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Glioblastoma is a lethal primary brain tumor lacking effective therapy. The secluded onset site, combined with the infiltrative properties of this tumor, require novel targeted therapies. In this scenario, the use of oncolytic viruses retargeted to glioblastoma cells and able to spread across the tumor cells represent an intriguing treatment strategy. Here, we tested the specificity, safety and efficacy of R-613, the first oncolytic HSV fully retargeted to EGFRvIII, a variant of the epidermal growth factor receptor carrying a mutation typically found in glioblastoma. An early treatment with R-613 on orthotopically transplanted EGFRvIII-expressing human glioblastoma significantly increased the median survival time of mice. In this setting, the growth of human glioblastoma xenotransplants was monitored by a secreted luciferase reporter and showed that R-613 is able to substantially delay the development of the tumor masses. When administered as late treatment to a well-established glioblastomas, R-613 appeared to be less effective. Notably the uninfected tumor cells derived from the explanted tumor masses were still susceptible to R-613 infection ex vivo, thus suggesting that multiple treatments could enhance R-613 therapeutic efficacy, making R-613 a promising oncolytic HSV candidate for glioblastoma treatment.

Syncytium formation in cells that express herpes simplex virus glycoprotein B (gB), gD, gH, and gL is blocked by gK (E. Avitabile, G. Lombardi, and G. Campadelli-Fiume, J. Virol. 77: 6836-6844, 2003). Here, we report the results of two series of experiments. First, UL20 protein (UL20p) expression weakly inhibited cell-cell fusion. Coexpression of UL20p and gK drastically reduced fusion in a cell-line-dependent manner, with the highest inhibition in BHK cells. Singly expressed UL20p and gK localized at the endoplasmic reticulum and nuclear membranes. When they were coexpressed, both proteins relocalized to the Golgi apparatus. Remarkably, in cells that coexpressed UL20p and gK, the antifusion activity correlated with a downmodulation of gD, gB, gH, and gL cell surface expression. Second, gB Δ867 has a partial deletion in the cytoplasmic tail that removed endocytosis motifs. Whereas wild-type (wt) gB was internalized in vesicles lined with the endosomal marker Rab5, gB Δ867 was not internalized, exhibited enhanced cell surface expression, and was more efficient in mediating cell-cell fusion than wt gB. The antifusion activity of UL20p and gK was also exerted when gB Δ867 replaced wt gB in the cell fusion assay. These studies show that the gB C tail carries a functional endocytosis motif(s) and that the removal of the motif correlated with increased gB surface expression and increased fusion activity. We conclude that cell-cell fusion in wt-virus-infected cells is negatively controlled by at least two mechanisms. The novel mechanism described here involves the concerted action of UL20p and gK and correlates with a moderate but consistent reduction in the cell surface expression of the fusion glycoproteins. This mechanism is independent of the one exerted through endocytosis-mediated downmodulation of gB from the plasma membrane.

