Higher Levels of Neutralizing Antibodies against KSHV in KS Patients Compared to Asymptomatic Individuals from Zambia

Kaposi sarcoma-associated herpesvirus (KSHV) is the etiologic agent for Kaposi Sarcoma (KS), the most common cancer diagnosed in HIV- infected patients. The role of neutralizing antibodies in KS pathogenesis and in KSHV infected individuals is not clearly understood. The goal of this study was to investigate and compare the prevalence and titers of neutralizing antibodies in plasma samples from KS patients and KSHV infected asymptomatic individuals from Zambia, a KS endemic region in sub-Saharan Africa. Plasma samples (N = 267) consisting of KS patients (group 1) and asymptomatic individuals (group 2) were collected from Lusaka, Zambia. A flow cytometry based quantitative neutralization assay utilizing recombinant KSHV expressing GFP was used to detect KSHV neutralizing antibodies. Our results show that the overall prevalence of neutralizing antibodies in KS patients (group 1) was 66.7% which was significantly higher than the prevalence of 6.5% present in KSHV infected asymptomatic individuals (group 2). Total antibody titers as well as neutralizing antibodies titers were found to be significantly higher among KS patients. It is likely that higher neutralizing antibodies prevalence and titers in KS patients result from higher levels of antigenic stimulation over time. This study is first to compare prevalence and titers of neutralizing antibodies in participants with and without disease from a KSHV endemic region.     

Cytomegalovirus Vaccine Strain Towne-Derived Dense Bodies Induce Broad Cellular Immune Responses and Neutralizing Antibodies That Prevent Infection of Fibroblasts and Epithelial Cells

Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.     

B cell repertoire analysis identifies new antigenic domains on glycoprotein B of human cytomegalovirus which are target of neutralizing antibodies

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.     

Single-cell analysis of Kaposi’s sarcoma-associated herpesvirus infection in three-dimensional air-liquid interface culture model

The oral cavity is the major site for transmission of Kaposi’s sarcoma-associated herpesvirus (KSHV), but how KSHV establishes infection and replication in the oral epithelia remains unclear. Here, we report a KSHV spontaneous lytic replication model using fully differentiated, three-dimensional (3D) oral epithelial organoids at an air-liquid interface (ALI). This model revealed that KSHV infected the oral epithelia when the basal epithelial cells were exposed by damage. Unlike two-dimensional (2D) cell culture, 3D oral epithelial organoid ALI culture allowed high levels of spontaneous KSHV lytic replication, where lytically replicating cells were enriched at the superficial layer of epithelial organoid. Single cell RNA sequencing (scRNAseq) showed that KSHV infection induced drastic changes of host gene expression in infected as well as uninfected cells at the different epithelial layers, resulting in altered keratinocyte differentiation and cell death. Moreover, we identified a unique population of infected cells containing lytic gene expression at the KSHV K2-K5 gene locus and distinct host gene expression compared to latent or lytic infected cells. This study demonstrates an in vitro 3D epithelial organoid ALI culture model that recapitulates KSHV infection in the oral cavity, where KSHV undergoes the epithelial differentiation-dependent spontaneous lytic replication with a unique cell population carrying distinct viral gene expression.     

The gammaherpesvirus 68 latency-associated nuclear antigen homolog is critical for the establishment of splenic latency

Open reading frame 73 (ORF 73) is conserved among the gamma-2-herpesviruses (rhadinoviruses) and, in Kaposi's sarcoma-associated herpesvirus (KSHV) and herpesvirus saimiri (HVS), has been shown to encode a latency-associated nuclear antigen (LANA). The KSHV and HVS LANAs have also been shown to be required for maintenance of the viral genome as an episome during latency. LANA binds both the viral latency-associated origin of replication and the host cell chromosome, thereby ensuring efficient partitioning of viral genomes to daughter cells during mitosis of a latently infected cell. In gammaherpesvirus 68 (gammaHV68), the role of the LANA homolog in viral infection has not been analyzed. Here we report the construction of a gammaHV68 mutant containing a translation termination codon in the LANA ORF (73.STOP). The 73.STOP mutant virus replicated normally in vitro, in both proliferating and quiescent murine fibroblasts. In addition, there was no difference between wild-type (WT) and 73.STOP virus in the kinetics of induction of lethality in mice lacking B and T cells (Rag 1(-/-)) infected with 1000 PFU of virus. However, compared to WT virus, the 73.STOP mutant exhibited delayed kinetics of replication in the lungs of immunocompetent C57BL/6 mice. In addition, the 73.STOP mutant exhibited a severe defect in the establishment of latency in the spleen of C57BL/6 mice. Increasing the inoculum of 73.STOP virus partially overcame the acute replication defected observed in the lungs at day 4 postinfection but did not ameliorate the severe defect in the establishment of splenic latency. Thus, consistent with its proposed role in replication of the latent viral episome, LANA appears to be a critical determinant in the establishment of gammaHV68 latency in the spleen post-intranasal infection.     

