A cellular function is required for pseudorabies virus envelope glycoprotein processing and virus egress.

The mouse L-cell mutant gro29 is defective for egress of herpes simplex virus type 1 (HSV-1) virions and is significantly reduced in HSV-1 glycoprotein export (B. W. Banfield and F. Tufaro, J. Virol. 64:5716-5729, 1990). In this report, we demonstrate that pseudorabies virus (PRV), a distantly related alphaherpesvirus, shows a distinctive set of defects after infection of gro29 cells. Specifically, we identify defects in the rate and extent of viral glycoprotein export, infectious particle formation, plaque formation, and virus egress. The initial rate of viral glycoprotein synthesis was unaffected in gro29 cells, but the extent of export from the endoplasmic reticulum to the Golgi apparatus was impaired and export through the Golgi apparatus became essentially blocked late in infection. Moreover, by using a secreted variant of a viral membrane protein, we found that export from the Golgi apparatus out of the cell was also defective in gro29 cells. PRV does not form plaques on gro29 monolayers. A low level of infectious virus is formed and released early after infection, but further virus egress is blocked. Taken together, these observations suggest that the gro29 phenotype involves either multiple proteins or a single protein used at multiple steps in viral glycoprotein export and virus egress from cells. Moreover, this host cell protein is required by both HSV and PRV for efficient propagation in infected cells.     

Temporal morphogenesis of herpes simplex virus type 1-infected and brefeldin A-treated human fibroblasts

BACKGROUND: Insights in the herpesvirus-cell interactions are of general cell biology interest, especially to studies of intracellular transport, and of considerable significance in the efforts to generate drugs, vaccines, and gene therapy. However, the pathway of virus particle egress and maturation is a contentious issue. MATERIALS AND METHODS: The intracellular transport was inhibited in cultured herpes simplex virus type 1 (HSV-1) infected human fibroblasts by brefeldin A (BFA). The virus-cell interactions including the viral envelopment, transport of HSV-1 virions, and transport of viral glycoprotein D (gD-1) and glycoprotein C (gC-1) were studied by titration assay, immunoblot, immunofluorescence light microscopy, and immunogold electron microscopy of cryosections. RESULTS: gD-1 and gC-1 were synthesized and normally transported to the plasma membranes of untreated HSV-1 infected host cells. BFA (1 microg/ml medium) effectively blocked the transport of the glycoproteins to the plasma membranes and affected the tubulin and vimentin of the cytoskeleton. Viral particles and glycoproteins accumulated in the perinuclear space and the endoplasmic reticulum of BFA treated cells. Withdrawal of BFA influence up to 9 hr resulted in restored tubulin and vimentin, transport of glycoproteins to the plasma membranes, and steady release of infectious viral particles to the extracellular space superior to the cellular assembly of new virions. The ultrastructural data presented support that the primary envelopment of viral particles occur at the nuclear membranes containing immature glycoproteins followed by multiple de-envelopments and re-envelopments of the virions during the transport and maturation in the endoplasmic reticulum and the Golgi complex. CONCLUSIONS: BFA-induced changes include the cytoskeleton with significant effect on HSV-1 maturation and egress. The data support a multiple-step envelopment of HSV-1 in a common pathway of glycoprotein synthesis and virion egress.     

Dysregulation of Autophagy in Murine Fibroblasts Resistant to HSV-1 Infection

The mouse L cell mutant, gro29, was selected for its ability to survive infection by herpes simplex virus type 1 (HSV-1). gro29 cells are fully susceptible to HSV-1 infection, however, they produce 2000-fold less infectious virus than parental L cells despite their capacity to synthesize late viral gene products and assemble virions. Because productive HSV-1 infection is presumed to result in the death of the host cell, we questioned how gro29 cells might survive infection. Using time-lapse video microscopy, we demonstrated that a fraction of infected gro29 cells survived infection and divided. Electron microscopy of infected gro29 cells, revealed large membranous vesicles that contained virions as well as cytoplasmic constituents. These structures were reminiscent of autophagosomes. Autophagy is an ancient cellular process that, under nutrient deprivation conditions, results in the degradation and catabolism of cytoplasmic components and organelles. We hypothesized that enhanced autophagy, and resultant degradation of virions, might explain the ability of gro29 to survive HSV-1 infection. Here we demonstrate that gro29 cells have enhanced basal autophagy as compared to parental L cells. Moreover, treatment of gro29 cells with 3-methyladenine, an inhibitor of autophagy, failed to prevent the formation of autophagosome-like organelles in gro29 cells indicating that autophagy was dysregulated in these cells. Additionally, we observed robust co-localization of the virion structural component, VP26, with the autophagosomal marker, GFP-LC3, in infected gro29 cells that was not seen in infected parental L cells. Collectively, these data support a model whereby gro29 cells prevent the release of infectious virus by directing intracellular virions to an autophagosome-like compartment. Importantly, induction of autophagy in parental L cells did not prevent HSV-1 production, indicating that the relationship between autophagy, virus replication, and survival of HSV-1 infection by gro29 cells is complex.     

