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Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection Cheung, Peter 1991

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BREFELDIN A ARRESTS T H E MATURATION A N D EGRESS OF HERPES SIMPLEX VIRUS PARTICLES DURING INFECTION By PETER CHEUNG  B.Sc,  The University of British Columbia, 1986  A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF T H E REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department  We  of Microbiology)  accept this thesis as conforming to the required standard  T H E UNIVERSITY OF BRITISH C O L U M B I A January  1991  © Peter Cheung,  1991  In  presenting this  degree at the  thesis in  University of  partial  fulfilment  of  of  department  this thesis for or  by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my 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  DE-6 (2/88)  KA-re,  tq«f /  ii  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 B F A , 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  DMEM  Dulbecco modified Eagles medium  endo H  Endoglycosidase H  ER  endoplasmic  effects  reticulum  GalNAc  ^-acetylgalactosamine  Glc  glucose  GlcNAc  iV-acetylglucosamine  h.p.i.  hours post infection  HSV-1  herpes simplex virus type 1  Man  Mannose  MOI  multiplicity of infection  PBS  phosphate  PFU  plaque forming units  RSB  reticulocyte  STEM  scanning transmission electron  TCA  trichloroacetic acid  buffered  saline  standard buffer microscope  T A B L E OF CONTENTS:  ABSTRACT  ii  ABBREVIATIONS  iv  T A B L E OF CONTENTS  v  LIST OF FIGURES  vi  ACKNOWLEDGMENT  vii  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 D N A replication, assembly and egress:  7  D. Function of viral glycoproteins:  11  III. Importance of host cell glycosylation machinery to HSV-1 infection: A . Chemical inhibitors of glycosylation HSV-1  14 affect  propagation:  14  B. Cell lines defective in glycoprotein processing also affect growth of HSV-1: IV. Experimental Rationale:  15 17  MATERIALS A N D METHODS  20  RESULTS  25  DISCUSSION  46  REFERENCES  61  vi  LIST OF FIGURES:  Figure 1.  Schematic diagram of the ultrastructure of HSV-1  Figure 2.  Schematic diagram of the egress pathway of  HSV-1 Figure 3.  3  9 Effects of B F A on release of P F U into growth  medium  26  Figure 4. Effects of B F A on viral release  28  Figure 5.  30  Figure 6.  Effects of B F A on nucleocapsid assembly. Electron microscopy of BFA-treated and untreated  cells Figure 7.  34 Immunofluorescence analysis of HSV-1 gD in  infected cells treated with B F A Figure 8.  Determination of the rate of viral release following  removal of B F A Figure 9.  42  Determination of BFA-sensitivity during viral  propagation Figure 10.  38  44 Schematic diagram of an intranuclear pocket of  enveloped particles seen in BFA-treated cells  50  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 ( C M V ) , varicellazoster virus ( V Z V ) , Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7) are the that infect humans.  herpesviruses  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, H H V - 6 , and H H V - 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 immunocompromised individuals. herpesviruses of  viruses  The medical significance of human  makes them one of the most intensely  studied group  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. U L T R A S T R U C T U R E OF HSV-1 VIRIONS: The virion of HSV-1, like all other herpesviruses, consists of four distinct morphological components (figure 1): 1) an electronopaque core containing the viral D N A , 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 10  6  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  DNA.  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 H S V - 1 .  1.  viral D N A core  2.  icosahedral capsid  3.  viral envelope  4.  glycoprotein spikes  5.  tegument  4  II. O V E R V I E W 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 (Smith and DeHarven 1973).  such as C M V  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  (WuDunn and Spear 1989).  glycosaminoglycans  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 p H , 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 D N A 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 m R N A 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 infected cell.  in the  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: A l l herpesvirus genes are transcribed in the nucleus using host cell R N A pol II (Constanzo, Campadelli-Fiume et al. 1977), and viral mRNAs are translated in the cytoplasm by free or membranebound 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,  V P 16, induces the expression of immediate-early (IE) genes in the absence of prior viral protein synthesis.  V P 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 V P 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 V P 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 D N A and structural proteins are accumulated in the nucleus, capsid assembly begins by cleaving the concatameric progeny D N A , 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 E R 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.  A n alternative model suggests  that enveloped viral particles in the perinuclear space do not move to the E R .  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 E R 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  PM  plasma  G  Golgi  ER  endoplasmic  PN  perinuclear  INM  inner  nuclear  membrane  ONM  outer  nuclear  membrane  N  nucleocapsids  V  enveloped  Pathway 1: (solid arrows) enveloped  membrane apparatus reticulum space  virion  Nucleocapsids in the nucleus are  as they bud through the inner nuclear membrane into  the perinuclear space.  Enveloped particles are then transported  through the host E R 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 E R and Golgi complex.  Mature glycoproteins then accumulate at the trans  Golgi membranes where cytoplasmic envelopment occur.  of  nucleocapsids  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). N-linked  A l l of these glycoproteins contain  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 E R 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 H S V  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 T H E HOST C E L L G L Y C O S Y L A T I O N 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  functioning of the host secretory  on the normal  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 HSV-1  glycosylation.  