Differential regulation of the attachment of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected human B cells to extracellular matrix by KSHV-encoded gB and cellular alphaV integrins

Kaposi's sarcoma-associated herpesvirus (KSHV) has two modes of replications: latent and lytic replications. Reactivation from latency is dictated, in part, by the cell cycle. Herein, we have attempted to delineate the importance of cell cycle in KSHV pathogenesis by exploring the expression pattern of cell-surface receptors during different phases of the cell cycle. αV integrin expression is augmented during S phase in fibroblasts, epithelial and KSHV-infected cells. Using a Matrigel system, we pioneer the concept that KSHV-infected primary effusion lymphoma cells can attach to extracellular matrix proteins. This attachment is mediated primarily via αV integrins or virally encoded gB, and occurs preferentially in cells from S phase or cells from S phase actively supporting a lytic infection respectively. Such an ability of infected B cells to attach to endothelial cells may also aid in the dissemination of infection. The keystone of this work is that for the first time, we describe the ability of KSHV-infected B cells to preferentially use cellular (αV) or viral (gB) receptors to specifically bind cells, depending upon the stage of the cell cycle and infection.     

Induction of mucosal and systemic neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) by oral immunization with bovine Papillomavirus-HIV-1 gp41 chimeric virus-like particles

Human immunodeficiency virus type 1 (HIV-1) envelope-specific neutralizing antibodies are generated late after initial infection, and the neutralizing antibody response is weak in the infected individuals. Administration of neutralizing antibodies such as 2F5 to HIV-1-infected individuals resulted in reductions in viral loads. Because HIV-1 is transmitted mainly via mucosa and because HIV-specific neutralizing antibodies reduce HIV-1 in infected individuals, a vaccine that can induce both mucosal and systemic HIV-1-specific neutralizing antibodies may be used to prevent and to treat HIV-1 infection. In this study, we made a bovine papillomavirus (BPV) L1-HIV-1 gp41 fusion protein in which ELDKWA of gp41 was inserted into the N terminus of BPV L1 (amino acids 130 to 136). Expression of the fusion protein in insect cells led to the assembly of chimeric virus-like particles (CVLPs). The CVLPs had sizes similar to those of BPV particles and were able to bind to the cell surface and penetrate the cell membrane. Oral immunization of mice with CVLPs induced gp41-specific serum immunoglobulin G (IgG) and intestinal secretory IgA. However, intramuscular immunization with the CVLPs resulted in similar amounts of gp41-specific IgG but low levels of secretory IgA. The antibodies specifically recognized the fixed HIV-1 gp41 on the cell surface. Importantly, the sera and fecal extracts from mice orally immunized with the CVLPs neutralized HIV-1(MN) in vitro. Thus, BPV-HIV-1 gp41 CVLPs may be used to prevent and to treat HIV-1 infection.     

Contribution of endocytic motifs in the cytoplasmic tail of herpes simplex virus type 1 glycoprotein B to virus replication and cell-cell fusion

The use of endocytic pathways by viral glycoproteins is thought to play various functions during viral infection. We previously showed in transfection assays that herpes simplex virus type 1 (HSV-1) glycoprotein B (gB) is transported from the cell surface back to the trans-Golgi network (TGN) and that two motifs of gB cytoplasmic tail, YTQV and LL, function distinctly in this process. To investigate the role of each of these gB trafficking signals in HSV-1 infection, we constructed recombinant viruses in which each motif was rendered nonfunctional by alanine mutagenesis. In infected cells, wild-type gB was internalized from the cell surface and concentrated in the TGN. Disruption of YTQV abolished internalization of gB during infection, whereas disruption of LL induced accumulation of internalized gB in early recycling endosomes and impaired its return to the TGN. The growth of both recombinants was moderately diminished. Moreover, the fusion phenotype of cells infected with the gB recombinants differed from that of cells infected with the wild-type virus. Cells infected with the YTQV-mutated virus displayed reduced cell-cell fusion, whereas giant syncytia were observed in cells infected with the LL-mutated virus. Furthermore, blocking gB internalization or impairing gB recycling to the cell surface, using drugs or a transdominant negative form of Rab11, significantly reduced cell-cell fusion. These results favor a role for endocytosis in virus replication and suggest that gB intracellular trafficking is involved in the regulation of cell-cell fusion.     

