereas the the second group requires D N A synthesis before it can be expressed. The expression of these five groups of viral genes is coordinately regulated such that genes belonging to the same family respond to similar regulatory mechanisms. In general, gene products serving similar functions in the HSV-1 life cycle are synthesized and regulated together, making the viral life cycle well organized and efficient. C. Viral D N A replication, assembly and egress: Replication of viral D N A requires many virally-encoded products and is thought to occur by a rolling circle mechanism using circular D N A templates (Roizman and Sears 1990). D N A synthesis can be detected at 3 h.p.i., and continues for another 9-12 hours (Roizman and Roane 1964; Roizman, Borman et al. 1965). When sufficient amounts of viral DNA and structural proteins are accumulated in the nucleus, capsid assembly begins by cleaving the concatameric progeny DNA, and packaging the genomes into empty 8 capsids (Roizman and Batterson 1986). These newly-formed nucleocapsids leave the host nucleus and journey through the cytoplasm to the cell surface for export (see Fig. 2). The assembled nucleocapsids accumulating in the nucleus exit this organelle by budding through the inner nuclear membrane into the perinuclear space (Nii, Morgan et al. 1969). Electron microscopy studies reveal that viral envelopes are derived from the inner lamellae of the nuclear membrane. The most widely accepted model of egress hypothesizes that enveloped particles in the perinuclear region are transported through the host ER and Golgi complex en route to the cell surface for release (Nii, Morgan et al. 1969; Johnson and Spear 1982). Enveloped particles in the nucleus contain immature glycoproteins on their membranes (Morgan, Rose et al. 1959), and these glycoproteins are processed as the particles traverse the host secretory system. An alternative model suggests that enveloped viral particles in the perinuclear space do not move to the ER. Instead, they de-envelope by fusing with the outer nuclear membrane and enter the cytoplasm as naked nucleocapsids (Stackpole 1969; Komuro, Tajima et al. 1989). Free viral glycoproteins are processed by the host ER and Golgi apparatus, and accumulate at the trans Golgi membranes where envelopment of the cytoplasmic nucleocapsids occur. Although the precise mechanism of egress is not clear, both models imply that the host cell secretory pathway plays a major role in the maturation of viral glycoproteins and the release of HSV-1 particles. Figure 2. Schematic diagram of the egress pathway of HSV-1 P M plasma membrane G Golgi apparatus ER endoplasmic reticulum PN perinuclear space I N M inner nuclear membrane ONM outer nuclear membrane N nucleocapsids V enveloped virion Pathway 1: (solid arrows) Nucleocapsids in the nucleus are enveloped as they bud through the inner nuclear membrane into the perinuclear space. Enveloped particles are then transported through the host ER and Golgi appartus for viral glycoprotein processing. Mature viral particles are released at the plasma membrane. Pathway 2: (dashed arrows) Nucleocapsids are also enveloped at the inner nuclear membrane. However, these particles in the perinuclear space de-envelope when they fuse with the outer nuclear membrane, and enter the cytoplasm as naked capsids. Free viral glycoproteins are processed as they traverse the host ER and Golgi complex. Mature glycoproteins then accumulate at the trans Golgi membranes where cytoplasmic envelopment of nucleocapsids occur. Enveloped virions are released at the plasma membrane. 11 D. Function of viral glycoproteins: HSV-1 encodes at least seven glycoproteins, designated gB, gC, gD, gE, gG, gH, and gl (Spear 1985; Kuhn, Kramer et al. 1990; Raviprakash, Rasile et al. 1990). Al l of these glycoproteins contain N-linked oligosaccharides, and several, such as gC and gD, contain O-linked sugars as well (Spear 1985). iV-linked glycosylation is initiated by the en bloc transfer of Glc3-Man9-GlcNAc2 glycans from dolicol phosphate lipid carriers to the nascent polypeptide chains (for review, see Kornfeld and Kornfeld 1985). After this transfer, the glycan chains are processed by ER and Golgi resident enzymes to yield high-mannose, complex, or hybrid forms of oligosaccharide side chains. The assembly of O -linked glycans starts with the transfer of GalNAc residues to the polypeptide chain, followed by the