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 It causes an early block in the N-glycosylation effect induced by tunicamycin.  to proteins.  process similar to the  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 Nlinked glycans precludes the envelopment and hence the yield of infectious H S V - 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 acetylglucosaminyl 1981).  transferase  II respectively  I and N(Vischer and Hughes  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 ricinresistant 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). to be defective  This cell line, termed gro29, was shown  in the transport and processing of glycoproteins  through the Golgi complex.  Although some infectious particles are  formed, they remain trapped in the cell cytoplasm. studies emphasize the dependence  A l l of these  of HSV-1 on host secretory and  glycoprotein processing functions for their normal propagation and egress.  IV. E X P E R I M E N T A L R A T I O N A L E : 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 E R 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 E R 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).  E R resident proteins are sequestered in the intermediate  compartment and returned to the E R 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 E R 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  18 pathway to the E R .  The anterograde movement of material from  the E R 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 E R . 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 endoglycosidase H digestion.  to  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 E R , the contents of the Golgi, including its resident enzymes, are redistributed to the expanded E R . Removal of B F A 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 E R 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 B F A 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 particles.  viral  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 K O S were obtained from D. Coen. A l l cells were grown at 3 7 ° 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.  also obtained from Bio/Can Scientific, Ontario, Canada.  B F A was  Anti-gD  monoclonal antibodies were obtained from M . Zweig and R. Philpotts.  Treatment of cells with B F A . 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.  at 2 h intervals for 20 h.  The extracellular medium was  sampled  The titres of infectious particles in each  sample were determined by infecting confluent cells with appropriated dilutions of virus.  monolayers  of Vero  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). 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% F B S , 4%  At  dialysed F B S , [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 m M NaCl, 10 m M Tris pH 7.8 for 1 h at 22,000 R P M in a Beckman SW41 rotor. from the bottom into 0.3 ml fractions.  Gradients were fractionated For determination of  radioactivity in insoluble material, 15% of each fraction to be analyzed was added to 50 p.g B S A followed by 1 ml of 10% cold T C A . Insoluble material was collected onto glass fibre filters after 1 h and radioactivity was  Isolation  determined by scintillation counting.  and quantitation of  nucleocapsids.  Monolayers of L cells were infected, treated with or without B F A , and labelled with [ S]methionine (100 u.Ci/ml) as described 35  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, p H 7.4, 150 m M NaCl, 1% NP-40, 1% sodium deoxycholate). were either sonicated or frozen and thawed three times.  Lysed cells 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 m M 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 S T E M .  23 Thin Section Electron microscopy. L cells were grown on Millicell H A inserts (Millipore Corporation) overnight prior to infection with H S V (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. Reichert-Jung ultracut E .  Thin sectioning was done on a  Sections were stained with uranyl acetate  then with lead citrate, and were examined and photographed using a Zeiss 10C S T E M .  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. for 30 min with a 1/100  Fixed cells were washed and incubated 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 antimouse IgG.  Coverslips were rinsed and mounted in 50% glycerol,  100 m M Tris, p H 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.. medium were collected at 4, 8, 12, and 18 h.p.i..  Samples of  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.  A l 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 H S V 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 B F A was added at 2 h.p.i..  Both  BFA-treated and untreated cells were incubated with 50 pCi/ml of [35S]methionine during infection.  from 2-18  h.p.i. to radiolabel virions assembled  The culture medium was harvested at the end of  26  •o  <D CO CB  0) LL Q.  75  o  Hours post infection  Figure 3.  Effects of B F A on release of P F U into growth medium.  Monolayers of L cells were infected with HSV-1 (MOI=10) and incubated from 2 h.p.i. with various concentrations of B F A as indicated The  extracellular medium was sampled to determine the P F U released  in the presence of B F A . The titres were measured by limiting dilution and plaque  assay.  the labelling period, and subjected to centrifugation on 5-40% dextran gradients to isolate virions.  The gradients were  fractionated and the amount of radioactivity in the T C A insoluble material of each fraction was measured.  As shown in Fig. 4, a peak  of radioactivity representing the released virions was seen in the control cell gradient but not in the B F A treated sample.  This  observation suggests that treatment of infected cells with B F A does result in a block of viral release.  Assembly of nucleocapsids is normal in BFA-treated cells. To determine whether nucleocapsids  form in BFA-treated  cells, monolayers were infected with HSV-1 and treated with B F A (3 pg/ml) from 2-18 h.p.i..  During this time, cells were labelled with  100 pCi/ml [35S]methionine proteins.  