Blocking antibody access to neutralizing domains on glycoproteins involved in entry as a novel mechanism of immune evasion by herpes simplex virus type 1 glycoproteins C and E

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC) blocks complement activation, and glycoprotein E (gE) interferes with IgG Fc-mediated activities. While evaluating gC- and gE-mediated immune evasion in human immunodeficiency virus (HIV)-HSV-1-coinfected subjects, we noted that antibody alone was more effective at neutralizing a strain with mutations in gC and gE (gC/gE) than a wild-type (WT) virus. This result was unexpected since gC and gE are postulated to interfere with complement-mediated neutralization. We used pooled human immunoglobulin G (IgG) from HIV-negative donors to confirm the results and evaluated mechanisms of the enhanced antibody neutralization. We demonstrated that differences in antibody neutralization cannot be attributed to the concentrations of HSV-1 glycoproteins on the two viruses or to the absence of an IgG Fc receptor on the gC/gE mutant virus or to enhanced neutralization of the mutant virus by antibodies that target only gB, gD, or gH/gL, which are the glycoproteins involved in virus entry. Since sera from HIV-infected subjects and pooled human IgG contain antibodies against multiple glycoproteins, we determined whether differences in neutralization become apparent when antibodies to gB, gD, or gH/gL are used in combination. Neutralization of the gC/gE mutant was greatly increased compared that of WT virus when any two of the antibodies against gB, gD, or gH/gL were used in combination. These results suggest that gC and gE on WT virus provide a shield against neutralizing antibodies that interfere with gB-gD, gB-gH/gL, or gD-gH/gL interactions and that one function of virus neutralization is to prevent interactions between these glycoproteins.     

Rhesus and human cytomegalovirus glycoprotein L are required for infection and cell-to-cell spread of virus but cannot complement each other

Rhesus cytomegalovirus (RhCMV), the homolog of human cytomegalovirus (HCMV), serves as a model for understanding the pathogenesis of HCMV and for developing candidate vaccines. In order to develop a replication-defective virus as a vaccine candidate, we constructed RhCMV with glycoprotein L (gL) deleted. RhCMV gL was essential for viral replication, and virus with gL deleted could only replicate in cells expressing RhCMV gL. Noncomplementing cells infected with RhCMV with gL deleted released intact, noninfectious RhCMV particles that were indistinguishable from wild-type RhCMV by electron microscopy and could be rescued by treatment of cells with polyethylene glycol. In addition, noncomplementing cells infected with RhCMV with gL deleted produced levels of gB, the major target of neutralizing antibodies, at levels similar to those observed in cells infected with wild-type RhCMV. Since RhCMV and HCMV gL share 53% amino acid identity, we determined whether the two proteins could complement the heterologous virus. Cells transfected with an HCMV bacterial artificial chromosome with gL deleted yielded virus that could replicate in human cells expressing HCMV gL. This is the second HCMV mutant with an essential glycoprotein deleted that has been complemented in cell culture. Finally, we found that HCMV gL could not complement the replication of RhCMV with gL deleted and that RhCMV gL could not complement the replication of HCMV with gL deleted. These data indicate that RhCMV and HCMV gL are both essential for replication of their corresponding viruses and, although the two gLs are highly homologous, they are unable to complement each another.     

Antigenic variants of herpes simplex virus selected with glycoprotein-specific monoclonal antibodies.

