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The role of glycosaminoglycans in binding of herpes simplex virus type 1 to mammalian cells Leduc, Yves 1993

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THE ROLE OF GLYCOSAMINOGLYCANS IN BINDING OF HERPES SIMPLEX VIRUS TYPE 1 TO MAMMALIAN CELLS  by  Yves Leduc B.Sc, McGill University, 1988  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER 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 December  1993  © Yves Leduc, 1993  In  presenting  degree freely  this  at the available  copying  of  department publication  of  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  I further  purposes  (Signature!  Department  of  J\  [ C £ o fr \ 0 i~ 0 <S y  The University of British Vancouver, Canada  Date  DE-6 (2/88)  Columbia  It  gain shall not  permission.  requirements that  agree  may  representatives.  financial  the  be is  that  the  Library  permission  granted  by  understood be  for  an  advanced  shall make for  the that  allowed without  it  extensive  head  of  my  copying  or  my  written  11  1.ABSTRACT Binding of herpes simplex virus type 1 (HSV-1) to the cell surfaces initially requires interactions between viral glycoproteins embedded in the viral envelope and cell surface glycosaminoglycans (GAGs). In this work, mutant cells derived from Ltk" murine fibroblasts and devoid of the cell surface GAG heparan sulfate (HS) (gro2C cells) or HS and chondroitin sulfate (CS) (sog9 cells) were used to study the importance of these GAGs in HSV-1 binding. HSV-1 plaque formation and binding, as assessed by the use of radiolabeled virions, were reduced by approximately 85% with gro2C cells relative to parental L cells. This indicated that cell surface HS is important but not essential for HSV-1 binding. Addition of soluble forms of HS and, to a lesser extent, CS types A, B (both 4-sulfated), and C (6sulfated) blocked adsorption of HSV-1 to gro2C cells and thereby inhibited plaque formation in a GAG concentration dependent manner. The high efficiency by which HS inhibited HSV-1 plaquing on gro2C cells relative to L cells indicated that HSV-1 binding to gro2C cells was "weaker" than to L cells. The inhibitory effect of CS type B on HSV-1 plaquing to gro2C cells showed that HSV-1 can interact with CS, albeit less efficiently than with HS, and suggested that HSV-1 binding to gro2C cells might involve cell surface CS. Further indications for the role of cell surface CS in HSV-1 binding came from the use of the gro2C derived, HS- and CS-deficient sog9 cells. Associated with this phenotype was a reduction in HSV-1 plaquing: 1% relative to L cells which suggests that cell surface CS is responsible for HSV-1 binding to CS-containing gro2C cells. These results indicate a predominant role for cell surface HS in HSV-1 binding and a secondary role for cell surface CS. Additional results obtained from studying the effects of synthetic anionic polysaccharides (dextran sulfate, DEAEdextran, and dextran T-500) on HSV-1 plaquing with L, gro2C and sog9 cells suggest that the HSV-1 interactions with GAGs are electrostatic in nature with minor requirements for the recognition of structural features.  Ill  TABLE OF CONTENTS Page 1. ABSTRACT  ii  2. LIST OF TABLES  vii  3. LIST OF FIGURES  viii  4. ACKNOWLEDGMENTS  x  5. INTRODUCTION  1  A.  The herpesviruses  1  B.  HSV structure  4  C  Proteoglycans  9  C.l. General structure and nomenclature  9  C.2. Heparan sulfate and chondroitin sulfate glycosaminoglycans  D  1 1  C.3. Core proteins  16  C.4. Roles and interactions  18  HSV lifecycle  20  D.l. Binding of HSV to cells  20  D.2. Penetration of HSV into cells  24  D.3. Gene expression and egress of HSV from infected cells  26  Pa E  Mutant cell lines used to study HSV-1  6. MATERIALS AND METHODS  28 32  A.  Abbreviations used  32  B.  Cells and viruses  32  C  Reagents  32  D.  Viral stock production  33  E.  Viral plaque assays  33  F.  Isolation of radiolabeled virus  33  G  Binding of radiolabeled virus to cells  34  H.  Penetration assay  35  I.  Plaque formation inhibition assays  35  J.  Determination of the chondroitin sulfate type on L cells  36  7. RESULTS A.  38  Role of heparan sulfate in HSV-1 infectivity  38  A.l. Selection of gro2C cells  38  A.2. Effect of heparan sulfate on HSV-1 binding  39  A.2.a. Wild type HSV-1 binding on gro2C cells  39  Pa A.2.b. GC" HSV-1 binding on gro2C cells  42  A.2.c Effect of soluble heparan sulfate on HSV-1 binding  42  A.3. Effect of heparan sulfate on HSV-1 penetration rate into L cells  46  A.4. Effect of soluble heparan sulfate on HSV-1 plaque formation B.  C  48  Role of chondroitin sulfate in HSV-1 infectivity  50  B.l. Effect of soluble chondroitin sulfate on HSV-1 plaque formation  50  B.2. Chondroitin sulfate type on L cells  53  B.3. Selection of sog9 cells  54  B.3.a. HSV-1 plaquing and GAG HPLC profile  54  B.3.b. Protein processing and transport  54  B.3.C. VSV propagation  57  B.4. HSV-1 binding on sog9 cells  57  B.5. Effect of soluble heparan sulfate on HSV-1 plaque formation with sog9 cells  59  Nature of the HSV-1 binding interaction: electrostatic vs sequence recognition  59  VI  Page C.l. Effect of dextran sulfate on HSV-1 plaque formation  61  C.2. Effect of DEAE-dextran on HSV-1 plaque formation  61  C.3. Effect of dextran T-500 on HSV-1 plaque formation  63  8. DISCUSSION  66  9. REFERENCES  75  Vll  2 . LIST OF TABLES Table  Page  1. Glycosaminoglycan groups  10  2. Properties of variant murine cell lines  56  3 . LIST OF FIGURES Figure 1.  2.  3.  4.  5.  Page  Diagrammatic representation of the herpes simplex virus virion structure  2  Diagrammatic representation of the herpes simplex virus lifecycle  7  Scheme of disaccharide sequences identified in heparin and heparan sulfate  13  Repeating disaccharide structure of chondroitin types A, B, and C  14  sulfate  Schematic drawing of the linkage region between a glycosaminoglycan chain and a serine residue of a core protein in a proteoglycan molecule  15  6.  Model of entry of HSV-1  21  7.  Anion exchange HPLC of cell-associated glycosaminoglycans (GAGs) derived from L, gro2C and sog9 cells  40  Binding kinetics of radiolabeled wild type HSV-1 on L and gro2C cells  41  Binding of increasing concentration of wild type HSV-1 to HEp-2, L, gro29, and gro2C cells  43  8.  9.  10. Binding kinetics of radiolabeled gC~ HSV-1 on L and gro2C cells  44  11. Binding of increasing concentration of gC~ HSV-1 to HEp-2, L, and gro2C cells  45  IX  Figure  Page  12. Effect of soluble heparan sulfate on biding of wild type and gC" HSV-1 virions on L, gro2C, and HEp-2 cells  47  13. Rate of penetration of HSV-1 into L and gro2C cells  49  14. Inhibition of HSV-1 plaque formation by soluble heparan sulfate  51  15. Inhibition of HSV-1 plaque formation by soluble chondroitin sulfate  52  16. Chondroitin sulfate type on L cells  55  17. Binding of increasing concentration of radiolabeled wild type HSV-1 to gro2C and sog9 cells  58  18. Inhibition of HSV-1 plaque formation by soluble heparan sulfate using sog9, L, and gro2C cells  60  19. Inhibition of HSV-1 plaque formation by soluble dextran sulfate using L, gro2C, and sog9 cells  62  20. Inhibition of HSV-1 plaque formation by soluble DEAE-dextran using L, gro2C, and sog9 cells  64  2 1 . Inhibition of HSV-1 plaque formation by soluble dextran T-500, dextran sulfate, and DEAE-dextran using sog9 cells  65  X  4 . ACKNOWLEDGMENTS I would like to express my thanks to Dr. Frank Tufaro for his unending patience, guidance and support during the course of this project. I gratefully acknowledge the University of British Columbia and the National Science and Engineering Research Council for their financial support. I would like to thanks Drs. Gerry Weeks and Pauline Jonhson for serving on my thesis committee. I am grateful to the members of the laboratory for their help, humor and understanding. Special thanks to Bruce Banfield and Leone Atkinson for their friendship, support, technical assistance and participation in numerous laughing sessions. I would like to express my sincere thanks to my parents, Rita Martel and Jean-Paul Beaudry, for their love and support throughout these demanding three years. A special warm thanks to Beverli Barnes for her constant love, patience, listening skills and unflinching support. You've kept me going with a smile.  1 5. INTRODUCTION A. The herpesviruses Herpetic diseases have been noted for centuries. Since these diseases are characterized by the spread of vesicle clusters on the skin (1), they were given the name herpes which is from the Greek herpein, meaning to creep. The herpesviruses are ubiquitous, comprising a very large group of over 80 different viruses. All species of animal life surveyed to date have yielded at least one distinct herpesvirus. All herpesviruses are potentially pathogenic and together they are responsible for a wide variety of diseases. One of the main characteristics of these viruses is that once infection has taken place, the viruses or their genome persist for life as latent infections at various locations in the body. The tissues in which latency is established, and where the virus is immunologically privileged, vary with the type of herpesviruses responsible for the infection (1). These latent infections are subject to reactivation at any time during life upon natural or iatrogenic provocation such as sexual intercourse, stress, fatigue and menstruation (2). The molecular events of reactivation are not well understood. Curiously, the clinical manifestations of reactivated infections may be quite different from the diseases caused by the primary infections. All herpesviruses resemble each other structurally and have biologic properties in common, particularly the hallmarks of latency and reactivation. They are among the largest of viruses, with a total diameter of approximately 150 to 200 nm. An inner core consisting of double stranded linear DNA intertwined with protein is surrounded by a icosahedral shaped nucleocapsid composed of 162 capsomeres. Around the nucleocapsid is a material of fibrous appearance called the tegument. A loose amorphous lipid envelope, derived from the host cell nuclear or plasma membrane and containing a number of viral glycoproteins, surrounds the tegument and the nucleocapsid (fig. 1). Based on common biological properties, the herpesvirus family is divided into three subfamilies: alphaherpesvirinae, betaherpesvirinae, and gammaherpesvirinae (3). The alphaherpesviruses [e.g. herpes simplexviruses types 1 and 2 (HSV-1 and HSV-2), pseudorabies virus (PrV), and varicella-zoster virus (VZV)] have a variable host range and short reproductive cycles. They spread rapidly in cultured cells derived from various tissues of a number of vertebrate species leading to  Linear DS DNA genome  Capsid  Tegument  Viral glycoproteins  Host-derived envelope  Figure 1. Diagrammatic representation of the herpes simplex virus virion structure.  3  destruction of the infected cells. Latent infections are established in neuronal ganglion cells. The betaherpesviruses [e.g. cytomegalovirus (CMV)] have a restricted host range and relatively long reproductive cycles. They spread slowly in cultured cells causing enlargement of the infected cells (cytomegalia). Latent infections are established in kidneys, secretory glands and other tissues. Gammaherpesviruses [e.g. Epstein-Barr virus (EBV)] have a very restricted host range infecting only lymphocytes in the family or order to which the natural host belongs. Most infections are arrested at a prelytic or lytic stage in that infectious particles are not produced. Latent infections are established in lymphoid tissues. Due to heterogeneity observed within the alphaherpesvirus subfamily, this group has been further divided into two genera: simplexvirus [HSV-1, HSV-2, bovine herpes virus type 1 (BHV-1), and PrV] and varicellavirus (VZV) (4). VZV has a more limited host range than members of the simplexvirus genera although not as restricted as the betaherpesviruses or gammaherpesviruses. Disease is caused in their natural host or only in closely related species and, unlike simplexviruses, standard types of cell cultures do not easily support productive infections even when derived from the natural host. Although PrV has been previously classified with the varicellaviruses (5), it has recently been suggested that this virus be considered a simplexvirus based on similarities observed in viral cell entry mechanisms (4). As for VZV, CMV, and EBV, humans are the sole known reservoir of infection by HSV. There are two serotypes of HSV, designated HSV-1 and HSV-2. Both are morphologically identical but differ in a number of biological, biochemical, clinical and genomic aspects. HSV-1 and HSV-2 have approximately 50% nucleic acid base-sequence homology (1). The most common clinical manifestations of HSV include keratitis and cutaneous lesions on the lips, oral cavity, the eyes, and the genitals (6). Rarely, the virus can induce more serious disease such as encephalitis and disseminated infections affecting several organ systems. HSV-1 is most commonly associated with facial lesions, keratitis and encephalitis in adults whereas HSV-2 is most commonly associated with genital lesions. However, both serotypes are not restricted to these specific locations. For example, approximately 60% to 90% of reported first episode genital herpes infections are caused by HSV-2, and 10% to 40% by HSV-1. The two serotypes cause clinically indistinguishable first episodes, however, patients with HSV-2 are much more likely to experience recurrences,  4  which will also be more frequent than if infected by HSV-1 (7). Infections with HSV-2 can also affect neonates who acquire the virus from the maternal canal at delivery (8). Disseminated infections are usually associated with immunosuppressed patients (e.g. AIDS), patients with skin disorders or severe infections, and neonates who do not have a fully functional immune system (1). A more complete understanding of the interactions between HSV and its host cell may yield critical information which could be used to elaborate new strategies for treating the various diseases caused by this medically important virus. B. HSV structure The inner core of HSV consists of a single molecule of double stranded linear DNA intertwined with protein (fig. 1). The DNA molecule is approximately 152Kbp in length and has a base composition of 68.3% G+C (9). Until recently, electron microscopy of embedded cells infected with HSV had shown that the viral DNA of mature virions was located within a short, compact, hollow cylinder or torus (10). However, it has been observed that variations in the embedding procedure sometimes resulted in the visualization of encapsidated DNA that appeared to consist of thin filaments forming a cage around an intricate elongated cylindrical core of thicker filaments with an intervening space between them (11). It was determined that the critical factor determining the configuration of encapsidated HSV-1 DNA molecules was the dehydrating agent used during the embedding procedure: ethanol or methanol produced the toroid shaped or the loose structures respectively. Although the logistics of this phenomenon are not clear, two hypothesis were suggested. The first relates to the presence of protein associated with the DNA. Alcohol treatment increases the stability of protein structure by strengthening the hydrogen bonds present in proteins. Prolonged dehydration of material in ethanol may result in the immobilization of protein and DNA within the viral nucleoid. The toroid structure may therefore represent an aggregation or collapse of the DNA induced by ethanol, whereas the inner cylindrical coil of large DNA fibers plus outer cage of thin DNA fibers may represent a less aggregated DNA, protected by the more polar methanol (11). The second hypothesis suggests that spermine, present in the DNA complex, may play a role in the condensation of HSV-1 DNA. Retained spermine, after ethanol treatment,  5  would enhance DNA condensation. Conversely, the greater solvent properties of methanol could partially release spermine thereby diminishing DNA condensation. Whichever of these two hypothesis is responsible for the observed differences of the DNA core structure, it is not known which type of structure more closely represents the actual in situ structure of the nucleoid. The 152Kbp DNA molecule consists of two covalently linked segments, designated long (L, 82% of the genome) and short (S, 18% of the genome). Each one of these segment consists of a unique sequence (UL and Us) flanked by a pair of distinct inverted repeats (RL and Rs). The RL and Rs regions each contain one well characterized gene encoding the transcriptional control proteins IE175 and IE110 respectively (9). Seventy two genes have now been recognized which encode 70 known distinct proteins. The protein shell, or capsid, surrounding the DNA genome is icosahedral in shape and consist of 162 capsomeres arranged in an orderly manner showing 5:3:2 axial symmetry. The capsomeres, appearing hexagonal when viewed in cross section, are hollow, elongated polygonal prisms with mean dimensions of 9.5 nm X 1.25 nm (12). The icosahedral structure has 20 triangular faces and 12 apices. Each of the 12 apexes is a single capsomere that has a fivefold rotation axis that passes through it and the center of the capsid. Each of the three axes with twofold symmetry making up one triangular face is composed of 3 capsomeres. Since each twofold symmetric axis is shared by 2 triangular faces, the total number of these axes is 30 (20 triangles X 3 twofold symmetric axes /2) for a total of 90 capsomeres. The threefold symmetric axes of each triangular face is made of 3 capsomeres. The HSV capsid is therefore made up of 12 capsomeres for the fivefold symmetric axes, 90 capsomeres for the twofold symmetric axes, and 60 capsomeres for the threefold symmetric axes of the triangular faces for a total of 162 capsomeres (12). The capsid is approximately 100 nm in diameter. Located between the capsid and the envelope is an electron-dense material of fibrous appearance when negatively stained called the tegument (13, 14) (fig. 1). The specific nature and function of the tegument is still unknown. However, it is known to harbor detectable amounts of a major viral transcriptional regulatory protein (14).  