to allow detection of nucleocapsid  Cells were then lysed in buffer containing NP40 and Na-  deoxycholate, which keeps nuclei intact while solubilizing other cellular components. same buffer.  The nuclei were pelleted and suspended in the  The nuclear and cytoplasmic extracts were sonicated  and centrifuged separately on 10-40% sucrose gradients to isolate nucleocapsids.  The amount of TCA-insoluble radioactivity in  gradient fractions of BFA-treated and untreated cells are shown in Fig 5.  Peaks of radioactivity representing nucleocapsids were  visible for both cytoplasmic (fraction 13, 10 ml gradient, 0.3 ml fractions) and nuclear enriched samples (fraction 5, 8 ml gradient, 0.5 ml fractions).  Based on the amount of radioactivity, the nuclei  contained approximately 20% of the total nucleocapsids in the cell. There was no significant difference in the number of cytoplasmic  28  0  2  4  6  8 10 12 14 16 18 20 22 24 26 28 30 32 Fraction Number  Figure 4.  Effects of B F A on viral release.  Monolayers of infected cells (MOI=10) were treated with 3 pg/ml of B F A at 2 h.p.i. and incubated along with untreated controls with 50 pCi/ml of [ S]methionine until 18 h.p.i.. 35  The extracellular medium was  harvested and centrifuged to pellet virions and cell debris.  Pellets  were suspended in PBS and centrifuged on a 5-40% dextran gradient to separate virions from other radioactive material.  The gradients were  fractionated from the bottom and amount of TCA-insoluble radioactivity in each fraction was determined by scintillation counting (see  Materials and Methods).  CPM=CPM x l O -  2  nucleocapsids isolated from BFA-treated and untreated cells (Fig. 5A), indicating that B F A did not impede nucleocapsid assembly, nor their movement to the cytoplasm.  Similarly, the number of  nucleocapsids was reduced only slightly in the nuclei of infected cells treated with B F A (Fig. 5B).  In experiments where whole cell  extracts were analyzed on sucrose gradients (Fig. 5C),  BFA-treated  and untreated cells contained similar peaks of radioactivity representing labelled nucleocapsids, whereas  the control gradient  did not.  B F A also affects the accumulation of cell associated virions. To evaluate the effects of B F A on the accumulation of intracellular infectious particles in infected cells, extracts  were  prepared from BFA-treated and untreated cells, sonicated, and the viral titres were measured by plaque assays on Vero cells.  Titres of  cell extracts harvested at 8 h.p.i. were approximately 0.0006, and 6 PFU/cell for B F A treated and non-treated cells.  Cell extracts  harvested at 18 h.p.i. yielded titres of approximately 0.0012, and 270 PFU/cell for B F A treated and non-treated cells.  The magnitude  of reduction in intracellular H S V seen in the 18 h.p.i. samples was similar to the reduction in the extracellular titres noted before.  It  appears from these results that B F A prevents the formation or accumulation of infectious particles in HSV-infected cells.  The lack of intracellular infectious virions in B F A treated cells is due to the lack of enveloped particles in cells. To investigate the cause of the apparent lack of infectious  30  Figure 5.  Effects of B F A on nucleocapsid assembly  Monolayers of infected cells (MOI=10) were treated with 3 pg/ml of B F A at 2 h.p.i. and incubated along with untreated controls with 100 pCi/ml of [ S]methionine until 20 h.p.i.. 35  Monolayers were washed  extensively and incubated with NP-40 lysis buffer (see and Methods).  Materials  After 15 min on ice, the nuclei were pelleted.  nuclear supernatants  and nuclear fractions were sonicated,  Post-  and urea  was added to 0.5 M to free nucleocapsids from cellular structures. Samples were centrifuged through a 10-40% sucrose gradient and fractions were collected from the bottom of the tubes.  The amount  of TCA-insoluble radioactivity in each fraction was determined by scintillation counting.  The data for nucleocapsids isolated from (A)  cytoplasm-enriched fractions, (B) nuclear-enriched, and (C) total cell extracts are  shown.  Nuclei  J 1  i  i 3  i  _i  i  5  i 7  i  i  i  9  Fraction Number  i 11  i  i 13  i  i_ 15  Total cell extract  Fraction  Number  particles in BFA-treated cells, electron microscopy techniques  were  employed to study the status of the viral particles present in B F A treated cells.  More specifically, we tested the possibility that this  lack of intracellular infectious particles was due to the absence of enveloped virions in the cell cytoplasm.  Cytoplasmic extracts of  BFA-treated and non-treated cells were collected, concentrated over sucrose pads and were negative  stained for direct examination  under the electron microscope.  Repeated trials of this experiment  showed that whereas naked nucleocapsids were seen in cytoplasmic extracts from BFA-treated cells, no enveloped particles were observed.  Unfortunately, it was difficult to concentrate intracellular  virus particles from both BFA-treated and non-treated cell extracts possibly due to the low amounts of viruses in infected cells. there were some difficulties in identifying positively particles using negative staining techniques.  Also,  enveloped  To overcome these  shortcomings, infected cells that were treated or mock-treated with B F A were thin sectioned for transmission electron microscopy examination.  Figure 6A, and B represents typical images observed  in BFA-treated or non-treated cells harvested at 8 h.p.i..  The  cytoplasm of non BFA-treated cells contained many enveloped particles accumulated in membrane vacuoles.  In contrast, there  was a complete lack of enveloped particles in the cytoplasm of B F A treated cells even though naked nucleocapsids were seen.  When  enveloped particles were observed in BFA-treated cells, these particles were enclosed in membranous pockets located inside the nucleus as shown in Fig. 6D.  In wild-type cells, similar pockets of  enveloped particles inside the nucleus were occasionally observed  34  Figure 6.  Electron microscopy of BFA-treated and untreated cells.  Monolayers of L cells were grown on Millicell H A inserts (Millipore) for 24 h prior to infection with HSV-1 (MOI=10).  Following  inoculation, fresh D M E M containing 10% FBS was added and the infection was allowed to proceed for 8, 12, or 18 h.  