Monoclonal antibodies specific for herpes simplex virus type 1 (HSV-1) glycoproteins were used to demonstrate that HSV undergoes mutagen-induced and spontaneous antigenic variation. Hybridomas were produced by polyethylene glycol-mediated fusion of P3-X63-Ag8.653 myeloma cells with spleen cells from BALB/c mice infected with HSV-1 (strain KOS). Hybrid clones were screened for production of HSV-specific neutralizing antibody. The glycoprotein specificities of the antibodies were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of radiolabeled infected-cell extracts. Seven hybridomas producing antibodies specific for gC, one for gB, and one for gD were characterized. All antibodies neutralized HSV-1 but not HSV-2. Two antibodies, one specific for gB and one specific for gC, were used to select viral variants resistant to neutralization by monoclonal antibody plus complement. Selections were made from untreated and bromodeoxyuridine- and nitrosoguanidine-mutagenized stocks of a plaque-purified isolate of strain KOS. After neutralization with monoclonal antibody plus complement, surviving virus was plaque purified by plating at limiting dilution and tested for resistance to neutralization with the selecting antibody. The frequency of neutralization-resistant antigenic variants selected with monoclonal antibody ranged from 4 X 10(-4) in nonmutagenized stocks to 1 X 10(-2) in mutagenized stocks. Four gC and four gB antigenic variants were isolated. Two variants resistant to neutralization by gC-specific antibodies failed to express gC, accounting for their resistant phenotype. The two other gC antigenic variants and the four gB variants expressed antigenically altered glycoproteins and were designated monoclonal-antibody-resistant, mar, mutants. The two mar C mutants were tested for resistance to neutralization with a panel of seven gC-specific monoclonal antibodies. The resulting patterns of resistance provided evidence for at least two antigenic sites on glycoprotein gC.     

Characterization of a Discontinuous Neutralizing Epitope on Glycoprotein B of Human Cytomegalovirus

Human cytomegalovirus (HCMV) is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and newborn infants infected in utero. The viral envelope glycoprotein B (gB) is an attractive molecule for active vaccination and passive immunoprophylaxis and therapy. Using human monoclonal antibodies (MAbs), we have recently identified antigenic region 4 (AD-4) on gB as an important target for neutralizing antibodies. AD-4 is formed by a discontinuous sequence comprising amino acids 121 to 132 and 344 to 438 of gB of HCMV strain AD169. To map epitopes for human antibodies on this protein domain, we used a three-dimensional (3D) model of HCMV gB to identify surface-exposed amino acids on AD-4 and selected juxtaposed residues for alanine scans. A tyrosine (Y) at position 364 and a lysine (K) at position 379 (the YK epitope), which are immediate neighbors on the AD-4 surface, were found to be essential for binding of the human MAbs. Recognition of AD-4 by sera from HCMV-infected individuals also was largely dependent on these two residues, indicating a general importance for the antibody response against AD-4. A panel of AD-4 recombinant viruses harboring mutations at the crucial antibody binding sites was generated. The viruses showed significantly reduced susceptibility to neutralization by AD-4-specific MAbs or polyclonal AD-4-specific antibodies, indicating that the YK epitope is dominant for the AD-4-specific neutralizing antibody response during infection. To our knowledge, this is the first molecular identification of a functional discontinuous epitope on HCMV gB. Induction of antibodies specific for this epitope may be a desirable goal following vaccination with gB.     

Cell surface expression of human cytomegalovirus (HCMV) gp55-116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response.

Cell surface expression of the human cytomegalovirus (HCMV) major envelope glycoprotein complex, gp55-116 (gB), was studied by using monoclonal antibodies and an HCMV gp55-116 (gB) recombinant vaccinia virus. HCMV-infected human fibroblasts and recombinant vaccinia virus-infected HeLa cells expresses three electrophoretically distinct proteins of Mr 170,000, 116,000, and 55,000 on their surface. These species have been previously identified within infected cells and purified virions. Two unique neutralizing epitopes were shown to be present on the cell surface gp55-116 (gB). Utilizing HeLa cells infected with the gp55-116 recombinant vaccinia virus as a specific immunosorbent, we have shown that approximately 40 to 70% of the total serum virus-neutralizing activity of a group of individuals with past HCMV infections was directed against this single envelope glycoprotein. The implications of this finding for vaccine development are discussed.     

Immunogenicity of herpes simplex virus glycoproteins gC and gB and their role in protective immunity.