Replication of herpes simplex virus: egress of progeny virus at specialized cell membrane sites

In the final stages of the herpes simplex virus 1 (HSV-1) life cycle, a viral nucleocapsid buds into a vesicle of trans-Golgi network (TGN)/endosome origin, acquiring an envelope and an outer vesicular membrane. The virus-containing vesicle then traffics to the plasma membrane where it fuses, exposing a mature virion. Although the process of directed egress has been studied in polarized epithelial cell lines, less work has been done in nonpolarized cell types. In this report, we describe a study of HSV-1 egress as it occurs in nonpolarized cells. The examination of infected Vero cells by electron, confocal, and total internal reflection fluorescence (TIRF) microscopy revealed that HSV-1 was released at specific pocket-like areas of the plasma membrane that were found along the substrate-adherent surface and cell-cell-adherent contacts. Both the membrane composition and cytoskeletal structure of egress sites were found to be modified by infection. The plasma membrane at virion release sites was heavily enriched in viral glycoproteins. Small glycoprotein patches formed early in infection, and virus became associated with these areas as they expanded. Glycoprotein-rich areas formed independently from virion trafficking as confirmed by the use of a UL25 mutant with a defect in capsid nuclear egress. The depolymerization of the cytoskeleton indicated that microtubules were important for the trafficking of virions and glycoproteins to release sites. In addition, the actin cytoskeleton was found to be necessary for maintaining the integrity of egress sites. When actin was depolymerized, the glycoprotein concentrations dispersed across the membrane, as did the surface-associated virus. Lastly, viral glycoprotein E appeared to function in a different manner in nonpolarized cells compared to previous studies of egress in polarized epithelial cells; the total amount of virus released at egress sites was slightly increased in infected Vero cells when gE was absent. However, gE was important for egress site formation, as Vero cells infected with gE deletion mutants formed glycoprotein patches that were significantly reduced in size. The results of this study are interpreted to indicate that the egress of HSV-1 in Vero cells is directed to virally induced, specialized egress sites that form along specific areas of the cell membrane.     

Herpes simplex virus nucleocapsids mature to progeny virions by an envelopment --> deenvelopment --> reenvelopment pathway

Herpes simplex virus (HSV) nucleocapsids acquire an envelope by budding through the inner nuclear membrane, but it is uncertain whether this envelope is retained during virus maturation and egress or whether mature progeny virions are derived by deenvelopment at the outer nuclear membrane followed by reenvelopment in a cytoplasmic compartment. To resolve this issue, we used immunogold electron microscopy to examine the distribution of glycoprotein D (gD) in cells infected with HSV-1 encoding a wild-type gD or a gD which is retrieved to the endoplasmic reticulum (ER). In cells infected with wild-type HSV-1, extracellular virions and virions in the perinuclear space bound approximately equal amounts of gD antibody. In cells infected with HSV-1 encoding an ER-retrieved gD, the inner and outer nuclear membranes were heavily gold labeled, as were perinuclear enveloped virions. Extracellular virions exhibited very little gold decoration (10- to 30-fold less than perinuclear virions). We conclude that the envelope of perinuclear virions must be lost during maturation and egress and that mature progeny virions must acquire an envelope from a post-ER cytoplasmic compartment. We noted also that gD appears to be excluded from the plasma membrane in cells infected with wild-type virus.     