6  Surrounding the capsid and tegument is a phospholipid bilayer envelope (fig. 1). After capsid assembly in the nucleus of the infected cell, the HSV virion acquires its envelope from the inner nuclear membrane as it exits from the nucleus (fig. 2) (15). Because electron microscopic images of all stages of the HSV envelopment process cannot be obtained, envelopment is thought to occur very rapidly. The envelope may vary in size though most are about 180 nm in diameter (12), forming, on fully intact virions, a tight sheath around the capsid. It is often observed that a portion of the envelope appears to form a tail-like projection (16). Very occasionally, envelopes have been seen enclosing two or more capsids (12). The envelope consists of inner and outer layers, corresponding to the electron-translucent and -opaque layers respectively observed by electron microscopy (16). Of the total approximate number of 30 to 40 distinct structural proteins encoded by HSV, six make up the icosahedral nucleocapsid while the rest are constituents of the tegument and the envelope (4). To date, 11 HSV glycoproteins have been identified: gB, gC, gD, gE, gG, gH, gl, gj, gK, gL, and gM (4, 6, 16-19). Of these, 8 (all but gj, gK, and gM) have been shown to be constituents of the virion envelope. These membrane viral glycoproteins are important determinants of viral pathogenicity. They are involved in various activities and processes of the HSV infection process such as adsorption to the cell surface and penetration inside the cell; envelopment, egress, and release processes; direct cell-to-cell spread of the virus and cell-to-cell fusion (17). Early electron microscopic studies revealed that the envelope was frequently seen to possess periodic projections at its periphery (12). Use of monoclonal antibodies coupled to colloidal gold has since permitted the identification of some of these projections as viral glycoproteins. These were seen as spikes of different shapes, size and appearances (20). gB spikes are about 14 nm long and have a flattened T-shaped top when seen in side view. The gB spikes are clustered in groups varying from small bulbous distentions from the envelope surface to long tail-like projections. Conversely, the gC structures are randomly distributed and widely spaced 24 nm spikes that are often too thin to resolve. gD structures appear comparatively shorter (8 to 10 nm) and irregularly clustered in patterns distinct from those formed by the gB spikes (20). Although the structural conformations of gE, gl, gH, and gL have not yet been observed and identified by electron microscopy, it is known that gE-gl and gH-gL form two hetero-oligomers present at the envelope surface (18). The presence of  7  Figure 2. Diagrammatic representation of the herpes simplex virus virion lifecycle. Symbols: CG, cis Golgi; MG, medial Golgi; TG, trans Golgi; TGN, trans Golgi network; N, nucleus; Cy, cytoplasm; ER, endoplasmic reticulum; PM, plasma membrane; ?, pathway unknown; 1,2, and 3, potential egress pathways of the virus. See text for details.  8  gM in the viral envelope has not been confirmed, but it has recently been shown to be present in virions and plasma membranes of infected cells (19). The initial steps of the HSV infection process can occur through pHindependent direct fusion of the virion envelope with the plasma membrane. This leads to disassembly of the virion and initiation of gene expression (21). Although the virus has sometimes been found to enter the cell by endocytosis, it is thought that this route leads to unproductive infections (21). It seems likely that any of the eight known viral envelope glycoproteins could interact with the cell surface and/or with other virion constituents to influence the two-step entry process of binding and penetration. Binding and penetration of HSV to a cell may require sequential and/or simultaneous couplings of the multiple viral glycoproteins with several sites or receptors on the cell surface (4). Although the specific molecular events taking place during binding and penetration and the precise roles of the viral glycoproteins have not been defined, sufficient information is currently available to shed some light on these multiple interaction processes. It should be noted that although differences exists, the process appears to be remarkably similar for HSV-1, HSV-2, PrV, and BHV-1 (4). Initial attachment of HSV to cells involves binding of gB and gC to cell-surface heparan sulfate (HS) proteoglycans (PG) (22-25). gB, along with gD and the gH-gL hetero-oligomer have been shown to be essential for entry of HSV at a step subsequent to initial attachment (26-30). Specifically, some authors have shown (29) through the use of biochemical approaches combined with qualitative and quantitative electron microscopy that gD mediates a stable attachment step in entry through interactions to a yet unidentified cell surface component. In addition, gH and presumably its hetero-oligomer counterpart gL, would be involved in an event that allows fusion initiation but not entry of the nucleocapsid into the cell (29). Because the main focus of this thesis is the study of the various interactions between HSV-1 and cell surface PGs occurring during the initial binding stages of infection, the nature and functions of PGs will be reviewed here.  9 C. Proteoglycans C.l. General structure and nomenclature Proteoglycans (PGs), formally known as mucoproteins, are macromolecules containing 90% to 95% carbohydrate in weight in the form of glycosaminoglycan (GAG) chains covalently bound to a protein core. GAGs are long, unbranched oligosaccharides, usually without sialic acid, composed of repeating disaccharide units consisting of hexosamine [Dglucosamine (GlcN) or D-galactosamine (GalN)] and either hexuronic acid [D-glucuronic acid (GlcA) and/or L-iduronic acid (IdoA)] or galactose units (in keratan sulfate only) (31). In contrast, glycoproteins usually contain from 1% to 60% carbohydrate in weight in the form of numerous, relatively short (generally less than 15 residues), branched oligosaccharide chains of variable composition, which often terminate with sialic acid. PGs come in all shapes and sizes, are widely distributed in animal tissues, and appear to be synthesized by virtually all types of cells, especially adherent cells. They exist intracellularly in secretory granule, at the cell surface, or in the extracellular matrix (32). Seven groups of GAGs have been distinguished based on their sugar residues, the type of linkage between them, and the number and location of the sulfate groups (table 1). These are hyaluronic acid (HA; the only unsulfated GAG and the only one that does not occur as a PG, i.e. it is not covalently linked to a protein core), heparan sulfate (HS), heparin, keratan sulfate (KS; the only GAG containing D-galactose instead of an hexuronic acid), chondroitin sulfate A (CSA; C4-sulfated), chondroitin sulfate B (CSB; C4-sulfated; also known as dermatan sulfate), and chondroitin sulfate C (CSC; C6-sulfated). As seen in table 1, the seven groups of GAGs make up two larger groups based on the type of hexosamine present in the repeating disaccharide unit. The glucosaminoglycan group includes HA, HS, heparin, and KS. The galactosaminoglycan group includes the three types of CS: A, B, and C. CSB, HS, and H contain both GlcA and IdoA units as their hexuronic acid constituents, whereas CSA, CSC, and HA have only GlcA. Although this simplistic mode of classification suggests clear cut boundaries between GAGs, the reality is much more complex. All GAGs, particularly the GlcA/IdoA copolymeric species, display considerable sequence heterogeneity both within and between the different GAG chains found in a particular PG. This variability in structure is due to incomplete GAG  10 Table 1. Glycosaminoglycan groups. Adapted from (31).  Repeating disaccharide (A-B)n Glycosaminoglycan  HAi  Molecular Weight  0.4 to  8X10  6  Hexuronic acid(B)  N-acetyl-Dglucosamine  D-glucuronic acid  HS2  0.5 to 1.2 X 10 4  N-acetyl-Dglucosamine  D-glucuronic and L-iduronic acid 3  H4  0.6 to 2.5 X 10 4  N-acetyl-Dglucosamine  D-glucuronic and L-iduronic acid  KS5  0.4 to 1.9 X 10 4  N-acetyl-Dglucosamine  D-galactose  0.5 to 5X 10 4  N-acetyl-Dgalactosamine  D-glucuronic acid  1.5 to 4X 10 4  N-acetyl-Dgalactosamine  D-glucuronic and L-iduronic acid  0.5 to 5X 10 4  N-acetyl-Dgalactosamine  D-glucuronic acid  CSA6 (4-sulfated)  CSB7 (4-sulfated; dermatan sulfate)  CSC8 (6-sulfated)  1 HA, Hyaluronic Acid HS, Heparan Sulfate 3 L-iduronic acid is produced by the epimerization of D-glucuronic acid at the position where the carboxyl group is located. 2  Hexosamine (A)  4 5 6 7 8  H, Heparin KS, Keratan Sulfate CSA, Chondroitin Sulfate type A CSB, Chondroitin Sulfate type B CSC, Chondroitin Sulfate type C  11 biosynthesis mechanisms which yield polymer sequences at various stages of modification (32). The result are GAG chains which vary mainly in sulfate substitution (both in number of residues per disaccharide unit and in positioning) and in the extent of C5 epimerization of GlcA to IdoA units. The variability between PGs is even greater. For a given GAG substituent (for example HS), the number of GAG chains on a protein core can vary from 1 to 100; the size and structure of the protein core is itself highly variable; and some PGs are composed of different types of GAG chains such as syndecan-1 (and possibly syndecan-3) which is a transmembrane PG that bears both HS and CS chains (33, 34). GAGs, and more so the whole PGs, are therefore considered by some to be the most highly variable macromolecules known which leaves the debate of appropriate nomenclature open to all. C.2. Heparan sulfate and chondroitin sulfate glycosaminoglycans Heparan sulfate and chondroitin sulfate GAGs exist as PGs, covalentiy bound to a variety of core proteins. HS is the most ubiquitous cell surface GAG. As a result of its biosynthesis (see below), HS contains the greatest structural variability than any other GAGs and is the most negatively charged structure produced by vertebrate cells (34). The name "heparin" is usually reserved for the intracellular heparin PG synthesized by mast cells. All other related GAGs are referred to as "heparan sulfate". Along with CS, HS can be found in all the chordates, in the mollusks, annelids, arthropods, and in the ancient organisms coelenterates (35). Both GAGs are found extracellularly as non-membrane bound PGs comprising the pericellular matrix and basement membrane, as membraneintercalated PGs at the cell surface, and intracellularly within storage vesicles of various secretory cells and possibly in the nucleus. As mentioned earlier, HS is a glucosaminoglycan with repeating disaccharide units composed of the hexosamines GlcNAc and both hexuronic acids GlcA and IdoA. Initial biosynthesis in the Golgi produces the simple polysaccharide chain [GlcA—Pi,4—> GlcNAc —0:1,4—>]n (32). From then on, a variety of enzymatic modifications occur: N-deacetylation and Nsulfation at various position of the GlcNAc residues; C5 epimerization of GlcA to IdoA units; C2-0-sulfation of IdoA; C6-sulfation of GlcN and/or GlcNAc and other, rarer O-sulfation at other carbon positions. As none of these modifications occur on the total length of the GAG chain, but rather in a seemingly random manner, the final products are chains with  12 considerable sequence diversity, primarily the result of variable sulfation (fig. 3). This sequence heterogeneity, in which each chain is distinct, presumably arises because the product of each modification is the substrate for the next step and the reactions do not go to completion. The three types of CS GAGs (fig. 4) are generated with modifications similar to those occurring with HS biosynthesis from an initial polymer with the structure [GlcA — p 13—> GalNAc—0:1,4—>]n (32, 36). The main difference with the enzymatic processes acting on HS's initial polymer is that in CS, GalNAc is not recognized by N-deacetylase and therefore remains acetylated. Because deacetylation is a prerequisite to N-sulfation, and N-sulfate groups are required for substrate recognition by the Osulfotransferase, the structural diversity of CS is much less pronounced than for the HS/heparin GAGs. Although the overall negative charge of HS is only slightly superior to CS, there is a great difference in the distribution of these charges. All three CSs have their negative charges uniformly distributed throughout the chain (1 sulfate group per disaccharide unit; fig. 4), whereas HS has localized highly sulfated regions (up to 4 sulfate groups per disaccharide unit; fig. 3) interspersed with unsulfated regions. Contrasting with the 17 (possibly 18) different hexuronic acid —> GlcN sequences and the 10 (possibly 12) GlcN —> hexuronic acid sequences identified in HS chains, CS has only 9 hexuronic acid —> GalNAc disaccharide units identified (32). These CS configurations are mainly consequences of the presence, in CSB, of both GlcA and IdoA units, and variable locations of O-sulfate groups elsewhere than on the acetylated carbon of GalNAc. All GAG chains that exist as PGs (excluding HA which exists as a free polysaccharide chain) are covalently linked to a core protein through a trisaccharide linkage region bound to a serine residue (fig. 5). Whether for CS or HS/heparin chains, the link region consists of a —P13—> Gal —P13—> Gal —Pi,4—> Xyl —p—> O—serine bridge (32). In both CS and HS GAGs, the xylose residue is phosphorylated at C2, whereas the second galactose from the protein core is O-sulfated only in CS chains. Chain biosynthesis is therefore initiated by xylosylation of serine residues by xylosyltransferase. Significant effort have gone into identifying a recognition sequence of amino acids around the target serine units that would indicate if a given serine will be used to anchor either CS- or HS-like GAG chains (33,37). Although present findings do not support the idea of a universal consensus sequence for GAG attachment two general sequences  13  CH 2 OH  CH 2 OH — O  G>cA  fdcA  OH  acA GIcA (2-OSOj J  -O °v  )  OH  kfoA  WoA  KM Ac  WoA(2-OS03)  IA^L W o A (/oj-vcn 2 - O S 0 3 )\  GlcNS0 3 CHjOSO^  CH2OS03 fGlcA OH  GlcA GIcA(2-OSOj) WoA  WoA HNAc  «doA(2-OS0 3 )  X ^ J [GlcA (2 OSO3)] { _ WoA HNSO,  WoA(2-OS03)  GlcNAc  acA  TGIcA  WoA(2-OS03) )  — O  r  ,,  OH  acA [GlcA(20SO-,)] WoA  HNSClj  _ WoA(2-OS0 3 )  GicNS0 3 (6-OS0 3 )  GlcNAc(6-OS03)  CH OH COO' •o  O  OH  HNSOj"  JdoA(2-OS0 3 )  GlcNS0 3 (3-OS0 3 )  OR  GlcA Structure of hexuronlc acid residues GlcA  WoA  ^IdoA^-OSC^}  GlcNS0 3 (3,6-dJ-OS0 3 )  Figure 3. Scheme of disaccharide sequences identified in heparin and heparan sulfate. The six variously substituted internal GlcN residues are combined with the HexA units indicated at CI and C4. Adapted from (32).  14  CHONDROITIN SULFATE A Alternating copoly</3-gluciironic acid-{l—3^ N-acetyl-/3-ga!actosamine-4-suIfat<Hl—4 J) O ii  CHONDROITIN SULFATE B (Dermatan sulfate) Alternating copoly(/3-iduronic acid-{l—3}N-acetyl-/3-galactosaniine-4-sulfate-[l—4])  (xNa+)  nHO-S-O  9"  C-OH  HO-  CH OH o« I 2  jfc?  '  R  O II  -H  (xNa+)  HO-S-O II I CH 2 OH O  NH I C - C H ,3 II O  O O: R  \OH HO-  o II  OH  C-OH  k°  -H  NH I C - C H 3, II O  CHONDROITIN SULFATE C Alternating copoly(/3-g1ucuronic acid-{l—3J-N-acetyl-j3-galactosamine-6-sulfate-{l—'4D  (xNa+)  O II CH?0-S-OH 2 II  HO-  Figure 4. Repeating disaccharide structures of chondroitin sulfate types A (4-sulfated), B (4-sulfated; also known as dermatan sulfate), and C (6sulfated). Adapted from (77).  15 Serine  Core protein  Link trisaccharide  Pl,3  —'  Glycosaminoglycan  Figure 5. Schematic drawing of the linkage between a glycosaminoglycan chain and a serine residue of a core protein in a proteoglycan molecule. The end of the glycosaminoglycan chain is covalently bonded to the serine through a "link trisaccharide" common to all proteoglycans. The glycosaminoglycan chain itself consists mainly of repeating disaccharide unit. The various potential monosaccharides A and B are described in Table 1. Adapted from (31).  16 are commonly recognized both involving serine-glycine residues. The first is a serine-glycine repeat followed, within 5 amino acids, by acidic amino acids. It was initially found in the CSPG serglycine (37). The second, found initially in the mostly HS PG syndecan, is a single serine-glycine that is preceded and often flanked by acidic residues (33). Authors of both studies point-out that variations in these observed sequences are common so that the precise structure required for initiation of chain elongation and polymer formation for HS or CS are not known. Pertinent questions regarding the regulation of GAG biosynthesis, remain unanswered. For instance, since all GAGs share the same trisaccharide link region to the protein core, what mechanism(s) determine(s) whether an initiated GAG chain will become a glucosaminoglycan (HS) or a galactosaminoglycan (CS)? As both CS and HS GAGs are known to share the same xylosyltransferase and galactosyl transferase necessary for the completion of the link region (32), it seems that the committing step would be the incorporation of the first hexosamine: GlcNAc or GalNAc. In CS, the GalNAc transferase involved in adding the first GalNAc to the link region appears to be different than the one involved in GalNAc additions within the rest of the GAG chain (32). If a unique GlcNAc transferase also exist for the incorporation of the first GlcNAc in HS chains, it would be tempting to speculate that control over the choice of the type of GAG chain synthesized could involve differential recognition, by these enzymes, of the link region. If this if the case, would the sulfated second galactose residue of the CS link region be involved since this is a structural difference with the HS link region? If no distinct recognition signals for the initiation of glucosaminoglycan versus galactosaminoglycan chain elongation are found within the link region or close to the target serine site, the selection may be under the control of more remote regions of the core protein. At this point in time, none of these questions can be answered with certainty. C.3. Core proteins Although many core protein structure have been identified, and preliminary classification into "families" have been attempted based on sequence information, none show obvious distinctive features. It is not possible at present, solely on the basis of sequence data, to predict if a protein will carry GAG substituents or not (32). The mechanisms that determine whether a protein will be converted into a PG are not known.  