Glycoprotein D (gD) interacts with two alternative protein receptors, nectin1 and HveA, to mediate herpes simplex virus (HSV) entry into cells. Fusion of the envelope with the plasma membrane requires, in addition to gD, glycoproteins gB, gH, and gL. Coexpression of the four glycoproteins (gD, gB, gH, and gL) promotes cell-cell fusion. gD delivered in trans is also capable of blocking the apoptosis induced by gD deletion viruses grown either in noncomplementing cells (gD −/− ) or in complementing cells (gD −/+ ). While ectopic expression of cation-independent mannose-6 phosphate receptor blocks apoptosis induced by both stocks, other requirements differ. Thus, apoptosis induced by gD −/− virus is blocked by full-length gD (or two gD fragments reconstituting a full-length molecule), whereas ectopic expression of the gD ectodomain is sufficient to block apoptosis induced by gD −/+ virus. In this report we took advantage of a set of gD insertion-deletion mutants to map the domains of gD required to block apoptosis by gD −/− and gD −/+ viruses and those involved in cell-cell fusion. The mutations that resulted in failure to block apoptosis were the same for gD −/− and gD −/+ viruses and were located in three sites, one within the immunoglobulin-type core region (residues 125, 126, and 151), one in the upstream connector region (residues 34 and 43), and one in the C-terminal portion of the ectodomain (residue 277). A mutant that carried amino acid substitutions at the three glycosylation sites failed to block apoptosis but behaved like wild-type gD in all other assays. The mutations that inhibited polykaryocyte formation were located in the upstream connector region (residues 34 and 43), at the α1 helix (residue 77), in the immunoglobulin core and downstream regions (residue 151 and 187), and at the α3 helix (residues 243 and 246). Binding of soluble nectin1-Fc to cells expressing the mutant gDs was generally affected by the same mutations that affected fusion, with one notable exception (Δ277-310), which affected fusion without hampering nectin1 binding. This deletion likely identifies a region of gD involved in fusion activity at a post-nectin1-binding step. We conclude that whereas mutations that affected all functions (e.g., upstream connector region and residue 151) may be detrimental to overall gD structure, the mutations that affect specific activities identify domains of gD involved in the interactions with entry receptors and fusogenic glycoproteins and with cellular proteins required to block apoptosis. The evidence that glycosylation of gD is required for blocking apoptosis supports the conclusion that the interacting protein is the mannose-6 phosphate receptor.

The full-length cDNA of the murine homolog of human nectin1delta (mNectin1delta), also known as human poliovirus receptor related 1 (PRR1) or herpesvirus entry mediator C, was cloned and showed a90% identity with its human counterpart. mNectin1delta is expressed in some murine cell lines, exemplified by NIH 3T3 and L cells, and in murine tissues. It mediates entry of an extended range of herpes simplex virus (HSV) strains, porcine pseudorabies virus (PrV), and bovine herpesvirus 1. A soluble form of the mediator blocked infectivity in mNectin1delta and human nectin1delta (hNectin1delta)-expressing cells, suggesting a physical interaction of the mediator with virions. The higher concentrations of soluble mNectin1 required to block infectivity relative to soluble hNectin1 suggest that the target of the two molecules is not identical. Entry of HSV, but not PrV, was blocked by soluble mNectin1delta in NIH 3T3 and L cells. Two features were unexpected. First, soluble mNectin1delta failed to physically interact with HSV glycoprotein D (gD) at a detectable level, although it interacted physically with virions. Second, coexpression of mNectin1delta and HSV gD did not restrict HSV or PrV infection, whereas coexpression of hNectin and gD did restrict infection, suggesting that mNectin1delta fails to be sequestered by HSV gD. We conclude that mNectin1delta serves as a species-nonspecific mediator for entry of the human and animal alphaherpesviruses. This activity, at least for HSV, is independent of a detectable binding to gD.

Earlier studies have shown that Kaposi sarcomas contain cells infected with human herpesvirus (HHV) 6B, and in current studies we report that both AIDS-associated and classic-sporadic Kaposi sarcoma contain HHV-7 genome sequences detectable by PCR. To determine the distribution of HHV-7-infected cells relative to those infected with HHV-6, sections from paraffin-embedded tissues were allowed to react with antibodies to HHV-7 virion tegument phosphoprotein pp85 and to HHV-6B protein p101. The antibodies are specific for HHV-7 and HHV-6B, respectively, and they retained reactivity for antigens contained in formalin-fixed, paraffin-embedded tissue samples. We report that (i) HHV-7 pp85 was present in 9 of 32 AIDS-associated Kaposi sarcomas, and in 1 of 7 classical-sporadic HIV-negative Kaposi sarcomas; (ii) HHV-7 pp85 was detected primarily in cells bearing the CD68 marker characteristic of the monocyte/macrophage lineage present in or surrounding the Kaposi sarcoma lesions; and (iii) in a number of Kaposi sarcoma specimens, tumor-associated CD68+monocytes/macrophages expressed simultaneously antigens from both HHV-7 and HHV-6B, and therefore appeared to be doubly infected with the two viruses. CD68+monocytes/macrophages infected with HHV-7 were readily detectable in Kaposi sarcoma, but virtually absent from other normal or pathological tissues that harbor macrophages. Because all of the available data indicate that HHV-7 infects CD4+T lymphocytes, these results suggest that the environment of the Kaposi sarcoma (i) attracts circulating peripheral lymphocytes and monocytes, triggers the replication of latent viruses, and thereby increases the local concentration of viruses, (ii) renders CD68+monocytes/macrophages susceptible to infection with HHV-7, and (iii) the combination of both events enables double infections of cells with both HHV-6B and HHV-7.