Identification and characterization of a mouse cell mutant defective in the intracellular transport of glycoproteins

We have isolated a mutant line of mouse L cells, termed gro29, in which the growth of herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) is defective. The block occurs late in the infectious cycle of both viruses. We demonstrate that HSV and VSV enter gro29 cells normally, negotiate the early stages of infection, yet are impaired at a late stage of virus maturation. During VSV infection of the mutant cell line, intracellular transport of its glycoprotein (G protein) is slowed. Pulse-chase experiments showed that oligosaccharide processing is impeded, and immunofluorescence localization revealed an accumulation of G protein in a juxtanuclear region that contains the Golgi complex. We conclude that export of newly made glycoproteins is defective in gro29 cells, and speculate that this defect may reflect a lesion in the glycoprotein transport apparatus.     

Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection.

Herpes simplex virus (HSV) requires the host cell secretory apparatus for transport and processing of membrane glycoproteins during the course of virus assembly. Brefeldin A (BFA) has been reported to induce retrograde movement of molecules from the Golgi to the endoplasmic reticulum and to cause disassembly of the Golgi complex. We examined the effects of BFA on propagation of HSV type 1. Release of virions into the extracellular medium was blocked by as little as 0.3 microgram of BFA per ml when present from 2 h postinfection. Characterization of infected cells revealed that BFA inhibited infectious viral particle formation without affecting nucleocapsid formation. Electron microscopic analyses of BFA-treated and untreated cells (as in control cells) demonstrated that viral particles were enveloped at the inner nuclear membrane in BFA-treated cells and accumulated aberrantly in this region. Most of the progeny virus particles observed in the cytoplasm of control cells, but not that of BFA-treated cells, were enveloped and contained within membrane vesicles, whereas many unenveloped nucleocapsids were detected in the cytoplasm of BFA-treated cells. This suggests that BFA prevents the transport of enveloped particles from the perinuclear space to the cytoplasmic vesicles. These findings indicate that BFA-induced retrograde movement of molecules from the Golgi complex to the endoplasmic reticulum early in infection arrests the ability of host cells to support maturation and egress of enveloped viral particles. Furthermore, we demonstrate that the effects of BFA on HSV propagation are not fully reversible, indicating that maturation and egress of HSV type 1 particles relies on a series of events which cannot be easily reconstituted after the block to secretion is relieved.     

Suppression of annexin A1 and its receptor reduces herpes simplex virus 1 lethality in mice

Herpes simplex virus 1 (HSV-1)-induced encephalitis is the most common cause of sporadic, fatal encephalitis in humans. HSV-1 has at least 10 different envelope glycoproteins, which can promote virus infection. The ligands for most of the envelope glycoproteins and the significance of these ligands in virus-induced encephalitis remain elusive. Here, we show that glycoprotein E (gE) binds to the cellular protein, annexin A1 (Anx-A1) to enhance infection. Anx-A1 can be detected on the surface of cells permissive for HSV-1 before infection and on virions. Suppression of Anx-A1 or its receptor, formyl peptide receptor 2 (FPR2), on the cell surface and gE or Anx-A1 on HSV-1 envelopes reduced virus binding to cells. Importantly, Anx-A1 knockout, Anx-A1 knockdown, or treatments with the FPR2 antagonist reduced the mortality and tissue viral loads of infected mice. Our results show that Anx-A1 is a novel enhancing factor of HSV-1 infection. Anx-A1-deficient mice displayed no evident physiology and behavior changes. Hence, targeting Anx-A1 and FPR2 could be a promising prophylaxis or adjuvant therapy to decrease HSV-1 lethality.     

Monensin inhibits the processing of herpes simplex virus glycoproteins, their transport to the cell surface, and the egress of virions from infected cells.

HEp-2 cells or Vero cells infected with herpes simplex virus type 1 were exposed to the ionophore monensin, which is thought to block the transit of membrane vesicles from the Golgi apparatus to the cell surface. We found that yields of extracellular virus were reduced to less than 0.5% of control values by 0.2 microM monensin under conditions that permitted accumulation of cell-associated infectious virus at about 20% of control values. Viral protein synthesis was not inhibited by monensin, whereas late stages in the post-translational processing of the viral glycoproteins were blocked. The transport of viral glycoproteins to the cell surface was also blocked by monensin. Although the assembly of nucleocapsids appeared to be somewhat inhibited in monensin-treated cells, electron microscopy revealed that nucleocapsids were enveloped to yield virions, and electrophoretic analyses showed that the isolated virions contained immature forms of the envelope glycoproteins. Most of the virions which were assembled in monensin-treated cells accumulated in large intracytoplasmic vacuoles, whereas most of the virions produced by and associated with untreated cells were found attached to the cell surface. Our results implicate the Golgi apparatus in the egress of herpes simplex virus from infected cells and also suggest that complete processing of the viral envelope glycoproteins is not essential for nucleocapsid envelopment or for virion infectivity.     