For treated  samples, B F A (3pg/ml) was added at 2 h.p.i. and remained in the medium for the duration of the incubation.  Cell monolayers were  then rinsed, fixed in glutaraldehyde, embedded and sectioned. images shown are for infected cells harvested at 8 h.p.i..  All  (A)  Cytoplasm of HSV-1 infected L cells containing enveloped viral particles.  (B) Cytoplasm of HSV-1 infected L cells treated with B F A  containing non-enveloped particles.  (C) Nucleus of HSV-1 infected L  cells containing enveloped particles in an intra- or peri-nuclear region.  (D) Nucleus of HSV-1 infected L cells treated with B F A  containing enveloped particles in the perinuclear space.  (E)  Perinuclear space of BFA-treated cells containing enveloped particles.  (F) Viral particle in the perinuclear space of BFA-treated  cells.  nu, nucleus; v, enveloped virus particles; n, nucleocapsid; pn, perinuclear space; c, cytoplasmic invagination.  (Fig. 6C), however, their frequency was rare compared to cells treated with B F A .  The conclusion drawn from these electron  microscopy studies suggests that absence of infectious particles within B F A treated cells are due to the lack of enveloped virions accumulating in the cytoplasm of infected cells.  It is possible that  the pockets of enveloped particles seen in both BFA-treated and non-treated cells represent accumulation of enveloped virions in the distended perinuclear space of infected cell nuclei. these virions are infective remains unclear.  Whether  The apparent higher  incidence of intranuclear pockets of enveloped particles observed in BFA-treated cells may be a reflection of the blockage to normal viral traffic in the cell cytoplasm induced by B F A .  B F A alters the distribution of viral glycoproteins. To study the effect of B F A on the distribution of HSV-1 glycoproteins, indirect immunofluorescence was done on infected cells using monoclonal antibodies directed against HSV-1 gD.  Cell  monolayers were infected and treated with B F A (3 pg/ml) from 2 h.p.i. onward.  At 10 h.p.i., B F A was removed and cells were incu-  bated in BFA-free medium for various lengths of time (see Fig. 7). To detect gD, cells were fixed and subsequently stained with an anti-HSV-1 gD monoclonal antibody.  Examination of control cells  (Fig. 7A) revealed punctate staining of gD at the periphery of the infected cells.  Juxtanuclear staining characteristic of the Golgi  complex was also evident.  The plasma membrane was  well-defined  in these cells, indicating that gD was present in this membrane.  The  perimeter staining likely represents  that  virion-containing vacuoles  3 8  Figure 7.  Immunofluorescence analysis of HSV-1 gD in infected  cells treated with BFA.  Cells were grown on glass coverslips for 3 days to allow for good adherence.  Monolayers were infected with HSV-1 (MOI=10).  At 2  h.p.i., 3 pg/ml of BFA was added to the medium (B,C, and D) or omitted for controls (A).  At 13 h.p.i., the medium was replaced with  fresh medium without BFA.  Sample monolayers were incubated at  3 7 ° C for 0 min (B), 30 min (C), or 3 h (D) and then fixed with formaldehyde.  Control cells (A) were fixed at 16 h.p.i..  gD was  detected using a monoclonal antibody and a fluorescein-conjugated (A,C) or rhodamine-conjugated (B,D) secondary antibody.  •if  *"  i *•  were en route to the plasma membrane. Treatment of infected cells with B F A induced a drastic alteration in the distribution of gD (Fig. 7B).  In contrast to control  cells, gD staining was homogeneous throughout the cell cytoplasm. Furthermore, gD was not detectable in the nuclear or plasma membranes.  Instead,  a fine, evenly-distributed reticular staining  characteristic of the E R was evident. evident,  The Golgi complex was not  suggesting that it was dispersed in BFA-treated infected  cells or that gD was not present in Golgi membranes. Many studies have concluded that the effects of B F A on protein secretion and dissolution of the Golgi complex are reversible within minutes after removal of B F A (see introduction).  Evidence  that some of the effects of B F A are reversible in HSV-1 infected cells can be found in Fig 7C and 7D.  By 30 min after removal of B F A  (Fig. 7C), regions of discrete staining were visible in a roughly perinuclear ring.  This suggests that gD which was previously  recycled into the E R was sorted and re-distributed into the reorganising Golgi membranes.  Some ER staining was also evident  as indicated by the reticular staining surrounding the nucleus.  By 3  h after removal of B F A (Fig. 7D) juxtanuclear staining was evident in a pattern characteristic of the Golgi complex.  Small regions of  punctate staining were also present at the cell periphery, suggesting that the pathway of virion egress was functioning to some extent. The plasma membrane was also clearly stained by this time, indicating that gD was transported to this membrane.  41 The effects of B F A on HSV-1 propagation are not fully reversible. To investigate whether the ability to secrete infectious particles was regained after B F A was removed, cells were infected, treated with B F A at 2 h.p.i. and incubated for a further 16 h.  At  this time, B F A was washed out and the amount of infectious virions released over the next 3 h into the extracellular medium was determined.  The results of this analysis (Fig. 8) revealed that cells  treated with non-inhibitory concentrations of B F A (0.03  and 0.1  u.g/ml) released large quantities of infectious particles into the medium within 30 min after the removal of B F A . Controls with no B F A were indistinguishable from 0.03 u,g/ml B F A . However, this viral production was not matched in the monolayers treated with higher concentrations of B F A . In these cells, there was a lag in the appearance of P F U in the extracellular medium.  Even though we  observed a gradual increase in extracellular P F U during the 3 h after B F A removal in the 1 u.g/ml samples, only 0.1% of the normal amount of extracellular P F U released into the medium. The data shown indicate the most virus that we have detected during the 3 h release from the effects of 1 p.g/ml B F A . yielded as few as 10  4  Other experiments  P F U during this period.  