A monoclonal antibody, designated as MAb 6E2, specific for human herpesvirus 6 variant B (HHV-6B) was derived from the spleen of a mouse immunized with lysates of HHV-6B(Z29) cord blood mononuclear cells. MAb 6E2 reacts by immunofluorescence with all the HHV-6B strains tested (Z29, CV, Hashimoto and SF) and fails to react with variant A prototypes, GS and U1102. The immunofluorescence staining was punctate and localized to the cytoplasm. The protein reacting with MAb 6E2 was identified as protein 48 000 in apparent Mr value by immunoaffinity chromatography of lysates of HHV-6B-infected mononuclear cells. © 1995.

Herpes simplex virus (HSV) entry into the cell involves the fusion of the virion envelope with a cellular membrane and delivery of capsid and tegument proteins to the cytoplasm. Our understanding of this phenomenon has greatly increased in recent years. On the virus side, the multipartite nature of the entry-fusion machinery (made of the glycoproteins gD, the heterodimer gH/gL and gB) entails a mechanism of gD activation promoted by the gD encounter with one of its receptor; and cross-talk among the entry-fusion glycoproteins, which culminates in gB activation and fusion execution. On the cell side, machineries and signalling activities are put in place. The number of known receptors and sentinels is increasing. The cell routes the virus through alternative entry pathways by means of routing factors, exemplified by αVβ3-integrin and paired immunoglobulin-like type 2 receptor alpha. Of the signalling events, a key one is the immediate host response to incoming virions. Unexpectedly, this is in part triggered by the same virion components and some cellular factors that also promote virus entry. Hence, a link is emerging between two phenomena so far considered as distinct.

The gD, gB, and gH/gL glycoprotein quartet constitutes the basic apparatus for herpes simplex virus (HSV) entry into the cell and fusion. gD serves as a receptor binding glycoprotein and trigger of fusion. The conserved gB and gH/gL execute fusion. Central to understanding HSV entry/fusion has become the dissection of how the four glycoproteins engage in cross talk. While the independent interactions of gD with gB and gD with gH/gL have been documented, less is known of the interaction of gB with gH/gL. So far, this interaction has been detected only in the presence of gD by means of a split green fluorescent protein complementation assay. Here, we show that gB interacts with gH/gL in the absence of gD. The gB-gH/gL complex was best detected with a form of gB in which the endocytosis and phosphorylation motif have been deleted; this form of gB persists in the membranes of the exocytic pathway and is not endocytosed. The gB-gH/gL interaction was detected both in whole transfected cells by means of a split yellow fluorescent protein complementation assay and, biochemically, by a pull-down assay. Results with a panel of chimeric forms of gB, in which portions of the glycoprotein bracketed by consecutive cysteines were replaced with the corresponding portions from human herpesvirus 8 gB, favor the view that gB carries multiple sites for interaction with gH/gL, and one of these sites is located in the pleckstrin-like domain 1 carrying the bipartite fusion loop.