17 Even the presence of the previously described putative GAG recognition sequences does not ensure their use. Core proteins may possess other functional domains such as signal sequences designed for anchoring the PG to the cell surface (see below) or for interactions with other molecules. Domains for the latter include binding elements that may be involved in interactions among extracellular matrix molecules and between cells and extracellular matrices. Some of these domains include fibronectin and collagen binding sites (32), lectin related sequences at the C-terminal that bind various sugars (galactose, fucose, GINAc) (38, 39, 40), epidermal growth factor (EGF)-like repeats (32, 39), and HA binding sites (41). There are a number of different ways in which PGs associated with the cell surface may be attached. One way, represented by the PG syndecan, involves anchoring to the plasma membrane through a hydrophobic core protein stretch that is intercalated in the lipid bilayer (33, 34). Other core proteins are bound to the cell surface through a phosphatidyl-inositol linkage while a third type of anchorage is mediated by cell surface receptors that specifically recognize certain core proteins (32). Finally PGs may be retained at the cell surface through interactions involving the GAG chains instead of the core proteins. The size of the core proteins vary enormously from 14 to over 600 kDa (32). Based on incomplete information which include size, location with respect to the plasma membrane, and types of GAG chains, five families can be distinguished. Note that numerous PG species fall outside this preliminary scheme since their core protein may bear no resemblance to those within this nomenclature pattern. The families are: (I) Large extracellular CSPGs that can interact with HA which include the cartilage PG aggrecan, the fibroblast PG versican (39, 41), and a 4-sulfated CSPG released from platelets (42). (II) Small extracellular PGs substituted with only one or two GAG chains (CS or KS). This family includes decorin (also known as PGII and PG40) (41, 43, 44) and biglycan (also known as PGI) (41, 44). Decorin contains one CS chain attached close to its N-terminus while biglycan contains two (41, 44). Both are found in many connective tissues and bone. (Ill) HSPGs of the extracellular matrix and/or the basement membrane. (IV) Membrane-intercalated cell surface PGs composed of both CS and HS GAG chains. Syndecan is the main representative of this family and is itself a group of four closely related PGs (32, 33, 34). (V) Intracellular PGs, such as serglycin (41), that are heavily substituted with CS and/or heparin GAG chains. As with GAGs, the  18 proper nomenclature of PGs as yet to be developed and will likely arise when, or if, analysis of core protein structures reveal additional similarities. C.4. Roles and interactions The roles of PGs are highly diverse ranging from mechanical functions for maintaining tissue structure to more dynamic roles in cell adhesion and motility, to very complex and poorly understood influences on cell differentiation and morphogenesis (32). The mechanical functions are best illustrated by the highly sulfated major rat cartilage CSPG that composes, along with collagen, the cartilage matrix. Its high sulfate content imparts on it a high charge density which strongly competes with solvent thereby functioning to resist compression in the joints (38, 40). This PG versatility results from their potential for multiple simultaneous interactions with other molecules and gives them the ability to function as a multi purpose "glue" in cellular interactions. In order to fully exert their actions, PGs generally depend on the concerted and cooperative contributions of both the GAG chains and the core protein. However, there are instances where the biological effects are exerted solely by the GAG component or, conversely, by the core protein. The simplest form of cooperation between the protein core and the GAG chains is the scaffold property of the core protein for appropriate immobilization and spacing of the GAG chains. When attached to the cell surface, the protein core's anchoring function may be essential for the appropriate positioning of a GAG-bound ligand. Therefore, although most of the biological functions of PGs may be specifically attributed to either the protein core or the GAG, proper three dimensional positioning, dependent on the presence of all PG components, may be essential for efficient activity. Most of the effects on cell processes by GAG chains depend on binding proteins. For example, HS's binding repertoire comprises diverse proteins found in the cellular microenvironment, including extracellular matrix components, peptide growth factors, cell adhesion molecules, lipolytic enzymes, protease inhibitors, circulating lipoproteins, viral coat proteins (such as gC and gB of HSV; see below), nucleases, DNA and RNA polymerases, and transcription factors. The binding of GAG chains to protein is predominantly via electrostatic interactions between the highly anionic sulfate groups and clusters of basic amino acids arranged in a  19 three-dimensional array on the protein. The sequences BBXB and BBBXXB, where B is a basic amino acid and X any other amino acid, have been proposed as consensus sequences for such proteic sites (41). These type of non-covalent interactions vary from relatively low affinity and specificity to highly specific interactions depending on the nature of the protein and GAG sequences involved. The degree of sulfation, the size and/or the number of GAG chains attached to a single core protein will influence the intensity of the binding interactions (41). A high degree of sulfation and a high number of large GAG chains in heparin binds the strongest to fibronectin (32). It seems to be that the large molecules are capable of entering into simultaneous multiple interactions. Similarly, many CS chains on a PG molecule provides a greater valency and strengthens otherwise weak interactions of this GAG with fibronectin and collagen (41). The affinity for protein and the superior binding capacity of HS compared to CS has been ascribed, in addition to the degree of sulfation, to the presence of IdoA units which are believed to impart conformational flexibility to the GAG chain (32, 34). These groups would allow the chain to alter its shape and the spatial orientation of the highly sulfated localized regions of HS ensuring efficient electrostatic alignment to occur. The HS GAG chains of the transmembrane PG syndecan are involved, through protein interactions, in binding multiple components of the cellular microenvironment that themselves interact with a cell surface receptor such that they can be considered as co-receptor, integral membrane components that are structurally distinct from high affinity receptors. HSPGs can therefore bind ligands and participate in the formation of a receptor complex that is required for the ligand's physiological action. For instance, fibroblast growth factor (FGF) binds the HS component of syndecan while also associating with its high affinity protein receptor. In a similar manner, it is now believed that HSV initially interacts with the HS GAG of a yet unidentified PG through gC and gB so as to permit secondary interactions to take place between other viral glycoproteins, such as gD and gH, and cell surface protein receptors. GAG chains are involved in cell adhesion through the previously mentioned interactions of HS with fibronectin and collagen (33, 41). This auxiliary mechanism to the more specific integrin-mediated adhesion also involves the core protein (32, 39, 40 ,41, 43). The core protein of the large rat cartilage CSPG also has a HA binding domain that interacts with link protein, a HA binding protein that strengthens the PG-HA interactions and  20  results in the formation of stable aggregates promoting efficient adhesion (40, 41). Other PGs such as the cartilage CSPGs, aggrecan (32) and the fibroblast PG versican (39, 41) possess HA binding sites within their core proteins. Intracellular heparin/HS PGs have been shown to be involved in the regulation of cell proliferation (32,41) possibly by regulating transcription factor activity and gene expression. Some of the growth effects exerted by PGs are likely to be mediated through interactions between GAGs and FGF. As mentioned earlier, the HS of syndecans binds FGF. The HS-FGF complex enhances FGF's activity by protecting it from degradation and by creating a reservoir for cells to use (41). The PG-mediated insolubilization of this growth factor permits the concentration of the growth factor's activity and directs it into a geometry appropriate for the architecture of the tissue. This action may be of general significance for the observed growth and differentiation effects of PGs as well as the initial attachment mechanisms of HSV to the cell surface. D. HSV lifecycle Entry of HSV is generally divided into two sequential events: binding and penetration. Both of these processes require sequential and/or simultaneous coupling of many or all of the 8 known viral envelope glycoproteins [gB, gC, gD, gE, gG, gH, gl, and gL (4)] with several sites or receptors on the cell surface. Since the actual number of viral envelope glycoproteins is not yet known and the precise roles of the known ones is still unclear, it is likely that their functions may also involve other important processes such as egress of the virus from the infected cell. To date, the viral glycoproteins known to participate in the entry of HSV-1, PrV, and BHV-1 can be grouped into 5 families of proteins which appear to be conserved among all the simplexviruses: gB, gC, gD, gH, and gL. D.l. Binding of HSV to cells The binding of HSV to cells appears to involve multiple mechanisms of differing efficiencies involving several viral glycoproteins and cellular components. Based on information indicating that binding itself involves HS-dependent and -independent steps, the process can be subdivided into initial and stable attachment (fig. 6).  21  Figure 6. Model of entry of HVS-1. (a) Initial attachment; (b) stable attachment; (c) fusion initiation; (d) fusion bridge expansion; (e) nucleocapsid release. The proposed rearrangements of virion envelope, tegument, and nucleocapsid in relationship to the cell plasma membrane are diagrammed. Adapted from (29).  22  Initial attachment of HSV to cells involves the interaction of gC and gB on the virus with cell surface HS (fig. 6a) (22-25, 45-48). The evidence for the involvement of gC include: (I) HSV deletion mutants devoid of gC (gC") that are significantly impaired in binding to cells (24, 46-48); (II) monoclonal antibodies (MAb) specific for HSV-1 gC can block binding (4648); (III) HSV-1 gC can bind to heparin-affinity columns in physiological saline (24); and (IV) neomycin, an aminoglycoside that can inhibit HSV infectivity in part by inhibiting binding, has differential effects on HSV-1 and HSV-2 and the gene responsible for this difference maps to the coding region of gC (4, 45, 46) which is known to differ in sequence between the two serotypes (6; see below). HSV mutants gC" can still bind to cells, albeit inefficiently: approximately 10% of wild type HSV (24). It is though that gB can also mediate the binding of HSV-1 to HS, and therefore complement gC, as gB has heparin-binding activity independent of gC (4). Mutants devoid of both gC and gB can practically not bind at all to HEp-2 cells (4) and the interaction of gB with HS during initial attachment is not required when gC is present (gB" viruses) (24) suggesting that gB's role in this step is important only in the absence gC which has higher HS affinity. In addition to a significant decrease in the number of gC" viruses bound to cells, gC~ particles also showed reduced rates of adsorption on BHK (46) and green monkey kidney cells (47) and a reduced rate of penetration on HEp-2 cells (24). It is important to note that the use of gC" HSV-1 with different cell lines yields different results. The gC" HSV-1 used for the work presented in this thesis revealed no significant difference in binding, relative to wild type HSV-1, when using either Ltk" murine fibroblast or HEp-2 cells. It is not clear whether the use of deletion HSV particles to study viral binding reveals information about the normal steps of entry or about alternative mode of binding used only in the absence of the deleted gene and/or for specific cell types but not others. GC and gB form the most prominent envelope spikes on the surface of HSV (20, 29). This enables them to be the first viral components to come into contact with the ubiquitous cell surface HS. As mentioned earlier, functional interactions between proteins and GAGs are predominantly, but not exclusively, electrostatic in nature and involve relatively high local concentrations of basic amino acids in proteins (41, 6). A feature common to both serotypes of gC and gB (i.e. from HSV-1 and -2) is a cluster of basic amino acids in the vicinity of a very hydrophilic region near the N-  23  terminus of each protein (6). The basic amino acid stretches in gC and gB are highly variable. The variation lies between gC and gB but also between the different forms expressed by HSV-1 and HSV-2. These amino acid differences may reflect differences in HS affinity between gB and gC and may also account for the apparent non-identity of receptors for HSV-1 and HSV-2 in that both gCs may recognize different structural features of HS. If this proves to be the case, sequence recognition of specific sections of HS may, in addition to electrostatic interactions, plays a role in initial binding of HSV to the cell surface. Additional evidence for the requirement of HS-like GAGs for initial attachment of HSV to cells include: (I) preincubation of HSV with soluble forms of heparin or HS inhibits binding proportionally to the amount of GAG used (6, 23-25, 47, 48) and (II) the loss or alteration of cell surface HS reduces binding (23, 25). The intensity of binding inhibition by soluble heparin or HS depends on a number of variables: the cell line used in the study, the presence of gC in the virion, and the concentration of heparin or HS used. At least three different approaches have been used to alter the amount of cell surface GAGs in studies of HSV binding: (I) enzymatic treatment of cell with heparitinase, heparinase or chondroitin lyase (23); (II) growth of cells in sodium chlorate to inhibit sulfation of all GAGs (4); and (III) use of mutant cells defective in the synthesis and/or transport to the cell surface of HS only or other GAGs or defective in sulfation of HS (25, 49). The results obtained in all three approaches can be summarized by stating that HSV's binding was consistently reduced on cells lacking HS whether this was achieved by enzymatic treatment or by the use of cells mutants deficient in HS production. These cells were consequently more resistant to HSV infection following treatment. The resistant phenotypes of cells reduced in HS sulfation as a consequence of sodium chlorate treatment further indicates that the binding of HSV to cell surface GAGs mainly involves electrostatic interactions. I will further discuss the use of PG cell mutants in a latter section. Although it can be concluded that HS is important in the initial attachment of HSV to the cell, it remains to be determined whether any HSPGs can serve as receptor or whether a particular variety of HSPG is mainly required in the interactions. Partial reduction in HSV-1 binding to baby hamster kidney (BHK) cells after treatment with phosphatidylinositol (PI) specific phospholipase C suggest that at least Pl-linked PGs may be included among the PGs used by the virus (50).  