The relative antigenicity of the individual herpes simplex virus type 1 (KOS) glycoproteins gC and gB was analyzed in BALB/c mice by using KOS mutants altered in their ability to present these antigens on cell surface membranes during infection. The mutants employed were as follows: syn LD70 , a non-temperature-sensitive mutant defective in the synthesis of cell surface membrane gC; tsF13 , a temperature-sensitive mutant defective in the processing of the precursor form of gB to the mature cell surface form at 39 degrees C; and ts606 , an immediate early temperature-sensitive mutant defective in the production of all early and late proteins including the glycoproteins. By comparing the relative susceptibility to immunolysis of mouse 3T3 cells infected at 39 degrees C with wild-type virus, presenting the full complement of the glycoprotein antigens, gC, gB, and gD, with target cells infected with mutants presenting only subsets of these antigens, we determined that a major portion of cytolytic antibody contained in hyperimmune anti-herpes simplex virus type 1 (KOS) mouse antiserum was directed against glycoproteins gC and gB. The relative immunogenicity of wild-type and mutant virus-infected cells also was compared in BALB/c mice. Immunogen lacking the mature form of gB induced a cytolytic antibody titer comparable to that of the wild-type virus, whereas that lacking the mature form of gC showed a 70% reduction in titer. The absence of the mature cell surface forms of gB and gC in immunogen preparations resulted in a 4- to 15-fold reduction in in virus neutralizing titer. Animals immunized with ts606 -infected cells (39 degrees C) induced relatively little virus-specific cytolytic and neutralizing antibody. Analysis of the glycoprotein specificities of these antisera by radioimmunoprecipitation showed that the antigens immunoprecipitated reflected the viral plasma membrane glycoprotein profiles of the immunogens. The absence of the mature forms of gC or gB in the immunizing preparation did not appreciably affect the immunoprecipitating antibody response to other antigens. Mice immunized with wild-type and mutant virus-infected cells were tested for their resistance to intracranial and intraperitoneal challenge with the highly virulent WAL strain of herpes simplex virus type 1. Despite the observed alterations in serum virus-specific antibody induced with the individual immunogens, all animals survived an intraperitoneal challenge of 10 50% lethal doses. However, differences in the survival of animals were obtained upon intracranial challenge.(ABSTRACT TRUNCATED AT 400 WORDS)     

Herpes simplex virus 1 protein kinase Us3 phosphorylates viral envelope glycoprotein B and regulates its expression on the cell surface

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). As reported here, we attempted to identify the previously unreported physiological substrate of Us3 in HSV-1-infected cells. Our results were as follows. (i) Bioinformatics analysis predicted two putative Us3 phosphorylation sites in the viral envelope glycoprotein B (gB) at codons 557 to 562 (RRVSAR) and codons 884 to 889 (RRNTNY). (ii) In in vitro kinase assays, the threonine residue at position 887 (Thr-887) in the gB domain was specifically phosphorylated by Us3, while the serine residue at position 560 was not. (iii) The phosphorylation of gB Thr-887 in Vero cells infected with wild-type HSV-1 was specifically detected using an antibody that recognized phosphorylated serine or threonine residues with arginine at the −3 and −2 positions. (iv) The phosphorylation of gB Thr-887 in infected cells was dependent on the kinase activity of Us3. (v) The replacement of Thr-887 with alanine markedly upregulated the cell surface expression of gB in infected cells, whereas replacement with aspartic acid, which sometimes mimics constitutive phosphorylation, restored the wild-type phenotype. The upregulation of gB expression on the cell surface also was observed in cells infected with a recombinant HSV-1 encoding catalytically inactive Us3. These results supported the hypothesis that Us3 phosphorylates gB and downregulates the cell surface expression of gB in HSV-1-infected cells.     

Linker-insertion nonsense and restriction-site deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1.

To study the effects of missense, nonsense, and deletion mutations of the gB glycoprotein gene of herpes simplex virus type 1, a gB-transformed cell line was isolated that, after virus infection, would express sufficient quantities of gB from the cellular chromosome to complement temperature-sensitive gB mutants. The transformed cell line was then used as a permissive cell to transfer two gB mutations from plasmid to viral DNA. One of the mutants, K082, harbored an HpaI linker insertion that introduced one new amino acid and a chain terminator codon within amino acid residue 43. The other mutant contained a 969-base-pair deletion in a part of the gene that includes the membrane-spanning region; a correspondingly shorter gB polypeptide was detected by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation of infected-cell extracts with four pooled monoclonal antibodies. No polypeptide was observed from K082-infected cells. The shortened gB polypeptide was efficiently processed and secreted into the growth medium. Each of the four monoclonal antibodies precipitated full-length gB, and three of the four precipitated the shortened polypeptide. Enveloped virus particles could be purified after infection of nonpermissive cells with either mutant virus. Virus particles appeared to possess normal polypeptide and glycopeptide profiles except for the absence of gB. Therefore, the presence of gB is not essential for viral assembly, including envelopment. Recombinants in virus stocks grown on the gB-transformed cells occurred at frequencies on the order of 10(-7) to 10(-5), compared with a frequency of approximately 10(-2) in mixed infections with the two mutants.