Cell lines that constitutively express herpes simplex virus 1 (HSV-1) glycoprotein H (gH-1 ) failed to synthesize the mature form of gH and accumulated a precursor-like form of the glycoprotein, which was retained intracellularly, most likely in RER. Fine-structure analysis of the oligosaccharides present in recombinant gH revealed oligosaccharides processed by RER enzymes; sialylated complex-type and biantennary oligosaccharides, which are assembled in the trans-Golgi, were absent. A small fraction had the characteristics of oligosaccharides processed by the early mannosidases of the Golgi. These findings suggest that a defect in the transport out of RER to the Golgi may account for the intracellular retention of the immature form of gH in cells that express the glycoprotein constitutively. Upon superinfection of cells expressing gH-1 with HSV-2, recombinant gH-1 underwent maturation, indicating that a viral function is required to attain full processing of gH. The known HSV glycoproteins do not appear to carry out this function, since in cells infected with deletion mutants in gD, gG, gE, and gE-gl, with a spontaneous gC- mutant, or with a temperature-sensitive mutant in gB, maturation of gH occurred independently of the presence or of the maturation of the single glycoproteins tested. The present findings together with previous observations on HSV, human CMV, and the EBV homologue of gH suggest that inability of gH to undergo full processing in the absence of viral protein (s) is a property of gH.

The multipartite entry-fusion system of herpes simplex virus is made of a quartet of glycoproteins-gD, gB, gH.gL-and three alternative gD receptors, herpesvirus entry mediator (HVEM), nectin1 and modified sites on heparan sulphate. This multipartite system recapitulates the basic steps of virus-cell fusion, i.e. receptor recognition, triggering of fusion and fusion execution. Specifically, in addition to serving as the receptor-binding glycoprotein, gD triggers fusion through a specialised domain, named pro-fusion domain (PFD), located C-terminally in the ectodomain. In the unliganded gD the C-terminal region folds around the N-terminal region, such that gD adopts a closed autoinhibited conformation. In HVEM- and nectin1-bound gD the C-terminal region is displaced (opened conformation). gD is the tool for modification of HSV tropism, through insertion of ligands to heterologous tumour-specific receptors. It is discussed whether gD responds to the interaction with the natural and the heterologous receptors by adopting similar conformations, and whether the closed-to-open switch in conformation is a generalised mechanism of activation. A peculiar recombinant highlighted that the central Ig-folded core of gD may not encode executable functions for entry and that the 219-314 aa segment may be sufficient to trigger fusion. With respect to fusion execution, gB appears to be a prospective fusogen based on its coiled-coil trimeric structure, similar to that of another fusion glycoprotein. On the other hand, gH exhibits molecular elements typical of class 1 fusion glycoproteins, in particular heptad repeats and strong tendency to interact with lipids. Whether fusion execution is carried out by gB or gH.gL, or both glycoproteins in complex or sequentially remains to be determined.

A Myc epitope was inserted at residue 283 of herpes simplex virus type 1 (HSV-1) glycoprotein K (gK), a position previously shown not to interfere with gK activity. The Myc-tagged gK localized predominantly to the endoplasmic reticulum, both in uninfected and in HSV-infected cells. gK, coexpressed with the four HSV fusogenic glycoproteins, gD, gB, gH, and gL, inhibited cell-cell fusion. The effect was partially dose dependent and was observed both in baby hamster kidney (BHK) and in Vero cells, indicating that the antifusion activity of gK may be cell line independent. The antifusion activity of gK did not require viral proteins other than the four fusogenic glycoproteins. A syncytial ( syn ) allele of gK ( syn -gK) carrying the A40V substitution present in HSV-1(MP) did not block fusion to the extent seen with the wild-type (wt) gK, indicating that the syn mutation ablated, at least in part, the antifusogenic activity of wt gK. We conclude that gK is part of the mechanism whereby HSV negatively regulates its own fusion activity. Its effect accounts for the notion that cells infected with wt HSV do not fuse with adjacent, uninfected cells into multinucleated giant cells or syncytia. gK may also function to preclude fusion between virion envelope and the virion-encasing vesicles during virus transport to the extracellular compartment, thus preventing nucleocapsid de-envelopment in the cytoplasm.