have  These results suggest  that unlike the rapid recovery of cellular protein secretion post B F A removal (Doms, Russ et al. 1989), recovery of virion release to normal levels, if it does occur, requires a very long time.  A B F A sensitive period exists. To examine the possibility of a critical time period during the replication cycle that is most sensitive to the effects of B F A , B F A  0  1  2  3  4  Time after B F A removal (h)  Figure 8.  Determination of the rate of viral release following B F A  removal.  Cells were infected and treated as described in the legend to Fig. 3. 18 h.p.i., cell monolayers were washed thoroughly to remove B F A . Samples of the extracellular medium were taken at various times the HSV-1 titres were determined by limiting dilution.  was applied to infected cells in 4 h blocks at different times of the infection cycle.  Recovery from the effects of these 4 h B F A  exposures were measured at 20 h.p.i. by comparing the cumulative release of infectious virions post B F A treatment of treated and mock-treated cells.  Viral titres of the extracellular medium  collected at 20 h.p.i. of the test and control cells were determined and were expressed as percentages of viral titre of the mocktreated control cells. representative  Figure 9A shows the result obtained in a  experiment.  Although the actual percentages  of  viral titre fluctuated from experiment to experiment, the overall trend was identical to that shown in the representative  experiment.  Exposures of infected cells to B F A from 0-4 or 16-20 h.p.i. did not seem to significantly alter the release of viral particles from cells. Application of B F A to infected cells at 4-8, 8-12, or 12-16 h.p.i. appeared to reduce the subsequent release of virions by at least 60% of control cell viral release experiments.  Moreover, the  8-12  h.p.i. B F A treatment time period was repeatedly shown to be the time period most affected by B F A . This period corresponds to the exponential virus release phase of a typical infection, thus further emphasizing that B F A affects the egress mechanism of the viral life cycle.  44 Figure 9.  Determination of BFA-sensitivity during viral propagation.  Monolayers of cells were infected with HSV-1 (MOI=10) and treated with B F A (3u,g/ml) for 4 h intervals from 0 to 20 h.p.i.. monolayers were also analysed in parallel experiments.  Control Following  treatment with B F A , the monolayers were washed and the medium was replaced with fresh D M E M containing 10% FBS.  Samples of the  medium were taken at 20 h.p.i. and the HSV-1 titre was determined by plaque assay.  (A) Virus yield from BFA-treated cells expressed  as a percentage of the yield from untreated control cells. samples were analysed.  Duplicate  (B) Virus yield from untreated control cells  treated in the same manner as in part A .  Virus titres from samples  harvested at each time point were added together to reflect the cumulative yield during the 20 h of the experiment.  0-4  4-8  8-12  12-16  16-20  Hours post infection  0  4  8  12  Hours post infection  16  20  DISCUSSION  It has long been appreciated that herpes simplex virus (HSV) requires the host secretory apparatus for the maturation and egress of newly-synthesized  viral particles (Campadelli-Fiume and  Serafini-Cessi 1985).  Not only do viral glycoproteins depend on the  host cell E R and Golgi compartments for their processing, it is likely that enveloped  virions are transported through these same  organelles for their export out of the cell (Johnson and Spear 1982). In this report, the effects of Brefeldin A (BFA) on the propagation of HSV-1 in infected cells were investigated.  B F A is a drug that  inhibits the cellular transport of proteins from the E R to the Golgi complex.  By examining how the BFA-induced blockage to host cell  secretion affects the growth of HSV-1, we hope to further define the interactions between virus and host components  that are required  for the efficient propagation of this virus. Results from this study demonstrated that treatment of infected cells with B F A leads to a reduction in extracellular virus yields as compared to untreated control cells (Fig. 3).  Treatment of  infected cells with as little as 0.3 u.g/ml of B F A delayed the appearance of infectious particles into the extracellular medium by at least 8 hours.  Furthermore, higher concentrations of B F A such as  1 p.g/ml or above led to a blockage of the release of infectious virions.  This is illustrated by the seven order of magnitude  reduction in the cumulative P F U released from BFA-treated cells as compared to control cells at 18 h.p.i. (Fig. 3).  The transient  inhibitory effect seen with 0.3 |ig/ml of B F A is likely a reflection of B F A being metabolized by cells over long incubation periods to concentrations that are no longer effective propagation.  in inhibiting viral  To ensure that sufficient B F A was present during the  long experimental procedures, all subsequent  experiments  were  performed using 3 pg/ml of B F A . By labelling progeny viral particles assembled during HSV-1 infection with [ S]methionine, it was demonstrated that the lack of 35  plaque forming units in the extracellular medium was not due to the release of non-infectious viruses.  Instead, there was a blockage  to particle egress in BFA-treated cells (Fig. 4).  Further  investigations on the cause of this inhibition revealed that B F A caused a major reduction in the accumulation of infectious particles inside the infected cells (see results).  It appears that B F A interferes  with the formation of mature viral particles in host cells, and hence few viral particles are exported into the extracellular medium. The interference of the formation of infectious particles can occur at different parts of the virus life cycle.  It is unlikely that  B F A disrupts the assembly of viral nucleocapsids in the nucleus since similar amounts of assembled nucleocapsids were found in total cell extracts from BFA-treated and non-treated cells (Fig. 5). Furthermore, when cell extracts from BFA-treated cells were separated into fractions enriched for nuclei or cytoplasm, assembled nucleocapsids were found in both fractions.  This suggests that B F A  does not affect the assembly of nucleocapsids in the infected cell nucleus, nor does it impede the transport of these nucleocapsids into the cytoplasm.  