sequential addition of sugars to the growing glycan chain (Campadelli-Fiume and Serafini-Cessi 1985). Maturation of O-linked oligosaccharides likely occurs in the Golgi complex (Johnson and Spear 1983), although the process is not as well characterised as is Af-linked glycosylation. HSV-1 glycoproteins found on the surface of viral envelopes are involved in functions that mediate viral infectivity such as virus binding, penetration, and egress. Nucleocapsids without envelopes are not infectious because they lack these viral glycoproteins and therefore cannot interact with host membranes (Stein, Todd et al. 1970). Studies on viral mutants that have deletions in the genes for individual glycoproteins show that gC, gE, gG, and gl are dispensable for viral replication (Longnecker, Chatterjee et al. 1987; Longnecker and Roizman 1987; Weber, Levine et al. 1987), whereas gB, gD, and gH are required for virion infectivity (Cai, Person et al. 1987; Desai, Schaffer et al. 1988; Ligas and Johnson 1988). Except for gG and gl, which can associate to form immunoglobulin G Fc receptors (Johnson, Frame et al. 1988), no interactions of the other glycoproteins have been documented. gC, gB, gD, and gH have all been implicated to function in viral attachment or penetration (Little, Jofre et al. 1981; Gompels and Minson 1986; Fuller and Spear 1987; Kuhn, Kramer et al. 1990), but there is no clear model of their mode(s) of action. By studying the formation of complexes by HSV virion proteins with biotinylated cell membrane components, Kuhn et al (Kuhn, Kramer et al. 1990) found that gC, gB, and gD bind to surface components of various cell lines. Because it has been shown that gC is not essential for infectivity, it is likely that gC is only one of several viral glycoproteins that can mediate viral attachment to the host cell. Several pieces of evidence suggest that gB, gD, and gH are involved in the process of viral penetration. It has been shown that monoclonal antibodies directed against any one of these glycoproteins can block fusion of the virus with the host cell plasma membrane without having any significant effect on viral attachment (Fuller and Spear 1987; Highlander, Sutherland et al. 1987; Highlander, Cai et al. 1988; Fuller, Santos et al. 1989). In addition, virions lacking any one of these three glycoproteins fail to gain entry into the host cells to start an infection, despite the observation that they bind to cells efficiently. (Sarmiento, Haffey et al. 1979; Little, Jofre et al. 1981; Cai, Gu et al. 1988; Desai, Schaffer et al. 1988; Ligas and Johnson 1988). Johnson has shown that the initial adsorption of HSV-1 to cells apparently involves numerous sites and is difficult to saturate (Johnson and Ligas 1988). However, soluble gD can block virus entry (Johnson, Burke et al. 1990), suggesting that gD interacts with a smaller subset of cell surface molecules. It has been postulated that after the initial binding of the virion to heparan sulfate moieties of cell surface proteoglycans, gD binds tightly to specific receptors on the cell surface. The close apposition of viral and cellular membranes that ensues after attachment allows gH and gB to mediate fusion of the viral and host cell membranes (Campadelli-Fiume, Arsenakis et al. 1988; Johnson and Spear 1989; WuDunn and Spear 1989). Recently, Hajjar et al reported identifying the basic fibroblast growth factor (FGF) receptor as the cell surface receptor for HSV-1 recognition (Baird, Florkiewicz et al. 1990; Kaner, Baird et al. 1990). They found that antibodies directed against F G F inhibit the entry of HSV-1 into target cells. In addition, previously non-permissive cell lines can be rendered susceptible to HSV-1 infection by transfecting F G F receptors into these cells. This receptor is not the putative gD receptor because it does not appear to interact with gD. Instead, Hajjar's data suggest that F G F molecules are associated with the virion and facilitate the recognition and internalization of HSV-1 by the F G F receptor. It is possible that HSV-1 enters host cells via several independent mechanisms. The multiple models of virus entry and release underscores the complexity of the life cycle of HSV-1 . in. IMPORTANCE OF THE HOST C E L L GLYCOSYLATION MACHINERY TO HSV-1 INFECTION: A . Chemical inhibitors of glycosylation affect HSV-1 propagation: Glycosylation of viral proteins requires a large number of host enzymes to