A novel member of the nectin family, nectin1γ, was molecularly cloned. The cDNA has the same ectodomain as nectin1α and nectin1β, the two known transmembrane isoforms that serve as receptors for herpes simplex virus (HSV) entry into human cell lines (nectin1α and nectin1β, also called PRR1-HveC and HIgR, respectively). The 1.4-kb transcript, which originated by alternative splicing, is expressed in human cell lines, and appears to have a narrow distribution in human tissues. The sequence does not have a hydrophobic anchoring region, and the protein is secreted in the culture medium of cells transfected with the cDNA. Nectin1γ, purified from culture medium, can compete with membrane-bound nectin1β and reduce HSV infectivity. The expression of nectin1γ cDNA in cells resistant to HSV infection and lacking HSV receptors enables HSV to enter the cell, which implies that it is present at the cell surface. Thus, nectin1γ has the potential both to mediate and to reduce HSV entry into cells.

The murine nectin1alpha (mNectin1alpha), a homolog of human nectin1alpha (hNectin1alpha, or PRR1, HveC), mediates the entry of herpes simplex virus (HSV) into cells. Previously, we reported that the binding of hNectin1 to HSV glycoprotein D (gD) was readily detectable, whereas the binding of mNectin1 to gD was not detectable, thus raising the question whether mNectin1 mediates a gD-dependent or a gD-independent pathway of entry. Here we report comparative binding studies of murine- and human-nectin1alpha to virions and to gD. The assays consistently showed either a very weak binding or no detectable binding of murine nectin1alpha to gD. They included (i) binding of soluble mNectin1-Fc or hNectin1-Fc to virions and competition of the binding by soluble gD(Delta290-299t) and by monoclonal antibodies to gD; (ii) pull-down experiments of wt gD from lysates of infected cells; and (iii) ELISA binding of soluble gD(Delta290-299t) to cells expressing mNectin1 or hNectin1. In contrast to the binding studies, the entry studies readily showed that entry mediated by mNectin1 was dependent on gD. Thus, a gDnull (gD-/-) mutant virus was unable to enter mNectin1-expressing cells, and entry of wild-type virus was inhibited by antibodies to gD or soluble gD at similar concentrations. We infer that gD represents a weak ligand in the interaction between mNectin1 and virions, whereas it represents a strong and the major ligand for hNectin1. Yet gD is required in HSV-1 entry mediated by mNectin1alpha. We conclude that a high-affinity binding of the receptor to gD is not a requirement in the gD-dependent pathway of HSV entry to cells.

The receptors for entry of herpes simplex viruses 1 and 2 (HSV-1 and -2), widely expressed in human cell lines, are members of a subset of the immunoglobulin superfamily exemplified by herpesvirus entry mediator C (HveC) and the herpesvirus immunoglobulin-like receptor (HIgR). This report focuses on two members of this subset, herpesvirus entry mediator B (HveB), recently designated nectin2/PRR2α, and its splice variant isoform, nectin2/PRR2δ. Nectin2α and -δ share the ectodomain but differ in the transmembrane and cytoplasmic regions. HveB was reported to enable entry of HSV-1 carrying mutations in glycoprotein D (gD) and of HSV-2, but not of wild-type (wt) HSV-1. We report that (i) both nectin2α and -δ served as receptors for the entry of HSV-1 mutant viruses HSV-1(U10) and -(U21) and AP7 r that carry the Leu25Pro substitution in gD but not for HSV-1 mutants U30 and R5000 that carry the Ser140 or Ala185 substitution in gD. All of these mutants were able to overcome the block to entry mediated by expression of wt gD. (ii) Infection of cells expressing nectin2α or -δ required exposure to multiplicities of infection about 100-fold higher than those required to infect cells expressing HveC or HIgR. (iii) gD from HSV-1(U21) bound in vitro soluble forms of nectin2. The association was weaker than that to the soluble form of HveC/HIgR. Binding of wt HSV-1 gD to soluble nectin2 was not detectable. (iv) A major region of nectin2 functional in virus entry mapped to the V domain, located at the N terminus.