24  The next step, stable attachment, allows close association of virus with the cell in a manner that is more resistant to removal by heparin or high-ionic-strength washes, hence its reference as HS-independent (fig. 6b). This interaction involves gD with a yet unidentified cell surface receptor, and has been shown, in PrV, to be resistant to the addition of exogenous heparin (51). Viruses lacking gD or inactivated by anti-gD neutralizing Abs were less stably bound to cells (possibly by initial gB and/or gC binding to HS) than viruses containing gD, as judged by increasing sensitivity to elution with high-ionic-strength washes (29). Soluble gD, bound to cells, significantly reduced viral attachment when added to cells prior to or with the addition of virus (29, 52). Evidence supporting that gD interacts with a receptor other than HS comes from observations that (I) binding to cells of inactivated virions containing gD, but not those devoid of gD, can inhibit penetration but not binding of a homologous challenge virus (53); (II) a truncated form of soluble gD binds to cells and inhibits HSV entry but not binding (52); and (III) binding to cells of soluble gD is markedly reduced by treating cells with proteases but is not affected when cell surface HS were enzymatically removed (52). Electron microscopy studies confirmed that viruses inactivated by anti-gD neutralizing Abs attached to cells but were arrested at a step prior to initiation of a visible fusion bridge between the virus and the cell (29). D.2. Penetration of HSV into cells Penetration of HSV into cells, as defined as a step subsequent to initial binding to the cell surface which triggers fusion of the virion envelope with the plasma membrane (fig. 6c-e), requires multiple interactions, in a cascade like manner, involving various glycoproteins and cell surface components. These cell surface components, including the putative protein(s) interacting with gD, remain to be identified. Evidences implicating at least gB, gD, and gH in the penetration process were obtained mainly through studies using either blockage of specific viral glycoproteins by MAbs or viral mutants devoid of the relevant gene. These studies revealed that gB, gD, and gH are each required for viral penetration but not for initial binding of the virus to the cell (18, 22, 26, 27, 29, 30, 51, 52, 54, 55). After stable attachment is induced, an initial fusion bridge forms (fig. 6c) followed by a series of events that would result in lipid rearrangement, fusion bridge formation and fusion of the virion envelope with the plasma membrane (fig. 6c-e). The  25  mechanisms involved may have similarities with those required for virusinduced cell fusion as gB, gD, and gH are also known to be required for this event (4, 22, 55). Additional information linking gD with a step subsequent to binding of the virus include what is known as gD interference: cells transformed to express gD can be resistant to infection by the homologous virus used (56). Although the precise mechanisms regulating gD-mediating interference are not known at present, it is known that the resistance to infection is due to an inability by the virus to penetrate into gD-expressing cells and not to a failure to bind. Recent explanations for this phenomenon include interactions of cell-associated gD with viral gD and/or other envelope component(s) of the superinfecting virus that would prevent penetration. The involvement of gD in both stable attachment and initiation of penetration (fig. 6b, c) seems to imply two distinct functions for this glycoprotein (29, 51). The dual function of gD is supported by the observation that Abs to gD that block attachment of virus do not bind to the same sites as do Abs that neutralize penetration (57). This finding implies two potential functional domains in agreement with the two proposed functions. Although the precise role of gH (and also gB and gD) in penetration is not clear, this glycoprotein is essential for HSV infectivity (54), is involved in virus-induced cell fusion (18) and seems to be involved in fusion initiation or expansion (fig. 6c, d) (29). Electron microscopic observations revealed that virus treated with anti-gH neutralizing Abs attached to cells (27, 29) and could form a fusion bridge (in contrast to gD-blocked virions) but did not show the expansion of the fusion bridge or extensive rearrangement of the envelope and tegument observed with non-treated virions (29). These are only preliminary results and remain to be confirmed with the use, for instance, of virions devoid of gH. Although present in the virion envelope since anti-gH Abs neutralize virus (27, 29), gH has minimal exposure since colloidal gold immunolabelling while easily detected spikes containing gB, gC and gD, failed to detect gH in the virion envelope (20). This sequestered arrangement of gH is characteristic of a protein involved in lipid destabilization that would require triggering (by gD?) to interact with the cellular lipid bilayer (29). A study with virions devoid of gH and with a temperature-sensitive mutant (54) confirmed that gH is required for membrane fusion but not viral attachment. The exact functions of both gD and gH remain to be determined. At present, the  26  involvement of gD in stable attachment seems solidly established. Conversely, the penetration process is only beginning to be understood and the proposed functions of gD and gH in the process remain to be confirmed. Other HSV glycoproteins required for viral penetration could involve gL and gK. gL forms a hetero-oligomer with gH inside the infected cell and has been shown to be present in plasma membranes of infected cells, although it has not yet been shown to be present in the HSV envelope (18). The coexpression of gL and gH is required for proper post-translational processing of both glycoproteins and cell surface expression of gH (18). Since gL is required for virus replication, cell lines expressing gL were constructed to complement infection by mutant viruses devoid of gL for their production (30). GL-negative particles, purified from infected cells, were shown to be deficient in gH and were able to adsorb onto cells but not to penetrate and initiate infection (30). It has not yet been determined whether gL plays a direct role in HSV penetration or if it acts only indirectly as a requirement for proper gH expression. Because gK plays some role in controlling virus-induced cell fusion (4), it was suggested that it may also play a role in penetration. These and yet to be identified viral components could be required for bridge expansion and nucleocapsid release into the cytoplasm of the cell (fig. 6d and e respectively). The two step entry process of attachment and penetration is consistent with cell surface molecules as determinants of virus tropism. It provides advantages to virus survival by allowing use of different cell receptors that can vary in presence or quantity. However, it is obvious from this brief overview that to divide entry into two defined steps (or five as in fig. 6) is an oversimplification. Entry, from HS-dependent binding (initial attachment) to penetration and release of the nucleocapsid inside the cell's cytoplasm, is a process that involves multiple viral and cellular components interacting sequentially and/or simultaneously in a complex and, at present, unclear manner. D.3. Gene expression and egress of HSV from infected cells Once the nucleocapsid enters in the cytoplasm of the cell, it travels to the nucleus where, through a nuclear pore, the genome is released inside the nucleus (fig. 2). It is unclear what mechanism(s) is used by the virus to reach the nucleus but the use of cytoskeletal elements have been suggested (3).  27  Once the genome is in the nucleus, replication and a cascade of gene expression begins (3). At least five classes of HSV-1 genes are known and sequentially expressed by virally encoded transcription factors and host cell RNA polymerase II: a, Pi, P2, Yi, and 72- The immediate early genes of the cc class are first transcribed. After translation in the cytoplasm, a gene products reenter the nucleus as they are required for the expression of the p gene class. Peak levels of expression of the a genes are observed from 2 to 4 hours post infection (HPI) whereas the expression of the pi and p2 genes reaches their peak from 5 to 7 HPI. The gene products of both the pi and P2 genes are involved in viral genome replication and the only aspect distinguishing them is the requirement, by the Pi genes, of the 0C4 gene product. The y genes are the last group of genes to be transcribed and encode predominantly structural proteins. In contrast to the p genes which are involved in viral DNA replication but do not require it for their own expression, the y genes are dependent on concurrent viral DNA replication for their expression, the yj genes being more sensitive to this phenomenon than the yi genes. Before initiation of viral DNA replication, the linear genome is thought to circularize since it is by a rolling circle mechanism that replication proceeds. HSV genome circularization has been demonstrated in vitro (58). The product of replication is concatomeric DNA which requires cutting into full genome length before packaging in empty nucleocapsids can take place. All the above described events, apart from translation, take place within the nucleus of the infected cell. Once nucleocapsids have been filled with DNA genomes, they associate with a modified region of the inner nuclear membrane that presumably consists of viral tegument proteins (fig. 2). Budding through the inner nuclear membrane results in the acquisition of a tegument and an envelope. From this point on, two potential egress pathways have been suggested as outlined in fig. 2. In pathway 1, the virion keeps its envelope as it is transported through the Golgi complex and out of the cell. Immature envelope glycoproteins within the newly acquired envelope (59) are sequentially processed to their mature form as the whole enveloped virion is transported through the three distinguishable cisternae of the Golgi complex, and through what is thought to be the trans Golgi network (TGN). In a mechanism that may be similar for secretory proteins (60), the fully infectious virion is secreted outside the cell.  28  In the second pathway, the newly enveloped nucleocapsids deenvelope from the perinuclear space or the endoplasmic reticulum and are released "naked" into the cytoplasm. Viral glycoproteins are processed through the Golgi to their mature forms independently of the nucleocapsids which travels through the cytoplasm and rebuds into cytoplasmic membranes derived from the Golgi (TGN) (61). At this point, the nucleocapsids reacquire an envelope containing functional viral glycoproteins. The virus then buds out of the cell. These two pathway models arose from multiple studies involving several different alphaherpesviruses (HSV, PrV, and VZV). Studies investigating the effect of the drug monensin on HSV-1 infected cells suggested that HSV-1 required interactions with "early" (cis) Golgi components (60) indicating the use of pathway 1, whereas studies on VZV and PrV suggested that these viruses interact with "late" (trans) Golgi components (62, 63) indicating the use of pathway 2. Therefore, while it seems that different alphaherpesviruses may favor the use of one pathway over the other, further studies will be required to elucidate the mechanism(s) involved in egress. E. Mutant cell lines used to study HSV-1 Along with the use of viral deletion mutants and drugs, somatic cell mutants defective in known enzymatic functions have also been useful to investigate the cellular and viral requirements necessary for HSV infectivity. Esko et al (64) isolated Chinese hamster ovary (CHO) cell mutants defective in the synthesis of various GAGs by screening for clones that failed to incorporate radioactive inorganic sulfate into macromolecules (65, 66). Radiolabeled HSV was found to bind efficiently to wild-type CHO cells but not to any of three mutants defective in HS synthesis, despite the fact that one of these mutants produced CS, implying that this GAG, sulfated to a lesser extent than HS, does not play a role in the initial attachment interactions (25). I will later elaborate on the theory that CS may play a secondary role in attachment. CHO mutants have been highly useful in confirming the role of HS as a primary HSV attachment receptor. However, partly in part because CHO cells are one of the few cell types non-permissive for HSV replication, it has been difficult to further resolve the precise mechanism by which PGs play a role in viral infection or in other events of the viral life cycle.  29 An alternate approach to yield cell mutants resistant to HSV infection was developed by Tufaro et al (66). This approach used HSV as a selective agent and has yielded several HSV resistant mutant cell lines, two of which, gro2C and sog9, were used in this work. Because this technique as produced mutant cell lines defective in cellular functions essential for the virus' lifecycle but non-essential for the cell's survival (49, 67), it is thought that it will ultimately be useful for identifying targets for antiviral drugs which could block viral infection without affecting cell viability. Briefly the procedure involved mutagenesis of murine fibroblast Ltk" cell monolayers with ethyl methanesulfonate (EMS), followed by HSV-1 infection. Colonies of cells surviving exposure to HSV-1 were cloned and further analyzed for their ability to propagate HSV-1. These mutant cells were termed gro mutants (66). The HSV-1 resistant phenotype of one of these mutants, gro2C, was found to be directly related to its incapacity to bind the virus due to a yet unidentified disfunction in cell-surface HS synthesis and/or transport to the cell surface. Although HSV-1 plaquing on gro2C cells was reduced by approximately 85%, the plaque morphology was normal, and the virus released was infectious indicating that for fibroblast murine cells, HS enhances HSV attachment but is otherwise not essential at any stage of the lytic cycle in culture. Moreover, from the observation that gro2C cells are infectable, albeit inefficiently (15% of parental L cells) while HS is absent from their cell surface, it can be concluded that other cellular molecules appear to confer susceptibility to HSV. This is in contrast to CHO mutant cells defective in HS synthesis which did not show detectable residual binding (25). The difference in phenotype of the L cell and CHO mutants remain to be explained. Due to the nature of the selection protocol, the arising of a mutant like gro2C is, in itself, further proof for the role that cell-surface HS plays the HSV attachment. In contrast to the CHO mutants, this protocol specifically selected, from an HSV-1 permissive cell line, HSV1 resistant mutant cells that are deficient in synthesis and/or transport of a GAG directly related to the HSV infection process. In the first section of this work, I used gro2C cells to further our understanding of the relationship between cell surface HS and HSV-1. The HSV-1 permissivity of these cells allowed the use of plaque assays to study this relationship. Plaque assays are much more sensitive than binding assays using radiolabeled virions and may thus resolve levels of interactions not detectable with non-permissive CHO cells.  30  In preliminary experiments I demonstrated and confirmed that radiolabeled HSV-1 bound significantly less on gro2C cells than on parental L cells implying the importance of cell surface HS in binding. However, residual binding on gro2C cells again pointed-out that other cell surface components were implicated in HSV-1 binding besides HS. Interestingly, radiolabeled gC" HSV-1 particles bound with similar affinity as wild type virions to L and gro2C cells. In addition, both strains of virions were similarly inhibited in their binding to L and gro2C cells by increasing concentration of soluble HS. These results suggest that, for these cell lines at least, other viral components besides gC (gB?) can interact with cell surface and soluble HS and with yet unidentified cell surface components of gro2C cells. I assessed the penetration rate of bound HSV-1 to L and gro2C cells in order to establish the role of HS in this process. No detectable difference was observed implying that the decreased HSV-1 plaquing on gro2C cells is the result of deficient binding and not deficient penetration. This permitted me to use plaquing assays as measurement of binding efficiency. Soluble HS was significantly more efficient in hindering HSV-1 plaque formation with gro2C cells than with L cells. This indicated that the residual HVS-1 binding on gro2C cells is "weaker" in that much less soluble HS was required to inhibit plaque formation. Because cell surface CS is the only remaining cell surface GAG on gro2C cells, it was of interest, in the second section of this work, to investigate the possibility that CS could act as main HSV-1 binding receptor on gro2C cells and, maybe, as an alternative binding receptor on L cells. Previous studies dismissed the involvement of CS as an HSV-1 binding receptor. As mentioned, CHO mutants expressing only cell surface CS did not bind more radiolabeled HSV-1 particles than cell mutants devoid of both HS and CS (25); soluble CS could not inhibit HSV-1 binding on green monkey kidney (GMK) cells (68); and enzymatic digestion of cell surface CS on HEp-2 cells had no effect on HSV-1 plaquing (23). There are underlying problems with each of these experiments in their ability to detect the effect of CS on HSV-1 binding. First, CHO cells are nonpermissive to HSV-1 and, consequently, one must rely on low sensitivity binding assays using radiolabeled virus. Second, soluble CS interference with HSV-1 binding on normal GMK cells and HSV-1 binding on enzymatically digested CS on HEp-2 cells both have a similar bias: the presence of highly charged cell surface HS that could mask any detectable  31 interactions between HSV-1 and cell surface CS. The use, in this work, of highly sensitive plaque assays using the HSV-1 permissive, HS-deficient gro2C cell line permitted observation of HSV-l-CS interactions. Soluble CS types A, B, and C each had variable but detectable inhibitory effects on HSV-1 plaquing with gro2C cells while not affecting significantiy plaquing on L cells. These results indicate that HSV-1 can interact with soluble CS. However, this is not direct evidence that the residual HSV-1 infectivity with gro2C cells is a result of interactions with gro2C's cell surface CS. The role of cell surface CS in HSV-1 binding was investigated further by the use of sog9 cells. These cells are derived from gro2C cells that were subjected to a second round of HSV-1-resistant selection. Sog9 cells are highly resistant to HSV-1 infection, plaquing only 1% relative to L cells. This significant plaquing reduction is associated with the complete absence of the cell surface GAGs HS and CS, and was found to be resistant to the addition of high concentration of soluble HS. This suggested that the 1% residual HSV-1 binding on sog9 cells occurred independently of interactions between the HSV-1 envelope and cell surface GAGs, and that cell surface CS can facilitate HSV-1 infection, albeit with a lower efficiency thanHS. Furthermore, the effect of the synthetic sugar polymers dextran, dextran sulfate, and DEAE-dextran on HSV-1 plaquing with GAG deficient sog9 cells is discussed in an attempt to establish if the HSV-1 binding interactions are mainly electrostatic in nature and/or also require the recognition of structural features.  