process the growing oligosaccharide chains attached to the protein backbone (for review, see Kornfeld and Kornfeld 1985). By studying the effects of inhibitors that interfere at different steps of this process on the growth of HSV-1, it has been clearly demonstrated that the acquisition of infectivity and egress of newly-synthesized viral particles are dependent on the normal functioning of the host secretory system. One of the first glycosylation inhibitors tested for its effect on HSV-1 growth was tunicamycin (Pizer, Cohen et al. 1980; Peake, Nystrom et al. 1982). This drug acts as an analogue of UDP-iV-acetyl glucosamine, and disrupts the formation of the dolicol phosphate intermediate required in the initial step of Af-linked glycosylation. HSV-1 infected cells treated with tunicamycin fail to yield infectious progeny particles. As expected, only severely underglycosylated forms of viral glycoproteins accumulate in these cells. Electron microscopy revealed that in tunicamycin-treated cells, only a small number of enveloped particles are detected on the cell surface, suggesting that there is a defect in the process of envelopment of the virion. Another reagent that interferes with the propagation of HSV-1 in infected cells was studied by Campadelli-Fiume et al (Campadelli-Fiume, Sinibaldi-Vallebona et al. 1980). Benzhydrazone (BH) is a derivative of bis-amidinohydrazone which prevents the addition of high-mannose oligosaccharides to proteins. It causes an early block in the N-glycosylation process similar to the effect induced by tunicamycin. Once again, infected cells treated with B H do not accumulate mature viral glycoproteins, nor are infectious particles produced. Studies on the effects of tunicamycin and B H on HSV-1 infected cells suggest that glycosylation is a requirement for infectivity of HSV-1. However, other studies have shown that full maturation of viral glycoproteins is not essential for infectivity. Monensin is a monovalent cation ionophore which blocks the transit of membrane vesicles from the Golgi apparatus to the cell surface (Tartakoff 1983). Johnson and Spear demonstrated that monensin can inhibit the full processing and transport of HSV-1 glycoproteins as well as reducing the egress of virions from infected cells (Johnson and Spear 1982). They found that although viral glycoproteins are not fully processed in the presence of monensin, some infectious virions accumulate in the cytoplasm of monensin-treated cells. The studies highlighted so far demonstrate that while the absence of N-linked glycans precludes the envelopment and hence the yield of infectious HSV-1, the presence of fully-processed, complex-type glycans in not an obligatory requirement for herpesvirus infectivity. B. Cell lines defective in glycoprotein processing also affect growth of HSV-1: Cell lines that are defective in glycoprotein processing have also been shown to interfere with HSV-1 growth. Serafini-Cessi and Campadelli-Fiume found that two mutant baby hamster kidney 1 6 (BHK) cell lines that are resistant to killing by the galactose-binding lectin ricin, also interfered with the propagation of HSV-1 when infected with this virus (Campadelli-Fiume, Poletti et al. 1982; Serafini-Cessi, Dall'Olio et al. 1983). Ricin is a plant lectin that binds to cell surface oligosaccharides containing galactose (Vischer and Hughes 1981). Most mature Af-linked glycoproteins have galactose residues near the end of their oligosaccharide chains (Kornfeld and Kornfeld 1985). The two cell lines, Ricl4 and Ric21, were found to be defective in N-acetylglucosaminyl transferase I and N-acetylglucosaminyl transferase II respectively (Vischer and Hughes 1981). The lack of these two enzymes prevents the addition of N-acetylglucosamine to the growing glycan chain. This in turn prevents the incorporation of galactose, and results in the ricin-resistant phenotype. Campadelli-Fiume found that the release of virions from infected ricin-resistant cells is significantly reduced compared with infected wild type cells. However, fully infectious particles are found in the cell cytoplasm in spite of the lack of complete processing of viral glycoproteins in the mutant cells. Another mutant cell line defective in the release of HSV-1 particles was isolated by Tufaro et al (Tufaro, Snider et al. 1987; Banfield and Tufaro 1990). This cell line, termed gro29, was shown to be defective in the transport and