Earlier studies have shown that the Golgi apparatus was fragmented and dispersed in herpes simplex virus 1-infected Vero and HEp-2 cells but not in human 143TK- cells, that the fragmentation and dispersal required viral functions expressed concurrently with or after the onset of DNA synthesis (G. Campadelli-Fiume, R. Brandimarti, C. Di Lazzaro, P. L. Ward, B. Roizman, and M. R. Torrisi, Proc. Natl. Acad. Sci. USA 90:2798-2802, 1993), and that in 143TK- cells, but not Vero or HEp-2 cells, infected with viral mutants lacking the UL20 gene virions were glycosylated and transported to extracellular space (J. D. Baines, P. L. Ward, G. Campadelli-Fiume, and B. Roizman, J. Virol. 65:6414-6424, 1991; E. Avitabile, P. L. Ward, C. Di Lazzaro, M. R. Torrisi, B. Roizman, and G. Campadelli-Fiume, J. Virol. 68:7397-7405, 1994). Experiments designed to elucidate the role of the microtubules and of intact or fragmented Golgi apparatus in the exocytosis of virions showed the following. (i) In all cell lines tested (Vero, 143TK-, BHK, and Hep-2) microtubules underwent fragmentation particularly evident at the cell periphery and then reorganized into bundles which circumvent the nucleus. This event was not affected by inhibitors of viral DNA synthesis. We conclude that redistribution of microtubules may be required but is not sufficient for the fragmentation and dispersal of the Golgi apparatus. (ii) In all infected cell lines tested, nocodazole caused fragmentation and dispersal of the Golgi and a far more extensive depolymerization of the microtubules than was seen in untreated, infected Vero or HEp-2 cells. Taxol precluded the depolymerization of the microtubules and fragmentation of the Golgi in both infected cell lines. Neither nocodazole nor taxol affected the exocytosis of infectious virus from Vero, HEp-2, or 143TK- cells infected with wild-type virus. We conclude that the effects of nocodazole or of taxol are dominant over the effects of viral infection in the cell lines tested and that viral exocytosis is independent of the organization of microtubules or of the integrity of the Golgi apparatus. Lastly, the data suggest that herpes simplex viruses have evolved an exocytic pathway for which the UL20 protein is a component required in some cells but not others and in which this protein does not merely compensate for the fragmentation and dispersal of the Golgi apparatus.

Hyperimmune rabbit and mouse sera raised to human herpesvirus 7 (HHV-7)-infected cells and an immune human serum identified 20 [35S]methionine-[35S]cysteine-labelled proteins specific for HHV-7-infected cord blood mononuclear cells, ranging in apparent M(r) from 136K to 30K. The major proteins had apparent M(r) values of 121K, 100K, 87K, 85K, 60K, 51K, 46K, 42K, 40K and 36K. The human serum also identified seven [3H]glucosamine-labelled glycoproteins, with apparent M(r) values of 100K, 89K, 82K, 67K, 63K, 53K and 41K. Four monoclonal antibodies (MAbs) specific for HHV7-infected cells were derived. Two reacted with a family of five antigenically related polypeptides (87K, 85K, 70K, 61K and 57K in apparent M(r)), designated as the p85 complex. Two reacted with 121K and 51K M(r) proteins designated as p121 and p51, respectively. Human sera react with high frequency with the p85 complex and to a lesser extent with p121; hence these two proteins appear to be immunodominant for both humans and laboratory animals. The hyperimmune mouse serum and some of the MAbs showed some cross-reactivity with HHV-6A(U1102)- and 6B(Z29)-infected cells. The implications of cross-reactivity with respect to the human immune response to HHV-6 and -7 infections and prevalence analyses are discussed.