32  6. MATERIALS AND METHODS  A. Abbreviations used. BSA, bovine serum albumin; CPM, counts per minute; CS, chondroitin sulfate; CSA, chondroitin sulfate type A; CSB, chondroitin sulfate type B (dermatan sulfate); CSC, chondroitin sulfate type C; dH20, distilled water; DD, diethylaminoethyl (DEAE)-dextran; DS, dextran sulfate; DMEM, Dulbecco's modified Eagle medium; GAG, glycosaminoglycan; HPI, hour(s) post infection; HPLC, high performance liquid chromatography; hr(s), hour(s); HS, heparan sulfate; HSV, herpes simplex virus; FBS, fetal bovine serum; IgG, immunoglobulin G; MOI, multiplicity of infection; PBS, phosphate-buffered saline; pfu, plaque forming unit; PG, proteoglycan; SDS, sodium dodecyl sulfate; TCA, trichloroacetic acid; B. Cells and viruses. The parental murine fibroblasts cell culture used was the clone ID of LMtk". Gro2C are mutant cells derived from L cells that are partially resistant to infection by HSV-1. The procedure for isolation of gro2C was described previously by Gruenheid et al. (49). Briefly, Ltk~ murine fibroblast monolayers were mutagenized with EMS for 18 hrs and left to grow for 7 days in DMEM supplemented with 5% to 10% FBS before being infected with KOS HSV-1 (MOI=l). This resulted in the rapid death of most cells (within 72 hrs). HSV-1 resistant colonies formed at a ratio of 1 in 10 6 of the original population of mutagenized cells and were cloned and further analyzed for their ability to propagate HSV-1. These mutant cells were termed gro mutants (66). Sog9 are HSV-1 resistant mutant cells derived from gro2C using the same procedure as for the isolation of gro2C cells. Vero cells (monkey kidney fibroblasts) were obtained from S. McKnight. HEp-2 (human larynx epidermoid carcinomas) were obtained from W. Jeffries. All cells were grown at 37°C in DMEM supplemented with 5-10% FBS in a 5-6% C0 2 atmosphere. HSV-1 strain KOS was obtained from D. Coen (Harvard Medical School, Boston, MA). HSV-1 devoid of glycoprotein C (gC~ UL45+) was obtained from C. Brandt (University of Wisconsin). C. Reagents. [35S]sulfate (25-40 Ci/mmol), D-[6-3H(N)]glucosamine hydrochloride (40.2 Ci/mmol) and [methyl-3H]thymidine (20 Ci/mmol) were obtained from ICN. PBS used contained 10 mM Na2HP04, 15 mM KH2P04, 1.4 M NaCl and 25 mM KC1 (pH 7.4). CSA (from bovine trachea; 70% CSA, balance is CSC), CSB (from bovine mucosa; 85% CSB, balance is a  33  mixture of CSA and CSC), CSC (from shark cartilage; 90% CSC, balance is CSA), chondroitinase ABC , HS (from bovine intestinal mucosa), and chondroitin 4- and 6-sulfate disaccharides (sodium salts) were obtained from Sigma. Dextran T-500, DS and DD were obtained from Pharmacia. For use in plaquing assays, CSA, CSB, CSC, HS, dextran T-500, DS, and DD were dissolved at the appropriate concentration in PBS supplemented with 0.35 mM MgCh, 0.7 mM CaCl2 and 0.1% glucose. Chondroitinase ABC was dissolved in 0.01% BSA for frozen stocks. Tissue culture reagents were obtained from GIBCO Canada (Mississauga, Ontario, Canada). D. Viral stock production. Monolayers of Vero cells growing in large circular dishes (177 cm2) were washed twice with PBS and inoculated with wild type HSV-1 (MOI - 0.05) or gC" HSV-1 (MOI = 0.1). After 1 hr adsorption at 37°C, the inoculum was removed and replaced with DMEM containing 10% FBS. After 96 hrs of incubation at 37°C, the infected cells were scraped-off the dish and put in four 50 ml conical tubes along with the extracellular medium. Each tube was sonicated (W-385 Sonicator, Mandel Scientific Co. Ltd.) for four 30 second cycles (1 second pulses) in order to lyse all cells and release intracellular viral particles and subjected to low-speed centrifugation (2.5K rpm for 15 minutes at 4°C in an IECCentra 8R centrifuge) to pellet cell debris. The supernatant of all tubes were pooled and 0.5 ml aliquots were stored at -70°C. The viral titer was determined by standard viral plaque assay (see below) and estimated to be 2.1 X 108 pfu/ml. E. Viral plaque assays. Viral plaque assays were performed in duplicate. Confluent Vero cells in 6-well cluster dishes (Nunc; 9.6 cm2/well) were washed twice with PBS and inoculated with serial dilutions of virus (wild type or gC- HSV-1) in DMEM containing 5% FBS. After 1 hr adsorption at 37°C, the viral inoculum was removed and the cells were overlaid with DMEM containing 5% heat-inactivated FBS and 0.1% pooled human gamma globulin. Plaques were counted and visualized after 3 days by staining for 5 minutes with a 4% methylene blue solution in 70% methanol. F. Isolation of radiolabeled virus. Monolayers of Vero cells, growing in large circular dishes (177 cm 2 ), were infected with wild type or gC~ HSV-1 (MOI = 2 to 3) and incubated for 60 minutes at 37°C. The inoculum  34  was then removed and replaced with DMEM containing 2% dialyzed FBS and 50 /vCi [3H]thymidine/ml. After 3 days of incubation at 37°C to allow viral egress, the extracellular medium was removed and subjected to lowspeed centrifugation (2K rpm for 15 minutes at 4°C) to pellet cell debris. The supernatant was centrifuged for 2 hrs at 12,000 rpm in a Beckman SW41 rotor to pellet the virions. The resulting pellet was resuspended in 50 mM NaCl, 10 mM Tris (pH 7.8) overnight at 4°C. This material was sedimented through a 5 to 40% dextran gradient (Dextran T-10, Pharmacia), made in 50 mM NaCl, 10 mM Tris (pH 7.8), for 1 hr at 25,000 rpm in a Beckman SW41 rotor. Gradients were collected from the bottom of the tube into 0.5 ml fractions and those containing radiolabeled HSV-1 were identified by subjecting a small sample to analysis by liquid scintillation spectroscopy. Radioactive fractions were pooled, diluted 1:5 in PBS, and centrifuged for 1.5 hrs at 25,000 rpm in a SW41 rotor to pellet the virions. The resulting pellet was suspended in PBS supplemented with 0.35 mM MgCl2, 0.7 mM CaCl2 and 0.1% glucose, left overnight at 4°C and stored at -70°C. For determination of radioactivity in insoluble material, 50 jL7l of a virus preparation was added to 50 yg BSA followed by 1 ml of 10% cold TCA. Insoluble material was collected onto filters after 1 hr, and radioactivity was measured by scintillation spectroscopy. The titer of the purified virus was determined by plaque assay (see alcove). G. Binding of radiolabeled virus to cells. Confluent monolayers of L, gro2C, gro29, sog9, or HEp-2 cells in 24-well cluster dishes (Nunc) were pretreated for 60 minutes at 37°C with adsorption buffer (PBS, 0.35 mM MgCl2, 0.7 mM CaCh, 0.1% glucose, and 1% BSA) to block nonspecific virus adsorption. After cooling the cells for 20 minutes at 4°C, they were inoculated with [methyl-3H]thymidine-labeled wild type or gC" HSV-1 (from 103 to 4 X 10 4 TCA precipitable 3H CPM/well) in 0.3 ml of adsorption buffer. To determine the effect of soluble HS on radiolabeled wild type and gC" HSV-1 binding to L, gro2C and HEp-2 cells, a single concentration of radiolabeled virus (6.3 X 10 3 and 7.4 X 10 3 precipitable 3H CPM/well for wild type and gC" HSV-1) were mixed with increasing concentration of HS in cold adsorption buffer before inoculation. After an adsorption period at 4°C (0 to 12 hrs for the kinetics experiments and 5 hrs for the adsorption and HS experiments), the cells were placed on ice. The virus was removed and the cells washed twice with cold adsorption buffer and once with cold PBS. Cells were lysed in a total of 0.3 ml of lysis buffer (PBS, 0.35 mM  35  MgCl2, 0.7 mM CaCl2, 1% Triton X-100, and 1% SDS). The lysates were transferred to scintillation vials, dissolved in Ready Safe liquid scintillation cocktail (Beckman), and radioactivity was determined by liquid scintillation spectroscopy. Each point were performed in triplicates. Samples were corrected for small differences in the number of cells in each well. H. Penetration assay. The rate of virus penetration in L and gro2C cells was assessed by determining the rate at which adsorbed virus became resistant to inactivation by a low-pH citrate buffer. Confluent monolayers of L and gro2C cells in 6-well cluster dishes were cooled at 4°C for 30 minutes, washed twice with cold PBS and inoculated with wild type HSV-1 for a 2 hrs adsorption period at 4°C. The cells were placed on ice, the inoculum removed and washed three times in cold PBS to remove any unadsorbed particles. The cells were then overlaid with warm (37°C) DMEM containing 2% FBS and shifted to 37°C to allow viral penetration to proceed. At selected times after the temperature shift, duplicate experimental wells were treated for 1 minute with 2 ml of citrate buffer (40 mM citric acid, 10 mM KC1, 135 mM NaCl [pH 3.0]) and control wells with 2 ml of PBS supplemented with 0.35 mM MgCl2, and 0.7 mM CaCl2. The monolayers were then washed three times with warm PBS and overlaid with DMEM containing 1% FBS and 0.1% pooled human gamma globulin. Plaques were counted and visualized after 4 to 5 days by staining for 5 minutes with a 4% methylene blue solution in 70% methanol. Each experimental point was performed in duplicate. I. Plaque formation inhibition assays. Ninety five percent confluent monolayers of L, gro2C or sog9 cells, plated in 6-well cluster dishes, were washed twice with PBS and inoculated with HSV-1 diluted in PBS supplemented with 0.35 mM MgCh, 0.7 mM CaClz, and 0.1% glucose previously mixed with various concentrations of commercial PG (HS, CSA, CSB or CSC), dextran T-500, DS, or DD. After a 60 minutes adsorption period at 37°C, the inoculum was removed and the cells were thoroughly washed three times with PBS to remove unbound or lightly bound virus. The cells were then overlaid with DMEM containing 1% FBS and 0.1% pooled human gamma globulin. Plaques were counted and visualized after 4 to 5 days by staining for 5 minutes with a 4% methylene blue solution in 70% methanol. Each experimental point was performed in duplicate.  36 J. Determination of the chondroitin sulfate type on L cells. To identify the type of CS present on the cell surface of L cells, the sulfate and sugar backbone of GAG chains were radiolabelled to steady state with [ 35 S]sulfate and [ 3 H]glucosamine, purified by DEAE-Sephacel chromatography and ethanol precipitations, and anion exchange HPLC. Radiolabeled CS fractions isolated in this way were subjected to paper chromatography for separation of the 4- and 6-sulfated forms. Biochemical labeling of GAGs was performed by modifying a procedure described by Esko et al (65). GAGs were radiolabeled by incubating cells for 3 days with [35S]sulfate and D-[6-3H(N)] glucosamine hydrochloride (40 jL/Ci/ml each) in DMEM containing 10% FBS and modified to contain 1 mM glucose and no sulfate. The media was removed and the cells were solubilized with 1 ml of 0.1 M NaOH at 25°C for 15 minutes. Cell extracts were adjusted to pH 6.0 by the addition of concentrated acetic acid and pooled with the media. To eliminate the protein cores of the PGs, the samples were treated at 40°C for 12 hrs with 2 mg of pronase (Sigma) per ml in 0.32 M NaCl, 80 mM Na acetate containing shark cartilage CS (2 mg/ml) as the carrier. The radioactive GAGs were purified from cell debris by chromatography on DEAE-Sephacel (Pharmacia LKB) by binding in 50 mM NaCl followed by elution with 1 M NaCl. For HPLC analysis, the eluted GAGs samples were desalted by two consecutive ethanol precipitations. Four volumes of cold 100% ethanol was added and samples were rocked for 1 hr at 4°C. The precipitate was pelleted by centrifugation at 10K rpm for 20 minutes in a Sorvall RC-5B centrifuge using a SS-34 rotor. The pellet was resuspended in 1 ml of 0.5 M sodium acetate in 10% ethanol per radiolabeled tissue culture dish after the first ethanol precipitation and in 200 A/1 of dH20 per dish after the second. This final dH 2 0 radiolabeled GAG solution was stored at -20°C and used for HPLC anal} sis. Anion-exchange HPLC was performed using a TSK DEAE-3SW column (15 mm by 75 mm; Beckman Instruments). GAGs were eluted from the column by using a linear 50 mM to 1 M NaCl gradient formed in 10 mM KH2P04 (pH 6.0). All buffers contained 0.2% Zwittergent 3-12 (Calbiochem) to extend the life of the column. The GAGs in the peaks were identified by digestion of the sample with the relevant enzymes prior to chromatography. Fractions containing CS were pooled together and desalted by ethanol precipitation as described above. The resulting pellet was resuspended in a total of 50 A/1 of chondroitinase ABC buffer [15% 0.5 M Tris-HCl (pH 8.0), 15% 0.6 M sodium acetate and 15% 0.1% BSA] and digestions were done using 30  37  mU/ml of the enzyme for 12 hrs in a 40°C water bath. In addition to the radiolabeled sample, stock of commercial CSB (4-sulfated control) and CSC (6-sulfated control) (Sigma) were digested by chondroitinase ABC (100 jyg of each PG) in order to break down the GAG chains into disaccharide units. The sample and controls were then subjected to descending paper chromatography using a 30 cm by 10 cm strip of Whatman paper which had been pre-soaked in the running solvent [isobutyric acid : 1 N NH3 (5:3)] and dried. After a 5 to 6 hr run, the Whatman paper was dried overnight in a fume hood and GAG disaccharides were visualized by consecutively spraying with solution 1 (500 iA of saturated aqueous AgN03 diluted to 100 ml with acetone in which 2 to 4 ml of dH20 was then added until the precipitate dissolved) and solution 2 (0.5 N of aqueousmethanolic sodium hydroxide). The stained Whatman paper was heated to 100°C for 1 to 2 minutes. GAGs appeared as dark brown spots. The sample lane on the chromatogram was cut into squares (approximately 1 cm2), and the radioactivity in each was determined by liquid scintillation spectroscopy using Ready Safe liquid scintillation cocktail (Beckman). Radioactive spots were identified by comparison to unlabeled 4- and 6sulfated controls, which were run in parallel lanes.  38  7. RESULTS A. Role of heparan sulfate in HSV-1 infectivity A.l. Selection of gro2C cells As part of a broad study to investigate cellular components necessary for HSV-1 infection, Tufaro et al (66) developed a protocol for selecting HSV-1 resistant murine cells. Briefly, Ltk" murine fibroblast monolayers were mutagenized with EMS for 18 hours and left to grow in normal media for 7 days before being infected with KOS HSV-1 (MOI=l). This resulted in the rapid death of most cells (within 72 hrs). HSV-1 resistant colonies formed at a ratio of 1 in 10 6 of the original population of mutagenized cells. These colonies survived in media containing approximately 10 6 HSV-1 pfu/ml. Several of these HSV-1 resistant colonies were isolated and purified. There are many potential cellular mutations that could produce a cell with an HSV-1 resistant phenotype. Any mutation that is essential for one of the numerous stage of the viral infection process while being unnecessary for cellular survival could produce such a mutant cell line. One of the isolated HSV-1 resistant L cell-derived mutants, named gro2C, was found, after exposure to HSV-1, to be partially resistant to the virus: plaque formation was reduced by 85% compared to the control parental L cells (49). The plaques that formed in gro2C monolayers were morphologically wild type, suggesting that cell-to-cell spread of virus was not affected by the lesion of this mutant. Gro2C cells were therefore lacking in the production, transport and/or function of a cellular component and/or process important but not essential for the HSV-1 infection process. It was puzzling but interesting to note that when initially selected among other HSV-1 resistant cell lines, gro2C cells survived in a media containing a very high number of HSV-1 particles (see above), but that this seemingly complete HSV-1 resistant phenotype was not observed when gro2C cells were grown as a pure cell line. The reasons for these apparent contradictory observations will be examined below. The first step in the HSV-1 infection process is binding of the viral particle to the cell surface. This initial binding is known to involve cell surface and/or extracellular matrix HSPGs that interact electrostatically or through structural features recognition with one or several viral glycoproteins imbedded in the viral envelope. To verify if gro2C cells were  39 defective in the production or transport of specific PG to its cell surface, the sulfate and sugar backbone of GAG chains were radiolabeled to steady state with [35S]sulfate and [3H]glucosamine, purified by protease digestion, followed by DEAE-Sephacel chromatography and ethanol precipitation (49). Samples were then analyzed by anion exchange HPLC. GAG peaks were identified to be heparan sulfate (HS) and chondroitin sulfate (CS) by digestion with the appropriate enzymes (heparitinase, heparinase, chondroitinase AC and ABC) before HPLC analysis. As can be seen in figure 7 , L cells synthesized both HS and CS GAGs in approximately a 2:1 ratio. Gro2C cells however did not produce or effectively transport any detectable amount of HS either to their cell surface or as secreted HS in the media despite the presence of a normal complement of CS chains. This observation supports the hypothesis that gro2C is partially resistant to HSV-1 because the virus cannot effectively bind to the cell surface via HS PGs. This would explain the 15% residual HSV-1 plaquing on these cells. A.2. Effect of heparan sulfate on HSV-1 binding A.2.a. Wild type HSV-1 binding on gro2C cells To demonstrate the link between the presence of cell-surface HS and HSV-1 binding, binding assays, using [3H]thymidine-labeled HSV-1 virions exposed to confluent monolayers of L and gro2C cells, were performed. To estimate the adsorption of the virus to cells without permitting viral penetration and possible turnover of virus cell receptors, the assays were done at 4°C. Two approaches were used. The first was an attempt to determine the time required for HSV-1 to saturate all cell binding sites. For this kinetic binding assessment, monolayers of L and gro2C cells were exposed to a constant amount of purified radiolabeled HSV-1 for different times. At selected times, cells were washed thoroughly, lysed in a Triton X-100/SDS containing buffer and transferred to scintillation vials for quantitation of bound radioactivity. As shown in figure 8, 5 hrs was sufficient for the virus to approach binding equilibrium on gro2C cells whereas 12 hrs was not enough to attain the same state on L cells, although a binding plateau was beginning to form at that time. The second approach consisted of exposing L, gro2C, human HEp-2 and gro29 cells to increasing amounts of radiolabeled virus. Gro29 are  40 8000 -  •  L  HS  cells  6000 .  