Another explanation to account for the lack of cell-associated infectious particles is that B F A interferes with the envelopment of nucleocapsids.  It has already been shown that glycosylation  inhibitors such as tunicamycin disrupts the envelopment capsids (Peake, Nystrom et al. 1982). from infected cells were negatively  of viral  When cytoplasmic extracts  stained and examined by  electron microscopy, naked nucleocapsids, but not enveloped particles, were observed in BFA-treated cells.  To obtain more  evidence that BFA-treated cells lack enveloped particles in their cytoplasm, transmission electron microscopy was employed to study the viral particles in thin-sectioned cells.  Monolayers of B F A -  treated and untreated cells were infected, and prepared for electron microscopy at 8, 12, and 18 h.p.i..  In contrast to the abundance of  enveloped particles enclosed in cytoplasmic vesicles seen in control cells, no enveloped virions were present in the cytoplasm of B F A treated cells at any of the analysed time points (Fig. 6A and B). Similar amounts of non-enveloped nucleocapsids  were observed in  the cytoplasm of both BFA-treated and control cells, and this is consistent  with the results obtained in the radiolabeled  nucleocapsid  study.  Further analysis of the nuclei of BFA-treated cells revealed that the failure to detect enveloped particles in the cell cytoplasm was not due to a block in the mechanism of envelopment at the inner nuclear membrane.  Enveloped particles were frequently  found in BFA-treated cells, but they accumulate exclusively in membranous compartments within the nuclei (Fig. 6D-F).  These  intranuclear pockets of virions, which are often located adjacent to  nuclear membranes, are not likely to be invaginated cytoplasm because they do not contain ribosomes. represent  newly-enveloped  perinuclear space.  Instead, they probably  particles accumulating in the  distended  It has been reported that HSV-1 infection  induces the duplication of the nuclear membranes for the envelopment of newly made particles to occur (Poliquin, Levine et al. 1985).  Also, the presence of intranuclear compartments  containing enveloped particles has been documented for H S V infected cells (Dargan 1986).  When infected control cells were  examined by transmission electron microscopy, enveloped particles were seen in intranuclear structures (Fig. 6C), albeit their rare occurrence.  There is little doubt that virions are normally  enveloped at the inner nuclear membrane as they bud into the perinuclear space (Darlington and Moss 1969; N i i , Morgan et al. 1969).  These newly enveloped particles accumulate rarely at this  cellular location in wild type cells because they are immediately transported to their next destination, most likely to be the E R .  In  BFA-treated cells, since the normal transport of viral particles from the E R to Golgi is blocked, this may cause an aberrant accumulation of enveloped particles in the perinuclear space. Upon closer examination of the nuclei of BFA-treated cells, a high concentration of naked nucleocapsids in a separate compartment adjacent to the pocket of enveloped virions was observed (Fig 6E).  often  These structures are interpreted as enveloped  particles in' the perinuclear space surrounding an area of invaginated cytoplasm where naked nucleocapsids are seen (see Fig. 10).  and ribosomes  As mentioned before, enveloped particles  50  Figure 10.  Schematic diagram of an intranuclear pocket of  enveloped particles seen in BFA-treated cells.  Abbreviations:  N  nucleocapsid  V  enveloped  NU  nucleus  PN  perinuclear  CY  cytoplasm  virion  space  or cytoplasmic invagination  The pockets of enveloped particles seen in the nucleus of B F A treated infected cells are likely viral particles accumulating in the distended perinuclear space. separate particles.  Naked nucleocapsids are often seen in  membranous compartments adjacent to the  enveloped  They may represent enveloped viral particles that fused  with the outer nuclear membrane and are located in the invaginated cytoplasm as non-enveloped capsids.  appear to be detained abnormally in the perinuclear space of B F A treated cells.  These particles may fuse with the outer nuclear  membrane, de-envelope, as nucleocapsids.  and localise in the invaginated cytoplasm  Such an alternative method of egress from the  nucleus may provide a logical explanation for the presence of naked nucleocapsids and the lack of enveloped particles in the cytoplasm of BFA-treated cells. There are currently two models of viral egress proposed for H S V (see Fig. 2).  The first and most widely accepted model was  proposed by Morgan's group based on electron microscopy studies (Nii, Morgan et al. 1969).  This model suggests that newly-assembled  nucleocapsids acquire their viral envelopes as they bud at the modified inner nuclear membrane into the perinuclear space. Virions accumulated in this region exit from the cell via a process of "reverse phagocytosis".  Enveloped particles first move to the E R by  virtue of the contiguity of the nuclear membrane and E R , or they are transported specifically to the E R by membrane vesicles. Immature glycoproteins present on the viral envelopes are processed and modified as the particles traverse the ER-Golgi pathway en route to the cell surface.  This model is strongly  supported by the monensin study done by Johnson and Spear (Johnson and Spear 1982).  Monensin is known to block the  transport of proteins from the Golgi stacks to the cell surface by disrupting ionic gradients between cellular organelles.  Infected  cells treated with this drug not only inhibits the egress of virions, infectious particles are found in cytoplasmic vacuoles that are probably derived from Golgi membranes.  The second model of viral egress, proposed by Stackpole (Stackpole  1969), agrees that newly-assembled  nucleocapsid bud at  the inner nuclear membrane to acquire an envelope viral capsid.  around the  However, it predicts that enveloped particles in the  perinuclear space quickly fuses with the outer nuclear membrane, and are released as naked nucleocapsids into the cell cytoplasm. Free viral glycoproteins are made, transported into the E R , and processed as they traverse the E R and Golgi compartments.  