The UL20 protein of herpes simplex virus 1, an intrinsic membrane protein, is required in infected Vero cells in which the Golgi apparatus is fragmented for the transport of virions from the space between the inner and outer nuclear membranes and for the transport of fully processed cell membrane-associated glycoproteins from the trans-Golgi to the plasma membrane. It is not required in the human 143TK- cell line, in which the Golgi apparatus remains intact. We report the following. (i) The UL20 protein was detected in infected cells beginning at 6 h postinfection and was regulated as a gamma 1 gene. (ii) Pulse-chase experiments revealed no detectable alteration in the mobility of the UL20 protein in polyacrylamide gels. (iii) In both infected Vero and infected 143TK- cells, the UL20 protein was detected by immunofluorescence in association with nuclear membranes and in the cytoplasm. Some of the cytoplasmic fluorescence colocalized with beta-COP, a protein associated with Golgi-derived transport vesicles. UL20 protein was present in virions purified from the extracellular space but could not be detected in the plasma membrane. These results are consistent with the hypothesis that UL20 is a component of virion envelopes and membranes of virion transport vesicles and is selectively retained from the latter in a Golgi compartment.

Pulse-chase experiments in conjunction with quantitative immunoprecipitation have been used to study the time-course of conversion from precursor to mature form of herpes simplex virus 1 glycoproteins C, D and B (gC, gD, and gB). The experimental systems employed were two infected cell lines and cells that constitutively express gD or gB. The relative rates of conversion among the glycoproteins did not vary in the systems used; the rate of maturation of gC was about two-fold higher than that of gD which, in turn, was about one and a half-fold higher than that of gB. Treatment with phosphonoacetate which inhibits viral DNA synthesis and hence virion morphogenesis induced a striking increase in the time course of conversion of immature gC, gD, and gB to fully glycosylated forms when measured late in the infection. The model of HSV glycoproteins maturation as integral components of the virion envelope is discussed. © 1988.

Spontaneous small polykaryocytes were detected in a cell line designated BJ-o that harbors the Bam HI J fragment of herpes simplex virus 1 DNA and expresses constitutively glycoprotein D (gD). The fusion activity of BJ-o cells correlated with gD production and was drastically reduced following exposure of the cells to monoclonal antibody HD1 to gD. Studies on the characteristics and requirements of cell fusion dependent on gD led to the conclusion that the characteristics and requirements for gD-mediated fusion activity of BJ-o cells are similar to those previously reported for cell fusion induced by the virus in that (i) polykaryocytosis was not augmented by exposure to medium of low pH with or without prior exposure to trypsin, (ii) the number of polykaryocytes was reduced following removal of terminal sialic acid residues by neuraminidase, and (iii) the number of polykaryocytes was augmented by masking of high-mannose N-linked oligosaccharides with concanavalin A or with its reduced form, succinyl concanavalin A. This effect was reversed by competition with man nose.

Earlier studies have shown that herpes simplex viruses adsorb to but do not penetrate permissive baby hamster kidney clonal cell lines designated the BJ series and constitutively expressing the herpes simplex virus 1 (HSV-1) glycoprotein D (gD). To investigate the mechanism of the restriction, the following steps were done. First, wild-type HSV-1 strain F [HSV-1(F)] virus was passaged blindly serially on clonal line BJ-1 and mutant viruses [HSV-1(F)U] capable of penetration were selected. The DNA fragment capable of transferring the capacity to infect BJ cells by marker transfer contains the gD gene. The mutant gD, designated gDU, differed from wild-type gD only in the substitution of Leu-25 by proline. gDU reacted with monoclonal antibodies which neutralize virus and whose epitopes encompass known functional domains involved in virus entry into cells. It did not react with the monoclonal antibody AP7 previously shown to react with an epitope which includes Leu-25. Second, cell lines expressing gDU constitutively were constructed and cloned. Unlike the clonal cell lines constitutively expressing gD (e.g., the BJ cell line), those expressing gDU were infectable by both HSV-1(F) and HSV-1(F)U. Lastly, exposure of BJ cells to monoclonal antibody AP7 rendered the cells capable of being infected with HSV-1(F). The results indicate that (i) gD expresses a specific function, determined by sequences at or around Leu-25, which blocks entry of virus into cells synthesizing gD, (ii) the gD which blocks penetration by superinfecting virus is located in the plasma membrane, (iii) the target of the restriction to penetration is the identical domain of the gD molecule contained in the envelope of the superinfecting virus, and (iv) the molecular basis of the restriction does not involve competition for a host protein involved in entry, as was previously thought.