4000 .  HA 2000 -  CS 1  ^•B*  0> 6000  gro2C  cells  E i_  o o.  4000 -  2000  c =3 o (J  o -ItttumtAta 8000  6000  sog9  cells  4000 -  2000  #3*  mttf  60  20  Fraction  80  Number  Figure 7. Anion exchange HPLC of cell-associated glycosaminoglycans (GAGs) derived from L, gro2C and sog9 cells. Cells were labeled with 40 /iCi/ml each of [35S]sulfate (a) and [3H]glucosamine (•) for 3 days. Labeled GAGs were released from cell and medium proteoglycans and collected by preparative anion-exchange chromatography and ethanol precipitation. A portion (50 til) was then chromatographed by anionexchange HPLC, and the amount of radioactivity in each fraction was determined by liquid scintillation spectroscopy. The peaks enriched for hyaluronic acid (HA), heparan sulfate (HS) and chondroitin sulfate (CS) are identified above the tracing. Portions of this figure were previously published in (49).  Q. C  o  Z3  o .O D. CJ  Hours  post  infection  (HPI)  Figure 8. Binding kinetics of radiolabeled wild type HSV-1 on L (o) and gro2C (•) cells. Confluent monolayers of L and gro2C cells in 24-well cluster dishes were inoculated with constant concentration of [3H] labeled wild type HSV-1 (20700 CPM/well) for different times at 4°C. At selected times, cells were washed thoroughly, lysed and transfered to scintillation vials for quantitation of cell-bound radioactivity. The error bars represents standard deviations for each duplicate sample.  42  HSV-1 resistant cells, isolated along with gro2C, that produce 10% of the HS produced by L cells. Figure 9 shows that at equivalent concentration of input virus over the range tested, binding on gro2C and gro29 cells was approximately 14% and 71% respectively relative to L cells. Binding on the HEp-2 human cell line was more efficient than on L cells indicating that HSV-1 does not bind all cell lines with the same affinity. These results indicate that cell surface HS plays a major but is not essential role in HSV1 binding. A.2.b. GC~ HSV-1 binding on gro2C cells The HSV-1 glycoprotein C (gC), embedded in the virus envelope, has been shown to have high binding affinity to HS and therefore to act as the major viral component involved in initial viral binding. To study the effect that interaction(s) between HSV-1 gC and murine cell surface HS have on viral binding, the two binding assays described previously were performed using [3H]thymidine-labeled HSV-1 particles devoid of gC (termed gC~ HSV-1). In addition, these assays will indicate if HSV-1 can use viral component(s) other than gC for its interactions with HS and the yet unidentified cell component(s) utilized for binding on gro2C cells. Figures 10 and 11 show that (compare with fig. 8 and 9 ) binding of gC" HSV-1, within the range tested, is kinetically and quantitatively identical to binding of wild type HSV-1 on L, gro2C and HEp-2 cells. The differential binding between L and gro2C cells is also similar with both viral strains. These results indicate that under the condition tested, gC is not essential for HSV-1 binding on the mouse fibroblast L cell line, its derivative gro2C and human HEp-2 cells. HSV-1 must therefore use other viral glycoproteins, in addition to or instead of gC, to interact with HS and the yet unidentified cell component(s) utilized for binding on gro2C cells. The result obtained with HEp-2 cells is interesting in that it contradicts binding results previously published using this cell line and a gC~ HSV-1 strain (24). A.2.c. Effect of soluble heparan sulfate on HSV-1 binding The testing of commercial HS (Sigma) as a blocking agent of HSV-1 binding has two main purposes. The first is to compare the interactions of this soluble form of HS (termed sHS) with wild type and gC" HSV-1 to examine if gC plays any detectable role in this interaction and in HSV-1 binding. The second is to study the nature of HSV-1 binding by comparing  o  o o o 2  •o  c o CQ  Input  CPM/1000  cells  Figure 9. Binding of increasing concentration of radiolabeled wild type HSV-1 to HEp-2 ( •), L ( o), gro29 (A), and gro2C (•) cells. Confluent cell monolayers in 24-well cluster dishes were inoculated with the indicated increasing concentration of [3H] labeled wild type HSV-1 for 5 hours at 4°C. After the adsorption period, cells were washed thoroughly, lysed and transfered to scintillation vials for quantitation of cell-bound radioactivity. The error bar represents standard deviations for each duplicate samples.  10  Hours  post  infection  11  12  (HPI)  Figure 10. Binding kinetics of radiolabeled gC_ HSV-1 to L (o) and gro2C (• ) cells. The protocol followed was as described in fig. 8. The concentration of [3H] labeled gC" HSV-1 used was 28000 CPM/well.  tn  "53 u O O O Q_  T3 c  3 O  OQ  Input  CPM/1000  cells  Figure 11. Binding of increasing concentration of radiolabeled gC" HSV-1 to HEp-2 ( • ) , L (o), and gro2C (• ) cells. The protocol followed was as described infig.9.  46  the effects of sHS on virus binding to L, gro2C and HEp-2 cells. Differential effect of sHS could reveal differences in binding strategies used by the virus. This assay was similar to the ones described previously except that cell lines were exposed to constant amount of [3H]thymidine-labeled wild type or gC" HSV-1 which had been previously mixed with increasing concentration of sHS (0 to 15 /L;g/ml). As seen in figure 12, the comparable reduction of bound radioactivity between wild type and gC" HSV-1 on L and HEp-2 cells indicates that sHS interacts with both strains of HSV-1 so as to interfere with viral binding. Presumably, sHS coats wild type and gC" HSV-1 particles thereby blocking viral components necessary for binding interactions with cell surface molecules. As seen in the previous binding assays (fig. 8, 9, 10, and 11), gC" HSV-1 binding is quantitatively similar to the wild type strain. These observations indicate that other viral components besides gC can interact with HS. The residual binding of wild type and gC" HSV-1 on gro2C cells does not seem to be as sensitive to inhibition by sHS as binding to L and HEp-2 cells. It is tempting to conclude that binding of HSV-1 on gro2C is different than on L and HEp-2 cells in so far as it is unaffected by addition of sHS. However, these binding assays have a low sensitivity due to non-specific binding background signals. The low level of HSV-1 binding on gro2C, also reflected by the low plaquing efficiency on these cells, could fall within this limit so that the full extent of the effect of sHS on HSV-1 binding to gro2C cells cannot be assessed precisely by this method. Plaquing assays will attempt to reveal the effect of sHS on HSV-1 binding to gro2C cells. A.3. Effect of heparan sulfate on HSV-1 penetration rate into L cells The emerging model for the role of HS in HSV-1 infectivity is that it is the main cellular component initially recognized by the virus for binding. As mentioned, absence of HS on gro2C cells results in 85% decrease in plaque formation and in radiolabeled viral binding. However, after a gro2C cell is infected with the virus, the infection cycle proceeds normally. In contrast to L cells, HSV-1 infected gro2C cells secrete a higher number of infectious virus particles per cell (69). This suggest that although the charge interactions with HSV-1 effectively sequesters the virus on the L cell surface to initiate the infection process, the same interactions seems to occur when newly formed particles exit the cells  I  0  I  1  Heparan  I  5  sulfate  15  (/vg/ml)  Figure 12. Effect of soluble heparan sulfate on binding of radiolabeled wild type and gC" HSV-1 virions to L, gro2C, and HEp-2 cells. Confluent monolayers, growing in 24-well cluster dishes, were inoculated with a fixed concentration of [3H]thymidine-labeled wild type (6300 CPM/weU) or gC- (7400 CPM/well) HSV-1 previously mixed with the indicated concentration of commercial heparan sulfate (0 to 15 £/g/ml). Adsorption was for 5 hrs at 4°C afterwhich unbound virus was washed away with three PBS rinses. Cells were then lysed and transfered to scintillation vials for quantitation of cell-bound radioactivity. The error bar represents standard deviation for each duplicate samples.  48  thereby hindering their release into the environment. Following the observation that HS's interaction with HSV-1 may deter the release of newly formed viral particles from infected L cells, it was of interest to determine whether this initial HSV-l-HS interaction could also affect the penetration rate of the virus inside the cell. Although L cells can bind more HSV-1 particles per cell than gro2C cells, once a given particle was bound, was the penetration rate of that particle influenced by the different binding interactions probably utilized by these cell lines? To assess the penetration rates of HSV-1, confluent monolayers of L and gro2C cells were exposed to HSV-1 for 2 hr at 4°C, unbound particles were then washed off and cells were incubated at 37°C so that the infection cycle could proceed. At selected intervals, duplicate cultures were exposed to a low-pH citrate buffer, to inactivate any bound virus that had not yet penetrated the cell, or to PBS (controls). The more time a culture was left incubating between the temperature shift and the citrate wash, the more plaques that formed. The results for each time point are expressed as a percentage of the PBS control value. As seen in figure 13, the penetration rates of HSV-1 in L and gro2C cells are similar. This observation confirms that with respect to the murine fibroblast L cell line and its derived HS-deficient gro2C mutant cell line, cell surface HS plays no detectable role in HSV-1 penetration. This observation confirms that the decreased HSV-1 plaquing on gro2C cells is a result of inefficient HSV-1 binding only and not of other deficiencies affecting steps subsequent to binding. A.4. Effect of soluble heparan sulfate on HSV-1 plaque formation To examine further the interaction of HS with HSV-1, sHS was preincubated with the virus in order to study its effect on viral plaquing using L and gro2C cells. As it is suspected that the virus interacts with different cell surface components on L and gro2C cells, sHS may show differential effects on viral plaquing with these cells indicating alternate binding strategies. Duplicate L and gro2C monolayers, growing in 6-well cluster dishes, were infected with HSV-1 previously mixed with various concentration of sHS (from 0 to 20 /jg/ml) immediately before exposure to cells. Plaque formation was then monitored and experimental points obtained in the absence of sHS were taken as the 100% value for each cell line.  IIO-i 10090^^  c o +-» CO  80•  70•  60-  E l_ o **0} 3  ra D_  50•  40. 30•  201004 0  10  20  30  40  Time  50  60  70  80  90  100  (minutes)  Figure 13. Rate of penetration of HSV-1 into L and gro2C cells. Confluent monolayers of L ( o) and gro2C ( • ) cells growing in 6-well cluster dishes were inoculated with HSV-1 for a 2 hr adsorption period at 4°C. After removal of the inoculum and thorough washing of the cells, the dishes were shifted to 37°C to allow penetration to proceed. At selected times after the temperature shift, wells were treated for 1 minute with 2 ml of low pH citrate buffer or PBS (controls). The monolayers were then washed three times with PBS and incubated for 4 to 5 days to permit plaques to form. The number of plaques on the control cultures were essentially the same for all time points, and the average value for each point was taken as 100%. The error bars represent standard deviations for each duplicate sample.  50  Figure 14 shows that sHS was extremely efficient in inhibiting HSV-1 plaque formation on gro2C cells. HSV-1 plaquing was approximately 43 fold more sensitive to sHS with gro2C cells than with L cells: 0.3 £/g/ml of sHS inhibited plaquing on gro2C by 70% whereas 13 £/g/ml of sHS was required for similar plaquing inhibition with L cells. The inhibitory effect that sHS has on HSV-1 binding to gro2C cells could not be detected with the previously described binding assays due to the high non-specific background signal (fig. 12). This differential effect of sHS with L and gro2C cells was surprising. We had previously hypothesized that cells devoid of cell surface HS would interact with different HSV-1 components than cells possessing HS. Consequently, sHS was not expected to exert any inhibitory effect on HSV-1 plaquing to gro2C cells. These results prompt the development of a new hypothesis that could be further tested. We now believe that these observations indicate that binding of HSV-1 to gro2C cells involves similar viral components but is "weaker" than binding to L cells in that much less sHS was required to inhibit HSV-1 binding to gro2C cells. Because cell surface CS is the only remaining sulfated GAG on gro2C cells, it was of interest to investigate the possibility that it could act as the main HSV-1 binding receptor on gro2C cells and, maybe, as an alternative binding receptor on L cells. B. Role of chondroitin sulfate in HSV-1 infectivity B.l. Effect of soluble chondroitin sulfate on HSV-1 plaque formation Soluble CS (sCS) (Sigma) was used to assess if HSV-1 could interact with this GAG by measuring its effect on viral plaque formation using L and gro2C cells. The procedure was identical to the one followed in the sHS plaque formation inhibition assay (section A.4). All three forms of CS were assayed: CSA (4-sulfated N-acetylgalactosamine), CSB (also known as dermatan sulfate; 4-sulfated N-acetylgalactosamine with iduronic acid), and CSC (6-sulfated N-acetylgalactosamine) (see fig. 4). The use of all three CS forms permitted assessment of the influence that sulfate position and hexuronic type (glucuronic or iduronic acid) might have on HSV-1's interaction with sCS. As seen in figure 15, plaquing efficiency on gro2C cell monolayers was inhibited by 60%, 90%, and 50% by sCS types A, B, and C respectively. The effect of sCS with the parental L cells were less, ranging from 20%  s o •M  re  E  3  cr re  Heparan  sulfate  (/yg/ml)  Figure 14. Inhibition of HSV-1 plaque formation by soluble heparan sulfate. L (o) and gro2C (• ) cell monolayers were inoculated with HSV1 previously mixed with various concentrations of commercial heparan sulfate (0 to 20 /L/g/ml). After a 60 minute adsorption period at 37°C, the inoculum was removed and the cells were thoroughly washed three times with PBS to remove unbound or lighdy bound heparan sulfate and virus. The cells were then incubated for 4 to 5 days to permit plaques to form. Each experimental point represents the average of two to eight experiments in which each point were the average of duplicate cultures. The error bars represent standard deviations.  52  £ c  o  *•>  CO  E  <D  03  Chondroitin  sulfate  (/yg/ml)  Figure 15. Inhibition of HSV-1 plaque formation by soluble chondroitin sulfate. L (o) and gro2C (• ) cell monolayers were inoculated with HSV-1 previously mixed with various concentrations (0 to 20 £/g/ml) of commercial chondroitin sulfate of types A (4-sulfated), B (4-sulfated; dermatan sulfate), and C (6-sulfated). The protocol followed was as described in fig. 14.  53  increase with CSA and 20% decrease with CSB. The effect of sCS on HSV-1 plaquing with L cells may be less observable and more concealed due to the presence of highly charged cell surface HS. Because the Nacetylgalactosamine residues of CS remain acetylated during their cellular processing, they are less extensively sulfated, and the resulting structural diversity and net charge of the repeating disaccharide is much less than for HS moieties. The lower disaccharide sulfate negative charge of CS may be related to its less significant effect on HSV-1 plaquing as GAGs' electrostatic charge may be their most important attribute for efficient HSV-1 binding interactions. These sCS-HSV-1 interactions are more difficult to observe using L cells and less efficient in plaquing inhibition with gro2C cells than sHS. It is of interest that the CS type that is most structurally similar to HS (CSB due to the presence of iduronic acid residues; fig. 4) was the most effective in inhibiting HSV-1 plaque formation. Taken together, these results suggest that HSV-1 can interact with sCS in a manner that alters infection efficiency. However, these assays do not show direct evidence that the residual infectivity of HSV-1 on gro2C cells is a result of interactions with cell surface CS. B.2. Chondroitin sulfate type on L cells Because different interaction intensities were observed between HSV-1 and the three sCS types, and, consequently, because cell surface CS may play an important role in HSV-1 binding on gro2C cells, it was of interest to determine which CS type was most prevalent on the surface of L cells. The sulfate and sugar backbone of L cell GAG chains were radiolabeled to steady state with [35S]sulfate and [3H]N-acetylglucosamine, purified by protease digestion, DEAE-Sephacel chromatography and ethanol precipitation. Samples were then analyzed by anion exchange HPLC. Fractions corresponding to CS peak were then pooled and subjected to ethanol precipitation in order to concentrate the CS chains. The resulting sample was digested overnight with chondroitinase ABC to breakdown the CS GAG chains into disaccharide units. These radioactive CS disaccharides were then run on descending paper chromatography using Whatman paper as support and isobutyric acid/ammonia (5:3) as solvent. Commercially available 4- and 6-sulfated CS disaccharides, and chondroitinase ABC digested CSB and CSC were run alongside as visual markers. After a 5 hrs run, the paper strip was dried overnight in a fume hood and stained.  