Glycoprotein gE of herpes simplex virus type 1: effects of anti-gE on virion infectivity and on virus-induced fc-binding receptors.

An Fc-binding glycoprotein, designated gE, was detected previously in cells infected with herpes simplex virus type 1 (HSV-1) and in virion preparations isolated from infected cells. For the studies reported here, we purified gE from HSV-1 strain HFEM(syn) by affinity chromatography and preparative electrophoresis and then immunized a rabbit to produce an antiserum to glycoprotein gE. We found that this antiserum selectively precipitated gE and its precursors from detergent-solubilized extracts of HSV-1 strain HFEM(syn)-infected HEp-2 cells, from extracts of other cell lines infected with the same virus, and from extracts of HEp-2 cells infected with several other HSV-1 strains. The antiserum did not precipitate any proteins from uninfected cells. The several forms of gE detected by immunoprecipitation accumulated in variable quantities in different cells infected with the different virus strains and also varied slightly with respect to electrophoretic mobility, suggesting some differences in the gE's from different HSV-1 strains and some effects of the host cell on the nature and extent of post-translational processing. One of the electrophoretic forms of gE previously detected in purified preparations of virions could be precipitated by anti-gE from extracts of purified HSV-1 strain HFEM(syn) virions. Moreover, anti-gE neutralized HSV-1 infectivity, but only in the presence of complement. Finally, F(ab')2 fragments of the anti-gE immunoglobulin partially inhibited the binding of 125I-labeled immunoglobulin G to the Fc receptors on HSV-1-infected cells.     

Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors.

Herpes simplex virus (HSV) glycoprotein D (gD) plays an essential role in the entry of virus into cells. HSV mutants unable to express gD were constructed. The mutants can be propagated on VD60 cells, which supply the viruses with gD; however, virus particles lacking gD were produced in mutant-infected Vero cells. Virus particles with or without gD adsorbed to a large number (greater than 4 x 10(4] of sites on the cell surface; however, virions lacking gD did not enter cells. Cells pretreated with UV-inactivated virions containing gD (approximately 5 x 10(3) particles per cell) were resistant to infection with HSV type 1 (HSV-1) and HSV-2. In contrast, cells pretreated with UV-inactivated virions lacking gD could be infected with HSV-1 and HSV-2. If infectious HSV-1 was added prior to UV-inactivated virus particles containing gD, the infectious virus entered cells and replicated. Therefore, virus particles containing gD appear to block specific cell surface receptors which are very limited in number. Particles lacking gD are presumably unable to interact with these receptors, suggesting that gD is an essential receptor-binding polypeptide.     

Membrane proteins specified by herpes simplex viruses. III. Role of glycoprotein VP7(B2) in virion infectivity.

Experiments done with a temperature"sensitive mutant of herpes simplex virus type 1 (HSV-1) have revealed that one of the virisn glycoproteins, designated VP7(B2), is apparently not required for the production of enveloped virus particles, whereas it does play a critical role in virion infectivity. The mutant, designated HSV-1[HFEM]tsB5, fails to accumulate VP7(B2) at nonpermissive temperature and produces virions that lack detectable quantities of this glycoprotein and that have very low specific infectivity. The poor infectivity of the virions is most readily explained by failure of penetration into the host cell rather than by failure of adsorption to cells because it was shown that the VP7(B2)-deficient virions can bind to cells and that polyethylene glycol, an agent known to promote membrane fusion, can significantly enhance infectivity of the adsorbed virions.     