processing of glycoproteins through the Golgi complex. Although some infectious particles are formed, they remain trapped in the cell cytoplasm. Al l of these studies emphasize the dependence of HSV-1 on host secretory and glycoprotein processing functions for their normal propagation and egress. IV. EXPERIMENTAL RATIONALE : To further the understanding of the virus-host interactions governing the processes of viral maturation and egress, this study set out to investigate the effects of the fungal metabolite Brefeldin A (BFA) on the propagation of HSV-1 in infected cells. Research done in the last few years showed that B F A blocks the movement of proteins from the ER to the Golgi complex (Takatsuki and Tamura 1985; Misumi, Miki et al. 1986; Oda, Hirose et al. 1987). Although its mechanism of action has not been identified, Lippincott-Schwartz et al proposed that B F A acts by inducing the fusion of early Golgi compartments with the intermediate recycling compartment located between the ER and the Golgi (Lippincott, Donaldson et al. 1990). The intermediate compartment appears to be involved in the sorting of secreted and ER-resident proteins (Saraste and Kuismanen 1984; Lodish, Kong et al. 1987; Schweizer, Fransen et al. 1988). ER resident proteins are sequestered in the intermediate compartment and returned to the ER in membrane vesicles via a microtubule-dependent transport mechanism. Because it has been established that the movement of proteins between the intermediate compartment and the Golgi complex is facilitated by a microtuble-independent process (Armstrong and Warren 1990), it can be concluded that Golgi proteins are not recycled to the ER directly from the Golgi complex. In the presence of B F A , the intermediate compartment is induced to fuse with the Golgi apparatus. This allows proteins in the Golgi complex to gain access to the microtubule-dependent 1 8 pathway to the ER. The anterograde movement of material from the ER to the Golgi complex is slower than the BFA-induced retrograde flow of membranes, resulting in the disappearance and redistribution of cis and medial Golgi into the ER. The net effect of B F A is to block the movement of proteins through the secretory organelles. It has been shown that the treatment of cells with low concentrations of B F A has no effect on endocytosis, protein synthesis and lysosomal degradation (Misumi, Miki et al. 1986; Nuchtern, Bonifacino et al. 1989), despite the observation that the maturation of oligosaccharide moieties on newly-synthesized glycoproteins is inhibited (Doms, Russ et al. 1989). Analysis of protein transport in BFA-treated cells using V S V G protein shows that G protein is not transported to the plasma membrane as it normally is in untreated cells (Doms, Russ et al. 1989). Modification of G protein by cis and medial Golgi enzymes in BFA-treated cells is demonstrated by their partial resistance to endoglycosidase H digestion. However, addition of sialic acid, which is a trans Golgi event, is not observed. These results implicate that as the early Golgi compartments recycle into the ER, the contents of the Golgi, including its resident enzymes, are redistributed to the expanded ER. Removal of BFA results in the rapid flux of Golgi components out of the ER and the reorganization of the Golgi complex. Doms et al (Doms, Russ et al. 1989) reported that Golgi enzymes are completely recovered from the ER to the reformed Golgi by 10 min post B F A removal. Functional transport of proteins through the reorganising secretory organelles may take longer, but it is still a rapid process. G protein from recovering cells becomes completely endo H-resistant in 30 min after B F A removal, and the addition of sialic acid is completed in 45 min. This report demonstrates that B F A blocks the accumulation and egress of infectious HSV-1 in infected cells. This failure to yield infectious particles is not due to any disruption of the assembly or envelopment of nucleocapsids. Instead, there appears to be a blockage in the movement of enveloped particles into the cytoplasm. Although the cellular distribution of viral glycoproteins is perturbed by B F A , this alteration is reversed within 2 h after the removal of B F A from the growth medium. In contrast, the B F A -induced impediment to the release of viral particles is not reversible. These effects of BFA that have been observed are distinct from the effects of other