Mature  viral glycoproteins then accumulate and modify the trans Golgi membranes where envelopment nucleocapsids occurs.  of the cytoplasmic naked  Proponents of this model have shown by  transmission electron microscopy that one can find  structures  resembling Golgi membranes wrapping around naked capsids in the cytoplasm (Komuro, Tajima et al. 1989).  These structures however,  are more frequently found very late in the infection cycle (16 to 48 h.p.i.).  Since enveloped particles are first released at about 8 h.p.i.  (Smith and DeHarven 1973), this model of egress may represent a secondary pathway for virion release. The present study does not distinguish one model of viral egress from another because both models imply that the ER-Golgi pathway plays an important role in the propagation of H S V particles.  The BFA-induced recycling of cis/medial Golgi into the ER  can block the movement of enveloped particles as well as immature viral glycoproteins to the trans Golgi.  The net effect of both  situations can lead to the inhibition of viral egress.  In agreement  with both models, enveloped particles were observed at the perinuclear space of infected cells.  If these particles were to follow  the second model of egress, one would expect to see an abnormally high concentration of naked nucleocapsids in the cytoplasm since B F A should not affect the de-envelopment process of viral particles at the outer nuclear membrane.  This, however, was not observed in  the electron microscopy analysis.  Instead, high concentrations of  enveloped particles seem to accumulate aberrantly in the perinuclear space of BFA-treated cells.  It is possible that enveloped  virions mainly travel through the E R and Golgi for export.  Newly-  enveloped particles that are to be transported to the E R may be blocked by the BFA-induced retrograde movement of material from the Golgi complex to the ER.  Net anterograde movement of material  from the nucleus to the E R to the Golgi may be required for these viral particles to enter the host secretory pathway.  As  hypothesized earlier in this discussion, enveloped particles detained in the nucleus may fuse with the outer nuclear membrane and be released as nucleocapsids into the cytoplasm similar to the situation predicted by the second model of viral egress.  This seems to  further support the idea that both mechanisms of viral release are employed in normal infected cells, and that the blockage of one pathway shunts the egressing particles into the other route. Although no cytoplasmic enveloped particles were present in BFA-treated cells, viral glycoproteins were abundant in the cytoplasm of these cells.  The processing and distribution of HSV-1  gD was investigated as a representative of all viral glycoproteins since gD is one of the most important and well studied glycoproteins of H S V - 1 .  By using Western immunoblot techniques, processing of  HSV-1 gD in infected cells treated with B F A was found to occur even  though full maturation of this glycoprotein was not achieved (Cheung, Banfield et al. 1991).  In addition, some of the gD isolated  from BFA-treated cells were found to be processed to endo H resistant forms.  Resistance of glycoproteins to endo H digestion is a  marker for the processing of glycoproteins by cis/medial Golgi resident enzymes.  The presence of endo H-resistant forms of gD in  BFA-treated cells suggests that the cis/medial Golgi and their resident enzymes were recycled into E R in the presence of B F A . The lack of mature gD in BFA-treated cells indicates that no glycoproteins were transported past the combined ER/Golgi compartment to the trans Golgi membranes.  These results are in  agreement with observations made on the processing of V S V G proteins in BFA-treated cells by Doms et al (Doms, Russ et al. 1989). In addition to the alteration of glycoprotein processing, B F A also affected the distribution of viral glycoproteins in cells. Immunofluorescence  studies revealed that B F A blocked the  transport of gD to the plasma membrane (Fig. 7A,B).  Unlike gD in  control cells which localised to Golgi-like compartments (Fig. 7A), gD in BFA-treated cells was spread over the cytoplasm in a fine reticular distribution characteristic of E R structures (Fig. 7B).  This  again supports the hypothesis that B F A induces the Golgi and its contents to flow back to the E R compartment.  gD that is in transit in  the host cell secretory system is dispersed into the enlarged E R and is not able to move beyond this blockage.  It is in this combined  compartment where viral glycoproteins are processed by the mixture of E R and Golgi enzymes. One of the interesting characteristics of B F A is the rapid  recovery of protein secretion upon removal of B F A . was examined using immunofluorescence  to follow  This property the  redistribution of HSV-1 gD at various times post B F A removal. Within 30 minutes, the evenly dispersed gD began to relocalise to discrete perinuclear regions, suggesting that gD was transported to the reorganising Golgi structure (Fig. 7C).  After 2 to 3 h of recovery  from the effects of B F A , the distribution of gD was identical to the untreated control cells (Fig. 7D).  gD was evident in Golgi-like  compartments and on the plasma membrane, indicating that the transport of viral glycoproteins had also recovered.  These results  are in complete accordance with the results obtained by Doms et al on the redistribution of V S V G proteins post B F A removal (Doms, Russ et al. 1989).  Together, these results suggest that the recovery  of viral glycoprotein distribution and secretion, at least at a morphological level, is rapid upon the removal of B F A . Although the immunofluorescence  data showed  that the  effects of B F A on the distribution of viral glycoproteins were reversed within 2 to 3 h, the ability to release infectious virions was found to be not as easily reconstituted.  In following the release of  infectious particles from infected cells recovering from 16 h of exposure to B F A , it was observed that only a small amount (<0.1%) of viral particles were released by 3 h post B F A removal as compared to the mock BFA-treated cells (Fig. 8). similar experiment  using the non-glycosylated  In contrast, a human growth  hormone (hGH) showed that the secretion of h G H in BFA-treated cells was recovered within 30 min post B F A removal (Cheung, Banfield et al. 1991).  