Egress of alphaherpesviruses: comparative ultrastructural study

Egress of four important alphaherpesviruses, equine herpesvirus 1 (EHV-1), herpes simplex virus type 1 (HSV-1), infectious laryngotracheitis virus (ILTV), and pseudorabies virus (PrV), was investigated by electron microscopy of infected cell lines of different origins. In all virus-cell systems analyzed, similar observations were made concerning the different stages of virion morphogenesis. After intranuclear assembly, nucleocapsids bud at the inner leaflet of the nuclear membrane, resulting in enveloped particles in the perinuclear space that contain a sharply bordered rim of tegument and a smooth envelope surface. Egress from the perinuclear cisterna primarily occurs by fusion of the primary envelope with the outer leaflet of the nuclear membrane, which has been visualized for HSV-1 and EHV-1 for the first time. The resulting intracytoplasmic naked nucleocapsids are enveloped at membranes of the trans-Golgi network (TGN), as shown by immunogold labeling with a TGN-specific antiserum. Virions containing their final envelope differ in morphology from particles within the perinuclear cisterna by visible surface projections and a diffuse tegument. Particularly striking was the addition of a large amount of tegument material to ILTV capsids in the cytoplasm. Extracellular virions were morphologically identical to virions within Golgi-derived vesicles, but distinct from virions in the perinuclear space. Studies with gB- and gH-deleted PrV mutants indicated that these two glycoproteins, which are essential for virus entry and direct cell-to-cell spread, are dispensable for egress. Taken together, our studies indicate that the deenvelopment-reenvelopment process of herpesvirus maturation also occurs in EHV-1, HSV-1, and ILTV and that membrane fusion processes occurring during egress are substantially different from those during entry and direct viral cell-to-cell spread.     

Insertion mutants of herpes simplex virus have a duplication of the glycoprotein D gene and express two different forms of glycoprotein D.

We produced insertion mutants of herpes simplex virus (HSV) that contain two functional copies of genes encoding different forms of glycoprotein D (gD). These viruses have the gene for HSV type 2 (HSV-2) gD at the normal locus and the gene for HSV-1 gD inserted into the thymidine kinase locus. Results of immunoprecipitation experiments done with monoclonal antibodies revealed that both gD genes were expressed by these viruses, regardless of orientation of the inserted HSV-1 gD gene, and that maximal synthesis of both glycoproteins depended on viral DNA replication. This apparently normal expression of the inserted HSV-1 gD gene was from a DNA fragment (SacI fragment, 0.906 to 0.924 map units) containing nucleotide sequences extending from approximately 400 base pairs upstream of the 5' end of the gD mRNA to about 200 base pairs upstream of the 3' end. The glycoproteins expressed from both genes were incorporated into the surfaces of infected cells. Electrophoretic analyses of purified virions and neutralization studies suggest that both glycoproteins were also incorporated into virions. This nonpreferential utilization of both gene products makes these viruses ideal strains for the generation and characterization of a variety of mutations.     

Toxin resistance and reduced secretion in a mouse L-cell mutant defective in herpes virus propagation.

The mouse L-cell mutant gro29 was selected originally for its inability to propagate herpes simplex virus; it shows severe defects in virus egress and the transport and processing of viral glycoproteins after infection. In this report, we show that uninfected gro29 cells display pleiotropic changes in protein secretion, oligosaccharide processing, and sensitivity to the toxins ricin and modeccin. Specifically, the rate of secretion of a nonglycosylated protein, human growth hormone, was reduced 70% in gro29 cells compared with the parental L cells. A direct measurement of the transport capacity of Golgi membranes in a cell-free assay suggests that gro29 cells contain less functional Golgi than parental cells. Despite this deficiency, N-linked oligosaccharides were processed efficiently in mutant cells, although there were differences in the structure of the mature forms. Lectin intoxication assays revealed that gro29 cells were cross-resistant to killing by the cytotoxic lectins ricin and modeccin, but not to wheat germ agglutinin, Ricinus communis agglutinin RCA120, or leucoagglutinin. Fluorescence labeling using fluorescein-conjugated lectins showed that uninfected gro29 cells expressed relatively few ricin-binding molecules, suggesting a possible mechanism for toxin resistance. These studies provide evidence that the processes of protein secretion, lectin intoxication, and herpes virus maturation and egress may share a common cellular component.     

Truncations of the simian immunodeficiency virus transmembrane protein confer expanded virus host range by removing a block to virus entry into cells.