54  Because the amount of radioactive CS sample was insufficient for visual identification of the proper CS type, the sample lane was cut into small squares, each of which were numbered and placed into scintillation vials for quantitation of the radioactive signal. Square no. 4 corresponded to the 6-sulfated CS form and square no. 5 to the 4-sulfated forms. As can be seen in figure 16, the majority of the radioactive counts were present in square no. 5 indicating that L cells predominately produce CS of type A or B (both are 4-sulfated). B.3. Selection of sog9 cells B.3.a. HSV-1 plaquing and GAG HPLC profile Since gro2C cells are infectable by HSV-1, these were subjected to a second round of selection with HSV-1 in order to isolate mutants hopefully completely resistant to infection. These mutants would be defective in expression of additional components of the cellular machinery essential for the virus' lifecycle. Two variant cell lines derived from gro2C, termed sog7 and sog9, were found to be extremely resistant to HSV-1 infection. Plaque formation of HSV-1 on sog9 cells was 1% compared to wild type L cells (table 2). Analysis by anion exchange HPLC of [35S]sulfate and [3H]N-acetylglucosamine radiolabeled cellular GAG chains produced by sog9 cells revealed that these cells had additional defects in the processing of GAGs in that they were unable to synthesize either CS or HS (fig. 7). These results, in addition to previous observations that HSV-1 can interact with sCS, indicates that cell surface CS can play a role in the HSV-1 entry pathway. Thus, two independent, sequential selections for HSV-1 resistance led to defects in the GAG synthesis pathway emphasizing the importance of these molecules for the HSV-1 lifecycle. In order to ensure that the reduced HSV-1 infectability with sog9 cells is a result of reduced binding efficiency and not of other important cellular defects, B. Banfield tested whether protein secretion and processing and Vesicular Stomatitis Virus (VSV) propagation were normal in sog9 cells. B.3.b. Protein processing and transport It is well established that defects in processing or secretion of proteins could lead to alterations in susceptibility to HSV and to its subsequent propagation. VSV is an enveloped RNA virus which encodes a  A o  2 ••-> 55 o a.  w  o  3 tx w  Direction of solvent flow  «  c-i  •£« CO Q.  D)E  <-Origin to to  _0J Q.  to to  E  to  u  TO  to  300  B 4->  13 C  E  200-  l_  Q.  c O u X  100  2  3  4  Square  5  6  7  number  Figure 16. Chondroitin sulfate type on L cells. GAG chains of L cells were radiolabeled with [ 35 S]sulfate and [3H]glucosamine, purified by protease digestion, DEAE-Sephacel chromatography and ethanol precipitation. Samples were analysed by anion exchange HPLC and fractions corresponding to the CS peak were pooled, ethanol precipitated, digested with chondroitinase ABC and run on descending paper chromatography along with the indicated unlabeled controls as described in Materials and Methods. The sample lane was cut into numbered squares and subjected to liquid scintillation spectroscopy. Radioactive spots were identified by comparison to the migration position of unlabeled 4- and 6- sulfated controls. Figure 16A shows the paper chromatogram. Figure 16B shows the ^H counts of the radioactive sample squares with the migration position of the unlabeled 4and 6-sulfated CS controls. CSB, chondroitinase ABC digested chondroitin sulfate type B (4-sulfated); di-4S, 4-sulfated chondroitin sulfate disaccharides; di-6S, 6-sulfated chondroitin sulfate disaccharides; CSC, chondroitinase ABC digested chondroitin sulfate type C (6-sulfated).  56  Table 2. Properties of variant murine cell lines. All values are expressed as a percentage relative to the murine L cell line.  Chondroitin sulfate synthesis  HSV-1 plaquing efficiency  Variant Cell line  Heparan sulfate synthesis  L  100  100  100  gro2C  <0.2  100  15  sog9  <0.2  <0.2  1  57  single glycoprotein (G protein) modified by two, complex type N-linked oligosaccharides in wild type L cells. To test whether the secretion and processing of proteins was defective in sog9 cells, VSV label-chase experiments with [35S]methionine labeled G protein were performed by B. Banfield. These experiments revealed that there was no impediment to the synthesis and processing of VSV G protein in gro2C or sog9 cells when compared with wild type L cells, indicating that infection proceeded normally in both cell lines. Thus, the receptor for VSV was expressed and functional in sog9 cells, thereby eliminating the possibility that there was a gross alteration in cell surface architecture which precluded virus infection. Moreover, there was no impediment to N-linked oligosaccharide processing, suggesting that the proteins expressed at the cell surface were normal in this regard (70). B.3.C. VSV propagation To examine virus propagation and egress from infected cells, monolayers of L, gro2C, sog9 and sog7 cells were infected with VSV and protein products were radiolabeled with [ 3 5 S]methionine. SDS polyacrylamide gel electrophoresis was then performed and it was found that VSV propagation and egress were normal in gro2C, and sog9 cells, confirming that the secretory apparatus of sog9 functioned normally. These results indicate that sog9 cells are able to support the entry, replication, and egress of an unrelated enveloped virus. Therefore the reduced HSV-1 infectability with sog9 cells is a result of reduced binding efficiency and not of defects in other important cellular functions. B.4. HSV-1 binding on sog9 cells To demonstrate more clearly the link between the presence of cell surface CS and HSV-1 binding, binding assays, using [3H]thymidine-labeled HSV-1 virions exposed to confluent monolayers of gro2C and sog9 cells, were performed. The protocol followed was the same as the one described for the binding assays with wild type HSV-1 on L and gro2C cells using increasing amounts of radiolabeled virus (fig. 9). As seen in figure 17, radiolabeled HSV-1 bound with a slightly lower efficiency on sog9 cells than on gro2C cells (1.8 fold less). This decreased binding efficiency of 1.8 fold is considerably different than the observed 10 fold decrease in plaque formation of HSV-1 on sog9 cells relative to gro2C cells. As previously mentioned, these binding assays are not as  u  o o o Q_  O  TJ O 00  120  Input  CPM/1000  cells  Figure 17. Binding of increasing concentration of radiolabeled wild type HSV-1 to gro2C (•) and sog9 (n ) cells. The protocol followed was as described in fig. 9.  59  sensitive as plaquing assays due to non-specific binding background signal. The signals emitted by bound radiolabeled HSV-1 particles on gro2C and sog9 cells fall within this sensitivity limit so that the assay does not permit a precise assessment of any differential binding. B.5. Effect of soluble heparan sulfate on HSV-1 plaque formation with sog9 cells To test the hypothesis that the residual 1% infectivity of sog9 cells would occur independently of the interactions between the HSV-1 envelope and the cell surface GAGs and to circumvent the sensitivity limit of the binding assays, the sensitivity of HSV-1 plaquing to sHS on sog9 cells was analyzed in plaque assays by K. Schubert and B Banfield. Monolayers of L, gro2C, sog9 cells were infected with HSV-1 previously mixed with various concentration of sHS (from 0 to 90 jL/g/ml). Plaque formation was monitored and the experimental points obtained in the absence of sHS were taken as the 100% value for each cell line. Figure 18 shows quite clearly that HSV-1 plaque formation on sog9 cells was in no way reduced in the presence of high concentration of sHS, which indicates that the residual infection was initiated without engaging cell surface GAGs. The residual 1% infectivity reflects GAG-independent stable binding interactions. The results obtained with sog9 and gro2C cells strongly suggest that cell surface CS can facilitate HSV-1 binding albeit with a lower efficiency than cell surface HS. Additional interactions than the ones involving HSV-1 and cell surface GAGs may be important for steps subsequent to stable attachment on the cell surface. However, these later interactions are severely impaired in the absence of cell surface GAGs. C. Nature of the HSV-1 binding interaction: electrostatic vs sequence recognition The interactions between viral glycoproteins and PGs is thought to be mainly electrostatic. The high negative charge carried by sulfated GAGs has been shown to be essential in this interaction which may involve basic amino acids present in viral glycoproteins such as gC and gB, both of which known to have heparin and heparan sulfate binding affinities. Electrostatic interactions are reflected in the higher binding affinity of HSV-1 for HS than CS, the later having, on average, a lower number of sulfate residues per disaccharides than HS. Although this type of interaction is important in  60  10CH  90 8070-  ra  60-  E  5040-  0) 3  30-  ra  20-  10 0 30  Heparan  sulfate  0/g/ml)  Figure 18. Inhibition of HSV-1 plaque formation by soluble heparan sulfate using sog9 ( • ), L ( H), and gro2C ( • ) cells. The protocol followed was as described in fig. 14.  61 HSV-1 binding, specific structural features may also play a role. In an attempt to determine the relative importance of electrostatic interactions and structural features in HSV-1 binding, I tested the effects of charged synthetic dextran polysaccharides on HSV-1 plaquing. Being synthetic and different in structure than GAGs, any effect that charged dextran polymers may have on HSV-1 plaquing can be attributed to their electrostatic charges alone. C.l. Effect of dextran sulfate on HSV-1 plaque formation Dextran sulfate (DS) (Pharmacia) is a polyanionic sulfated derivative of Dextran T-500. It is composed of glucose residues containing an average of 1.9 sulfate groups per glucosyl molecule which makes for a very high negatively charged macromolecule. Because DS is charged throughout the length of the molecule, its net negative charge is higher then that of HS which has highly sulfated sections followed by non-sulfated ones. The experimental procedure followed was identical to the plaque formation inhibition assays performed with sHS on L and gro2C cells (fig. 14). Figure 19 shows that DS inhibited HSV-1 plaquing on L cells in a more efficient way than sHS (compare with fig. 14): 90.2% reduction in plaquing with 3 /ig/ml of DS while 85% reduction required 20 jwg/ml of sHS. Plaquing on gro2C cells was also inhibited by DS but to a significant lesser extent then with sHS: 87% reduced plaquing was achieved with 20 IJg/ml of DS by while only 1 IJg/ml of sHS was required for the same effect. The plaquing inhibition profile for gro2C cells was similar to the one obtained with CSA (fig. 15). The higher efficiency of sHS, relative to DS, to inhibit HSV-1 binding to gro2C cells suggests the need for specific structural features in these interactions. The results for these cell lines and DS is roughly opposite of the plaquing inhibition effect seen with sHS. Surprisingly, DS had a very high stimulatory effect on HSV-1 plaquing with sog9 cells. As little as 300 ng/ml of DS was sufficient to increase plaque formation by 20 to 25 fold with sog9 cells. A peak was reached at approximately 3 A/g/ml of DS; higher concentrations had less effect on HSV-1 plaquing. C.2. Effect of DEAE-dextran on HSV-1 plaque formation Diethylaminoethyl (DEAE)-dextran (DD) (Pharmacia) is also a dextran T-500 derivative. It is a non-sulfated glucose polysaccharide that has one covalently linked DEAE molecule per three glucosyl residues. The nitrogen  10000  1000-:  TO  E  100  Z3  n Q_  8  Dextran  10  12  sulfate  14  16  18  20  (/ig/ml)  Figure 19. Inhibition of HSV-1 plaque formation by soluble dextran sulfate using L ( o), gro2C (•), and sog9 (•) cells. The protocol followed was as described in fig. 14.  63  rich DEAE molecule conveys on the polymer a net positive charge. HSV-1 plaque formation inhibition assays were performed with L, gro2C, and sog9 cells following the same protocol as with DS. As seen in figure 20, DD had no observable effect on HSV-1 plaquing with L cells and a small inhibitory effect on gro2C cells (58.8% reduction with 12 ^ig/ml of DD). HSV-1 plaquing on sog9 cells was stimulated by DDto levels similar to those observed when using DS. However, the figure shows that the stimulatory effect with DD was not as effective has DS in that peak effects required 12 tJg/ml of DD instead of the 300 ng/ml required with DS. C.3. Effect of dextran T-500 on HSV-1 plaque formation Dextran T-500, the parent compound of both DS and DD, is relatively neutral in charge. It was used as a control molecule in an HSV-1 plaquing assay with sog9 cells to determine the extent of the role that electrostatic charges play in the observed increase in plaquing efficiency with these cells. The experimental procedure was identical as the one followed in the plaque formation inhibition assays performed with both DS and DD. Figure 21 shows the effect of all three dextran polymers on HSV-1 plaquing with sog9 cells. The curves for DS and DD w^re derived from the data presented in figures 19 and 20. Addition of dextran T-500 had no significant observable effect on HSV-1 plaquing with sog9 cells. The results obtained with these three dextran polymers on HSV-1 plaquing with sog9 cells indicate that the stimulatory effect observed is directly related to the electrostatic charges carried by the sugars. This is indicative of the importance that electrostatic interactions have in the binding of HSV-1 to cell surface GAGs.  10000  1000  £ c g re  100  E O"  10  DEAE-Dextran  12  14  16  18  20  0/g/ml)  Figure 20. Inhibition of HSV-1 plaque formation by soluble DEAE-dextran using L (o), gro2C (•), and sog9 (•) cells. The protocol followed was as described in fig. 14.  10000 TT  £ 1000-  c  o "+J  E 100 CO  0  2 4  6 8 10 12 14 16 18 20 22 24 26 28 30  Dextran  polysaccharide  G/g/ml)  Figure 21. Inhibition of HSV-1 plaque formation by soluble dextran T500 (•), dextran sulfate (•), and DEAE-dextran ( A ) using sog9 cells. The protocol followed was as described in fig. 14. Data for the dextran sulfate and DEAE-dextran curves are from the experiments shown in fig. 19 and fig. 20.  66 8. DISCUSSION A previous study reported the initial characterization of gro2C cells (49). Although it could not be concluded with certainty that gro2C cells synthesized no HS chains, the assays used were sensitive enough to detect 0.05% or less of the normal complement of HS (fig. 7). This extensive deficiency in synthesis and/or transport of HS to the cell surface of gro2C cells was accompanied with 85% reduction in HSV-1 infection efficiency. This indicated that although HS is important, HSV-1 need not to interact specifically with it to gain entry into mouse fibroblasts. Numerous studies have confirmed the importance of HS in initial binding of HSV to cell surfaces (6, 17, 22-25, 28, 34, 45-50, 68). However, to this date only two main approaches have been used to study this interaction with the use of cells devoid of cell surface HS: (I) enzymatic treatment of cells with heparitinase and heparinase (23) and (II) selection of CHO mutants defective in the synthesis of HS by screening for clones that failed to incorporate inorganic sulfate into macromolecules (25, 65, 66). The enzymatic treatment of human HEp-2 cells with heparitinase and heparinase reduced HSV-1 plaquing efficiency by approximately 88% (23). However, it is difficult, using such an assay, to precisely determine if all cell surface HS chains have been removed and if the exposed residual cell surface molecules have been disturbed or not by the enzymatic treatment. Consequently, the HSV-1 residual binding cannot be said to be a result of interactions with normal cell surface molecules in the absence of HS. The HS-deficient CHO mutant cell line pgsD-677 also showed considerable reduction in HSV-1 binding (25). However, due to CHO's nonpermissiveness to HSV-1, these results were obtained with the use of only low sensitivity binding assays using radiolabeled particles which do not make it possible to clearly determine the extent of HSV-1 residual binding, if any, to these cells. In this work, I initially confirmed, with a combination of plaquing and binding assays, the observed 85% decreases in HSV-1 plaquing efficiency on gro2C cells (table 2) and binding (fig. 8 and 9). This indicated that HSV-1 binds and infects gro2C cells, albeit inefficiently (15% of parental L cells) while HS is absent from their cell surface. Due to the difficulty in estimating precisely the binding reduction percentage using binding assays with radiolabeled virions, it was of interest to know if the reduction in HSV-1 plaquing resulted from inefficient binding only or from  67  other deficiencies in the infection process. No detectable difference between the penetration rate of HSV-1 into L and gro2C cells was observed (fig. 13) implying that the decreased HSV-1 plaquing with gro2C cells was the result of deficient binding and not deficient penetration. To support this, one-step growth experiments showed that virus gene expression and subsequent virus assembly and egress occurred normally after entry into gro2C cells (49). Moreover, observations that plaque morphology was normal and released virus was infectious further indicated that for fibroblast murine cells, HS enhances but is not essential for HSV-1 attachment to the cell surface in addition to not being essential at any other stage of the lytic cycle. The HSV-1 binding difference to gro2C versus CHO pgsD-677 cells is interesting. It is not yet known if removal of HS from these cells uncovered cell type differences in cell surface molecules or if the results are a reflection of different assays used. I suggest, and will elaborate below, that if differences between the cell surface molecules of gro2C and pgsD-677 cells exist, these may be in the different types of CS chains