It is likely that a long recovery period is  required before large amounts of virus are released because enveloped particles are not accumulated in the BFA-treated cell cytoplasm.  A n extended period of time may be needed for the  recovering cells to process  and transport  newly-synthesized  particles through the reorganising secretory  pathway.  It is also possible that infected cells treated with B F A for long periods of time may never fully regain their potential output of progeny viruses.  There may be cellular functions that are critical to  viral propagation at particular times of the HSV-1 infection cycle.  If  these functions are disrupted by B F A , fully productive viral infection will not occur.  Regarding this issue, this study  demonstrated that infected cells exposed to B F A for 4 h periods at different parts of the- infection cycle are affected differently as measured by the cumulative amount of virus released at 20 h.p.i.. Exposure to B F A at 4-8, 8-12, and 12-16 h.p.i. resulted in 60-75% reduction of viral output as compared to mock BFA-treated cells (Fig. 9).  In contrast, this effect was not observed when B F A was  applied to infected cells at 0-4, and 16-20 h.p.i.. To understand these results, it is important to know about the chronological order of viral events in an HSV-1 replication cycle. Using electron microscopy studies, Smith and DeHarven reported that the assembly of viral capsids first start at about 4 to 5 h.p.i. (Smith and DeHarven 1973).  Envelopment of particles at the  nuclear membrane starts at 6 h.p.i., and the release of infectious particles into the extracellular medium can first be detected at about 8 h.p.i..  As noted before, B F A does not alter viral protein  synthesis and D N A replication.  Since viral assembly does not occur  until 4 to 5 h.p.i., it is not surprising that exposure of infected cells to B F A at 0-4 h.p.i. does not affect the subsequent egress of viral particles.  When B F A was applied to infected cells at 16-20 h.p.i., a  small inhibitory effect on viral release was seen.  The continued  release of virions in the presence of B F A observed in this case suggests that matured viruses are accumulated in cellular locations not sensitive to B F A prior to egress from cells, and that B F A does not affect the actual viral release events. The most interesting results of this experiment are the reductions of viral release when B F A was added at 4-8, 8-12, and 12-16 h.p.i..  B F A added at 4-8 h.p.i. would probably affect the exit  of enveloped particles from the infected nucleus since envelopment of particles at the nucleus occurs at about 6 h.p.i..  Even though  these cells had 12 hours of recovery time from B F A , there was still a 60% reduction in the cumulative viral output at 20 h.p.i..  This  suggests that enveloped particles that are blocked at the nucleus are not able to egress from the infected cells even after the effects of B F A on the secretory pathway is reversed.  As suggested earlier,  enveloped particles blocked at the nucleus may de-envelope and enter into the cytoplasm as naked nucleocapsids.  In this case, their  infectivity will not be regained even after B F A is removed from the host cells. B F A applied at 8-12, and 12-16 h.p.i. affects a different part of the viral life cycle as assembled and presumably enveloped particles are already in transit through the secretory pathway when B F A was added.  Particles in the Golgi complex may be recycled into  the E R and be trapped there until B F A was removed and the  secretory system has recovered.  In a study reported at the 15th  International Herpesviruses Workshop, B F A was found to inhibit the egress of pseudorabies virus (PRV), which is another member of the herpesvirus family (Whealy, Robbins et al. 1990).  Electron  micrographs of P R V infected cells treated with B F A for short periods of time showed that enveloped particles were accumulated in the E R . Assuming that B F A applied to HSV-1 infected cells at 812 and 12-16 h.p.i. also leads to the accumulation of enveloped particles in the E R , one important question raised is that whether these particles resume their course of egress after B F A is removed or are they functionally dead and are never released from the cells? The lack of viral output measured at 20 h.p.i. seems to suggest that particles that are temporarily trapped are not able to regain their ability to egress from the infected cell.  These detained particles  may be degraded in the cytoplasm or they may be detoured to somewhere egress.  else and are unable to re-enter the normal route of  To fully address these possibilities, further studies  determining the fate of these trapped particles are required.  It is  possible to use Bromodeoxyuridine (Budr), which is a base analogue of thymidine, to pulse label newly synthesized viral D N A in infected cells.  Using commercially available antibodies directed against  Budr, one may be able to do immuno-labeling electron microscopy and follow the egress of the Budr labelled progeny H S V particles. Applying this technique on infected cells exposed to brief periods of B F A at different time points of the virus life cycle may lead to results that will uncover the fate of these trapped viral particles. Given the importance of the host secretory pathway to the  HSV-1 life cycle, it is not surprising to find that drugs such as B F A , which interfere with the secretion of proteins, also disturb the propagation of this virus. unexpected results.  Nevertheless, this study did yield some  Since B F A blocks the movement of proteins  from the E R to the Golgi complex, one would expect to see herpes particles accumulating at the E R of BFA-treated cells.  This,  however, is not the case when B F A is present early in the infection. There appears to be a complete lack of enveloped particles in the cell cytoplasm, and viral particles accumulate aberrantly in the nucleus instead.  This suggests that the mechanism of movement of  virions from the nucleus to the ER is also sensitive to the effects of BFA.  Another interesting result is the persistent inhibition of the  release of viral particles after B F A is removed.  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