glycosylation inhibitors on the propagation of HSV-1 in infected cells. B F A appears to affect the movement of viral particles at an early stage of the egress pathway, and prevents the acquisition of full infectivity. Further understanding of the viral-host interactions disrupted by B F A may serve to clarify the precise mechanism of egress and the requirements for infectivity of the newly-synthesized viral particles. The results of this study have been published in the Journal of Virology, 1991 (Cheung, Banfield et al. 1991). MATERIALS AND METHODS Cells and viruses. L cells and HSV-1 KOS were obtained from D. Coen. All cells were grown at 37° C in D M E M supplemented with 10% FBS in a 5% C O 2 atmosphere. Brefeldin A was a gift from W. Jefferies. B F A was also obtained from Bio/Can Scientific, Ontario, Canada. Anti-gD monoclonal antibodies were obtained from M . Zweig and R. Philpotts. Treatment of cells with BFA. Monolayers of L cells were infected with HSV-1 at an MOI of 10 PFU/cell. After 1 h of infection, the inoculum was removed and D M E M containing 10% FBS was added. At 2 h.p.i., the medium was removed and fresh medium containing B F A in various concentrations was added. The extracellular medium was sampled at 2 h intervals for 20 h. The titres of infectious particles in each sample were determined by infecting confluent monolayers of Vero cells with appropriated dilutions of virus. The inoculum was removed after 1 h and medium containing methocell was added. Plaques were counted after 3 and 5 d of infection. Harvesting of virus. Monolayers of L cells were infected with HSV-1 (MOI=10). At 2 h.p.i., the medium was changed to labeling medium made up of methionine-free D M E M , 1/10 volume of D M E M with 10% FBS, 4% dialysed FBS, [35S]methionine (50 u.Ci/ml), and B F A (3 u,g/ml). Infected cells were labelled for 16 h, after which the medium was removed and subjected to low-speed centrifugation to pellet cell debris. The supernatant was sedimented through a 5-40% Dextran T10 gradient formed in 50 mM NaCl, 10 mM Tris pH 7.8 for 1 h at 22,000 R P M in a Beckman SW41 rotor. Gradients were fractionated from the bottom into 0.3 ml fractions. For determination of radioactivity in insoluble material, 15% of each fraction to be analyzed was added to 50 p.g BSA followed by 1 ml of 10% cold T C A . Insoluble material was collected onto glass fibre filters after 1 h and radioactivity was determined by scintillation counting. Isolation and quantitation of nucleocapsids. Monolayers of L cells were infected, treated with or without B F A , and labelled with [35S]methionine (100 u.Ci/ml) as described before. At 18 h.p.i., cells were harvested by washing with cold PBS and incubated for 15 min with cold lysis buffer (lOmM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate). Lysed cells were either sonicated or frozen and thawed three times. 0.5M urea was added and the cellular debris was pelleted by low speed centrifugation. The supernatant containing the nucleocapsids was sedimented through a 10-40% sucrose gradient for 1 h at 22K rpm using a Beckman SW 41 rotor. 0.3 ml or 0.5 ml fractions were collected, precipitated with 10% cold T C A , and counted in a scintillation counter as described earlier. Determination of the titre of cell-associated virions. Monolayers of L cells were infected and 3 pg/ml of B F A was added at 2 h.p.i.. At 8 or 18 h.p.i., cells were washed thoroughly with PBS and were lifted with PBS containing 5 m M E D T A . Harvested cells were pelleted by low speed centrifugation and were suspended in RSB. The cell suspension was sonicated and centrifuged to remove cellular debris. The titres of virus in these samples were determined by plaque assay on Vero cell monolayers. Isolation of intracellular viral particles for Electron Microscopy. Infected monolayers of L cells were treated or mock-treated with B F A (3 pg/ml) from 2 to 18 h.p.i.. At 18 h.p.i., cells were washed with PBS and were lifted with PBS containing 5 mM E D T A . Harvested cells were pelleted at low speeds (600 rpm for 10 min) and were suspended in ice cold RSB. Cells were broken up by dounce homogenization and intact nuclei were removed by centrifugation (1000 rpm for 10 min). Cytoplasmic extracts were then sonicated and large debris were removed by medium speed centrifugation (4200 rpm for 5 min). Clarified extracts were layered over 20% sucrose and viral particles were