synthesized by these cells. The residual HSV-1 binding to gro2C cells therefore points-out that other cell surface components, besides HS, are implicated in the binding process. The initial binding of HSV to cells is known to involve the interaction of the HSV glycoproteins gC and gB with cell surface HS (22-25, 45-48). Both glycoproteins can bind heparin/HS molecules presumably through interactions between clusters of basic amino acids present at their Nterminus (4, 6) and the negatively charged sulfate groups of heparin/HS chains. Binding assays with gC" HSV-1 virions have shown a 90% reduction in virus binding (24). It has been suggested that the 85% reduction in HSV1 binding to gro2C cells may be related to non-existing gC-HS interactions with these cells (49) implying a critical function for gC in initial attachment. Unlike previous results (24) I have found that radiolabeled gC" HSV-1 particles bound with similar affinity as wild type virions to L and gro2C cells (fig. 10 and 11 compared to fig. 8 and 9; fig. 12). In addition, binding of gC" HSV-1 was as HS-dependent as wild type virions in that (I) addition of increasing concentration of soluble HS hindered binding to L, gro2C, and HEp-2 cells and (II) similar differential binding between L and gro2C cells were observed (fig. 12). These results suggest that, for these cell lines under the conditions tested, gC is not required for HSV-1 binding and that therefore other(s) HS-dependent components), in  68  addition to or instead of gC, interact with soluble HS, the cell surface HS of L cells, and the cell surface molecule(s) utilized for binding to gro2C cells. It is not known whether the same viral component(s) involved in gC~ binding to L cells are involved in binding to gro2C cells. The HS-dependent gB seems a likely candidate for substitution of gC functions. Because gC" HSV-1 virions have never been shown to bind cells with similar affinity as gC containing virions, gB's role in HSV binding has always been thought to be secondary. It could be that the cell surface of L cells may be so different than other cell types so as to permit greater involvement of gB in binding. Alternate means of HSV-1 binding have been proposed to account for binding variability between cell types (24). It has yet to be determined what role, if any, does gB or other viral components have in binding of gC" and wild type HSV-1 to L and HSdeficient gro2C cells. The use of gB" HSV-1 virions in binding assays with L and gro2C cells could be of future interest. The results presented in this thesis suggest that the cell surface molecules mainly responsible for the 15% residual HSV-1 binding to gro2C cells are CS GAG chains. HS is composed of a repeating disaccharide of Nacetyl- and/or N-sulfoglucosamine and a hexuronic acid whereas CS consists of N-acetylgalactosamine and hexuronic acid (table 1). HS chains are extensively modified through sulfation of glucosamine units and by Ndeacetylation and N-sulfation, leading to the formation of GAG chains with considerable sequence diversity and high concentrated region of negative charges (fig. 3) (32). In contrast, the modifications of CS are much less extensive than HS leading to less pronounced structural diversity and an even distribution of negative charges (fig. 4) (32). If HSV relies mainly on electrostatic interactions between its envelope glycoproteins and cell surface GAGs for binding, it seems likely that in the absence of HS, CS could serve as binding receptor. The HSV-1 resistant phenotypes of cells reduced in HS sulfation as a consequence of either sodium chlorate treatment (4) or genetic selection (25) supports the importance of electrostatic interactions in HSV binding. However, previous studies have dismissed the involvement of CS as an HSV-1 binding receptor. CHO mutants expressing only cell surface CS did not significantly bind more radiolabeled HSV-1 particles than cell mutants devoid of both HS and CS (25); soluble CS could not inhibit HSV-1 binding on GMK cells (68); and enzymatic digestion of cell surface CS on HEp-2 cells had no effect on HSV-1 plaquing (23). As mentioned in the  69 introduction, there are underlying problems with each of these experiments in their ability to detect the effect of CS on HSV-1 binding. First, CHO cell studies rely on low sensitivity binding assays with radiolabeled virus due to their HSV non-permissiveness. Second, both HSV-1 binding interference studies with soluble CS using GMK cells and HSV-1 binding studies on CS-digested HEp-2 cells have a similar bias: the presence of highly charged cell surface HS that could electrostatically mask or inhibit any detectable interactions between HSV-1 and cell surface CS. The use, in this work, of highly sensitive plaque assays using the HSV-1 permissive, HS-deficient gro2C cell line permitted observation of HSV-1CS interactions. Three lines of evidence pointed toward CS as being involved in HSV1 binding. First, soluble HS was significantly more efficient in hindering HSV-1 plaque formation with gro2C cells than with L cells (fig. 14). This differential effect of sHS with L and gro2C cells was surprising. We had previously hypothesized that cells devoid of cell surface HS would interact with different HSV-1 components than cells possessing HS. Consequently, sHS was not expected to exert any inhibitory effect on HSV-1 plaquing to gro2C cells. These results prompt the development of a new hypothesis that could be further tested. We now believe that these observations indicate that the residual HSV-1 binding to gro2C cells involves similar viral components but is "weaker" than binding to L cells in that much less sHS was required to inhibit HSV-1 binding to gro2C cells. Being the only remaining cell surface GAG on gro2C cells, the regionally electrostatically weaker CS (compared to HS) could be responsible for this "weak" HSV-1 binding. The second line of evidence came from further investigations which relied on the use of soluble CS types A, B, and C (fig. 4) in plaquing assays. All three types had variable but detectable inhibitory effects on HSV-1 plaquing with gro2C cells (fig. 15) indicating that HSV-1 can interact with this GAG. It is of interest to note that CSB (dermatan sulfate) had the strongest plaquing inhibition effect. Of all three CS types, it is the only one containing iduronic acid as one of its hexuronic acid constituents (table 1 and fig. 4) and is therefore more closely related structurally to HS than CSA and CSC. Iduronic acid residues impart on GAG chains bearing them a high degree of chain flexibility. This could permit efficient orientation of sulfate groups to protein ensuring stronger interactions than GAGs not possessing iduronic acid. It could therefore be that structural features may,  70  in addition to electrostatic interactions, play a part in the initial HSV-1 binding process. As suggested above, the lack of inhibitory effects of soluble CSs on HSV-1 plaquing with L cells may be due to the presence of cell surface HS that electrostatically mask or inhibit interactions between the virus and cell surface CS. Although these results show that HSV-1 can interact with soluble CS they do not provide evidence that the HSV-1 binding to gro2C cells is a result of interactions with cell surface CS. The isolation, from gro2C cells, of the HS- and CS-deficient cell line sog9 (fig. 7) permitted the direct assessment of the ability of cell surface CS to serve as a receptor for HSV-1 binding. The absence of any detectable GAGs on the cell surface of sog9 cells was accompanied by an approximate ten fold increase in HSV-1 infection resistance (table 2). Assays to determine if sog9 cells were physiologically impaired in aspects other than GAG synthesis revealed normal protein processing and transport and, apart from the lack of GAGs, normal cell surface architecture (70). These results suggest that the reduced HSV-1 infectability of sog9 cells is a direct consequence of reduced binding efficiency and not of defects in other cellular functions. The 15% residual binding of HSV-1 to gro2C cells was efficiently blocked by the addition of soluble HS (fig. 14) and CSB (fig. 15) and thus involved GAG-dependent viral glycoproteins. Following the removal of CS from the cell surface of gro2C cells, any observed HSV-1 infectivity would result from interactions between non-GAG cell surface components and viral glycoproteins that do not require GAGs. The observation that HSV-1 binding to sog9 cells was resistant to the addition of high concentrations of HS (fig. 18) indicated that the viral glycoproteins involved in this process promoted a very stable GAG-independent binding. In addition to being required for HSV-1 penetration into cells, gD is known to be involved in a GAG-independent stable attachment step which presumably occurs after initial viral interactions with cell surface GAGs and before fusion of the plasma membrane with the viral envelope (26, 29, 51-53, 55). This stable attachment allows for close association of the virus with the cell surface to promote viral penetration. It may be that the 1% residual HSV-1 binding to sog9 cells is the result of gD interactions with its yet unidentified protein receptor thereby inducing a stable GAG-independent attachment. Future experiments with gD" HSV-1 virions may elucidate the role of gD in HSV-1 binding to sog9 cells. The reduction from a 15% GAG-dependent HSV-1 binding with CS-containing gro2C cells to a 1% GAG-independent stable  71 binding with CS-deficient sog9 cells (fig. 7) strongly indicates that cell surface CS plays a role in HSV-1 binding even though the viral glycoproteins involved are unknown. Plaque formation inhibition assays using all three CS types indicated the HSV-1 does not interact with each type with the same affinity. As mentioned above, CSB, the CS most closely related structurally to HS, had the strongest plaquing inhibition effect suggesting that, in addition to interacting with the electrostatic charges of sulfate groups, HSV-1 glycoproteins may also preferentially require certain structural features for efficient binding to GAGs. This observation also suggests that cell types expressing different CS types may differentially utilize CS for the HSV-1 binding process. L cells predominantly produce a 4-sulfated CS (CSA or CSB) (fig. 16). Could the previously described HSV-1 binding variability to gro2C cells and the CHO mutant cells pgsD-677 (HS-deficient) be attributed to the synthesis of different CS types? This question cannot be answered at present. The results obtained with gro2C and sog9 cells help elucidate the functions of GAGs in HSV-1 binding to murine fibroblast cells. HS plays the major role in facilitating initial attachment of the virus to the cell surface. Absence of HS from the cell surface of L cells (gro2C cells) reduces binding by approximately 85% (table 2) underlining that HS is important but not essential for HSV-1 binding. This conclusion is strengthened by the fact that two mutant cell lines resistant to HSV-1 infection and deficient in HS synthesis arose by different selection procedure: pgsD-677 CHO cells (25, 64, 65) and gro2C murine fibroblast cells (49). This fact virtually eliminates the possibility that a second mutation that could account for the HSV resistant phenotype arose fortuitously in both cell lines during selection. In the absence of HS, cell surface CS can promote HSV-1 binding albeit less efficiently than HS. These interactions would account for the 15% residual HSV-1 binding to gro2C cells (table 2). It is not known whether CS can exert its role in HSV-1 binding when HS is also present on the cell surface (such as with L cells). HS's high efficiency to interact with HSV-1 may render interactions with CS either unnecessary or undetectable. In the total absence of cell surface GAGs, HSV-1 binding to murine fibroblast cells drops to 1% as seen with sog9 cells (table 2). This binding is very stable and, being GAG-independent, may involve interactions with gD or other HSV-1 glycoproteins required for the initial steps of penetration (gH?). These latter important interactions, although  72  not requiring GAGs, are severally impaired when initial HSV-GAG interactions do not take place in order to sequester a high number of viral particles to the periphery of the cell. This initial "concentration" of viruses would facilitate the recognition of cell surface receptors with viral glycoproteins, such as gD, to promote stable attachment and eventually penetration. The interactions between viral glycoproteins and PGs are thought to be mainly electrostatic in nature. This is reflected by the higher affinity of HSV-1 for HS than CS, the latter having on average less sulfate residues per disaccharides than HS. This type of interaction is important for HSV-1 binding as previously mentioned from observations that the HSV-1 resistant phenotype of some cells was caused by reduced HS sulfation (4, 25). However, as mentioned above, the HSV-1 plaque formation inhibition assays with soluble CS type A, B, and C revealed that type B's structural similarity to HS may be the reason for its strongest interaction with HSV-1. To determine the relative importance of electrostatic interactions and structural features in HSV-1 binding, I tested the effects of charged synthetic dextran polysaccharides on HSV-1 plaquing. Being synthetic and of different structure than GAGs, any effect on HSV-1 plaquing can be attributed to electrostatic interactions. The very highly negatively charged dextran sulfate (DS) polymer inhibited HSV-1 plaquing on L cells with very high efficiency (fig. 19) while the positively charged DEAE-dextran (DD) (fig. 20) and the relatively neutral dextran T-500 polymers (data not shown) had no detectable effect. With L cells, the effect of DS on HSV-1 plaquing was roughly seven fold more inhibitory than HS (fig. 14). The data from gro2C cells, at first glance, corroborates L cell results. The dextran polymer DS was the most efficient in hindering plaque formation, followed by DD and dextran T-500 (fig. 19). However, the observation that HS and CSB inhibited HSV-1 plaquing on gro2C cells with far greater ease than DS (fig. 14, 15 and 19) indicates that the HSV-1 interactions with gro2C cells depends to a greater extent on structural features of the GAGs than the HSV-1 interactions with L cells. The high HSV-1 plaquing stimulation on sog9 cells by DS and DD , but not dextran T-500 (fig. 21) also indicates interactions mainly dependent of electrostatic charges.  73  A number of studies have demonstrated the inhibitory effect of anionic polymers on viral infectivity. DS was shown to significantly reduce fusion to biological membranes of orthomyxo, influenza B, toga, and rhabdo viruses (71). The fusion of Sendai virus to liposomes (72) and erythrocyte ghosts (73), the binding of BK virus (74), CMV (75), and Sindbis virus (76) were also inhibited, in order of effectiveness, by DS, heparin, and CS. All of these effects were found to be dependent on the concentration of anionic polymer applied. The mechanism for binding and fusion inhibition was suggested to result from adherence of the negatively charged polymers to the viral surface thereby blocking viral binding or fusion epitopes (71-74). Such blockage prevented close contact between the viruses and membrane surfaces. The structure of HS may permit its sulfate groups to electrostatically cover HSV-1 particles and block viral binding receptors more efficiently than DS. When plaquing with gro2C cells, the efficient blocking of binding receptors by HS may be more effective in hindering virus-cell contacts than blockage by DS which, although possessing a higher overall negative charge than HS, may not have the required structure for efficient interactions between its sulfate groups and HSV-1's binding receptors. This would explain the greater effect of HS in HSV-1 plaquing inhibition with gro2C cells. Because the cell surface of L cells is more negatively charged than gro2C cells (due to the presence of HS), the negative charges of anionic-covered HSV-1 particles may play a greater role in being repulsed from the cell surface than blockage of specific receptors. Therefore, with L cells, DS would inhibit HSV-1 plaquing more effectively than HS. This hypothetical model to explain the still unknown mechanism(s) involved in the inhibition effects of anionic polymers on viral infectivity suggests the combined influence of electrostatic repulsion and specific blockage of viral receptors. Conversely, electrostatic attraction may be into play in the high increase, by DS and DD, of HSV-1 plaquing with sog9 cells (approximately 20 fold increase) (fig. 21). To my knowledge, it has never been reported that DS had stimulatory effects on viral infectivity. The significantly altered cell surface of sog9 cells may be the cause. The sog9 cell surface, being devoid of sulfated GAGs, is, relative to L and gro2C cells, positively charged. This cell surface may be effective in promoting close contact with negatively charged viral particles such as when covered with DS. Particles covered with the cationic DD may also have a negatively charge surface through the attraction of anionic salts present in the media.  74  The above descriptions are only speculations on the mechanisms involved in anionic polymer-HSV-1 interactions. Nevertheless, the results obtained with DS, DD, dextran T-500, HS, and CS indicate that efficient HSV-1 binding depends mainly on electrostatic interactions although specific structural features may facilitate these interactions.  75 9. REFERENCES  1.  Mindel, A. 1989. Herpes simplex virus. In J. Tinker (ed.), The Bloomsbury series in clinical science, 1 s t ed. Springer-Verlag (London), Berlin, Heildelberg.  2.  Mindel, A., D. M. Coker, A. Feherty, and P. William. 1988. Recurrent genital herpes: clinical and virological features in men and women. Genitourin. Med. 64:103-106.  3.  Roizman, B., and W. Batterson. 1986. Herpesviruses and their replication, p. 607-636. In B. N. Fields and D. M. 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