We have investigated how truncation of the cytoplasmic domain of the transmembrane (TM) glycoprotein of simian immunodeficiency virus (SIV) modulates the host range of this virus. Termination codons were introduced into the env gene of SIVmac239 which resulted in the truncation of the transmembrane protein from a wild-type 354 amino acids (TM354) to 207 (TM207) and 193 (TM193) amino acids. Expression of the wild-type and mutant env genes from a simian virus 40-based vector resulted in normal biosynthesis and processing of the glycoproteins to gp130 and gp41 or the truncated TM proteins (gp28 and gp27). When expressed on the surface of COS-1 cells, all three glycoproteins mediated fusion of both CEMX174 and HUT78 cells. Virions containing the wild-type and mutant glycoproteins were capable of efficient replication in macaque peripheral blood lymphocytes and CEMX174 cells; in contrast, only virions that contained TM207 were capable of rapid infection of HUT78 cells. Both truncated glycoproteins were capable of efficiently mediating infection of both CEMX174 and HUT78 cells by an env-deficient human immunodeficiency virus. The wild-type SIV glycoprotein, however, was unable to mediate human immunodeficiency virus infection of HUT78 cells when assayed with this system. An analysis of the protein composition of SIV released from infected CEMX174 cells showed that the mutant virions contained significantly higher levels of glycoprotein compared with the wild type. These results demonstrate that truncation of the SIV cytoplasmic domain removes a block at the level of glycoprotein-mediated virus entry into HUT78 cells and points to a role for glycoprotein density in determining virus tropism.     

Incorporation of CD4 into virions by a recombinant herpes simplex virus.

Two herpes simplex virus type 1 (HSV-1) recombinants were constructed by inserting the human CD4 gene into the HSV-1 genome between the gC promoter and the gC structural gene. These viruses, designated K delta T/CD4 and K082/CD4, synthesized a significant quantity of CD4. CD4 was expressed on the surface of infected cells at levels substantially higher than on the surface of HUT78 cells, a CD4+ cell line. Most significantly, a small but detectable quantity of CD4 was incorporated into virions produced by the recombinant viruses. This was demonstrated both by immunoprecipitation of CD4 from purified virions and by neutralization of the recombinant virions by OKT4 and complement. These results suggest that specific virion incorporation signals are not strictly required for inclusion of glycoproteins into HSV-1 virions. It may be possible to utilize this ability to alter the host range or tissue specificity of HSV-1.     

Structural analysis of precursor and product forms of type-common envelope glycoprotein D (CP-1 antigen) of herpes simplex virus type 1.

The type-common CP-1 antigen of herpes simplex virus type 1 (HSV-1) is associated in the infected cell with two components, a 52,000-molecular-weight glycoprotein (gp52 or pD) and a 59,000-molecular-weight glycoprotein (gp59 or D). The larger form (D) is also found in the virion envelope. It was postulated that pD is a precursor of D. We found that pD shared methionine and arginine tryptic peptides with D isolated from infected cell extracts. D isolated from infected extracts had the same trypric methionine peptide profile as D isolated from the virion envelope. Thus, processing of pD to D does not involve any major alterations in polypeptide structure. Furthermore, D did not share tryptic methionine peptides with the other major glycoproteins of HSV-1. Using [2-3H]mannose as a specific glycoprotein label, we found that pD, which is a basic protein (isoelectric point = 8.0) contained a 1,800-molecular-weight oligomannosyl core moiety and was processed by further glycosylation and sialyation to a more acidic and heterogeneous molecule D, which as a molecular weight of at least 59,000.     

Construction and properties of a mutant of herpes simplex virus type 1 with glycoprotein H coding sequences deleted.

A mutant of herpes simplex virus type 1 (HSV-1) in which glycoprotein H (gH) coding sequences were deleted and replaced by the Escherichia coli lacZ gene under the control of the human cytomegalovirus IE-1 gene promoter was constructed. The mutant was propagated in Vero cells which contained multiple copies of the HSV-1 gH gene under the control of the HSV-1 gD promoter and which therefore provide gH in trans following HSV-1 infection. Phenotypically gH-negative virions were obtained by a single growth cycle in Vero cells. These virions were noninfectious, as judged by plaque assay and by expression of beta-galactosidase following high-multiplicity infection, but partial recovery of infectivity was achieved by using the fusogenic agent polyethylene glycol. Adsorption of gH-negative virions to cells blocked the adsorption of superinfecting wild-type virus, a result in contrast to that obtained with gD-negative virions (D. C. Johnson and M. W. Ligas, J. Virol. 62:4605-4612, 1988). The simplest conclusion is that gH is required for membrane fusion but not for receptor binding, a conclusion consistent with the conservation of gH in all herpesviruses.