pelleted by ultracentrifugation for 30 minutes at 75K rpm using a Beckman T L A 100.2 rotor. Sediments were washed in PBS, repelleted, and were suspended in 20 pi of distilled water. Concentrated viral particles were negatively stained with 2% phosphotungstic acid (pH 7.4) on formvar coated 400 mesh copper grids and were examined using a Zeiss 10C STEM. 23 Thin Section Electron microscopy. L cells were grown on Millicell H A inserts (Millipore Corporation) overnight prior to infection with HSV (MOI of 10). At 2 h.p.i., fresh medium with or without B F A (3 p.g/ml) was added. At 8, 12, or 18 h.p.i., cells were rinsed with PBS, and fixed with 2.5% glutaraldehyde in 0.1 M NaCaC (pH 7.3). After 1 h of fixation, cells were rinsed with 0.1 M NaCaC and were post fixed in 1% OsCW 0.1 M NaCaC for 1 h. Samples were rinsed, dehydrated in increasing concentrations of ethanol and then in 100% propylene oxide, and were embedded in Epon Araldite. Thin sectioning was done on a Reichert-Jung ultracut E . Sections were stained with uranyl acetate then with lead citrate, and were examined and photographed using a Zeiss 10C STEM. Indirect immunofluorescence. Cells were grown on acid-etched glass coverslips for 2 d and infected with HSV-1 (MOI=10). At 2 h.p.i., medium containing 3 u,g/ml of B F A was added to the monolayers. At 10 h.p.i., the B F A -containing medium was removed and the monolayers were further incubated in normal medium for 0, 30, 60, or 90 minutes. Cells ready to be stained were first rinsed with PBS and fixed with 3.2% paraformaldehyde in PBS. Fixed cells were washed and incubated for 30 min with a 1/100 dilution of anti-gD monoclonal antibody. Primary antibody was washed off and cells were incubated for 30 min with a 1/100 dilution of FITC- or RITC-conjugated goat anti-mouse IgG. Coverslips were rinsed and mounted in 50% glycerol, 100 mM Tris, pH 7.8. Cells were photographed using a Zeiss microscope with epifluorescence optics. B F A chase experiment. Monolayers of L cells were infected with HSV-1 (MOI=10) and various concentrations of B F A were added at 2 h.p.i.. Samples of medium were collected at 4, 8, 12, and 18 h.p.i.. Medium containing B F A was then replaced with normal medium at 18 h.p.i.. Further samples were collected at 30, 60, 120, and 180 minutes after B F A removal. Al l samples were diluted serially and used to inoculate monolayers of Vero cells growing in 96-well dishes. Titres were determined from the highest dilution that showed cytopathic effect. B F A sensitive period. Parallel dishes of HSV-1 infected cells were treated with or without 3 pg/ml of B F A for 4 h between 0-4, 4-8, 8-12, 12-16, and 16-20 h.p.i.. At the end of each B F A treatment, monolayers were washed extensively and normal medium was added. Release of infectious viral particles post B F A treatment was determined by harvesting the extracellular medium at 20 h.p.i. for all dishes and determining their titres by plaque assays on Vero cells. RESULTS B F A inhibits HSV-1 propagation. To determine the effect of B F A on the propagation of HSV-1, monolayers of L cells were infected with HSV-1 and were exposed to various concentrations of B F A from 2 to 20 h.p.i.. To measure the release of infectious viral particles, growth medium of these monolayers were sampled every 2 h and their viral titres were determined on Vero cells. Initial results, shown in Fig 3, indicate that 1 pg/ml of B F A was able to effectively block the release of viral particles from infected cells. This resulted in a seven order of magnitude decrease in virus yield as compared to non-BFA treated cells. Subsequent studies reveal that even at 0.3 pg/ml, B F A can affect the release of viral particles by delaying the exponential release of HSV into the extracellular medium by at least 8 h (Fig. 3). B F A inhibits viral egress. Since the lack of infectious virai particles in the extracellular medium of B F A treated infected cells may not be due to a block in viral egress, but that viral particles released are defective in their infectivity, we also examined the export of particles into the growth medium using radiolabeled virions. Cell monolayers were infected with HSV-1 and 0 or 3 pg/ml of BFA was added at 2 h.p.i.. Both BFA-treated and untreated cells were incubated with 50 pCi/ml of [35S]methionine from 2-18 h.p.i. to radiolabel virions assembled during infection. The culture medium was harvested at the end of 26 •o