Syncytial Mutants Research Articles

Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site.

Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion.IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses.

The UL21 Tegument Protein of Herpes Simplex Virus 1 Is Differentially Required for the Syncytial Phenotype.

The initial goal of this study was to reexamine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in noncomplementing cells, even at a low multiplicity of infection (MOI), though a reduction in titer was observed. Also, increased proportions of empty capsids were observed in the cytoplasm, suggesting a role for UL21 in preventing their exit from the nucleus. Surprisingly, passage of the null mutant resulted in rapid outgrowth of syncytial (Syn) variants. This was unexpected because UL21 has been shown to be required for the Syn phenotype. However, earlier experiments made use of only the A855V syncytial mutant of glycoprotein B (gB), and the Syn phenotype can also be produced by substitutions in glycoprotein K (gK), UL20, and UL24. Sequencing of the syncytial variants revealed mutations in the gK locus, but UL21 was shown to be dispensable for UL20Syn and UL24Syn To test whether UL21 is needed only for the A855V mutant, additional gBSyn derivatives were examined in the context of the null virus, and all produced lytic rather than syncytial sites of infection. Thus, UL21 is required only for the gBSyn phenotype. This is the first example of a differential requirement for a viral protein across the four syn loci.IMPORTANCE UL21 is conserved among alphaherpesviruses, but its role is poorly understood. This study shows that HSV-1 can replicate without UL21, although the virus titers are greatly reduced. The null virus had greater proportions of empty (DNA-less) capsids in the cytoplasm of infected cells, suggesting that UL21 may play a role in retaining them in the nucleus. This is consistent with reports showing UL21 to be capsid associated and localized to the nuclei of infected cells. UL21 also appears to be needed for viral membrane activities. It was found to be required for virus-mediated cell fusion, but only for mutants that harbor syncytial mutations in gB (not variants of gK, UL20, or UL24). The machinery needed for syncytial formation is similar to that needed for direct spread of the virus through cell junctions, and these studies show that UL21 is required for cell-to-cell spread even in the absence of syncytial mutations.

RAGE deficiency predisposes mice to virus-induced paucigranulocytic asthma.

Asthma is a chronic inflammatory disease. Although many patients with asthma develop type-2 dominated eosinophilic inflammation, a number of individuals develop paucigranulocytic asthma, which occurs in the absence of eosinophilia or neutrophilia. The aetiology of paucigranulocytic asthma is unknown. However, both respiratory syncytial virus (RSV) infection and mutations in the receptor for advanced glycation endproducts (RAGE) are risk factors for asthma development. Here, we show that RAGE deficiency impairs anti-viral immunity during an early-life infection with pneumonia virus of mice (PVM; a murine analogue of RSV). The elevated viral load was associated with the release of high mobility group box-1 (HMGB1) which triggered airway smooth muscle remodelling in early-life. Re-infection with PVM in later-life induced many of the cardinal features of asthma in the absence of eosinophilic or neutrophilic inflammation. Anti-HMGB1 mitigated both early-life viral disease and asthma-like features, highlighting HMGB1 as a possible novel therapeutic target.

Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus.

Herpes simplex virus (HSV) is investigated not only as a human pathogen but also as a promising agent for oncolytic virotherapy. We previously showed that both the initial entry and subsequent lateral spread of HSV can be retargeted to cells expressing tumor-associated antigens by single-chain antibodies fused to a receptor-binding-deficient envelope glycoprotein D (gD). Here we introduced syncytial mutations into the gB and/or gK gene of gD-retargeted HSVs to determine whether viral tropism remained dependent on the interaction of gD with the target receptor. Entry and spread profiles of the recombinant viruses indicated that gD retargeting does not abolish the hyperfusogenic activity of syncytial mutations and that these mutations do not eliminate the dependence of HSV entry and spread on a specific gD-receptor interaction. These observations suggest that syncytial mutations may be valuable for increasing the tumor-specific spreading of retargeted oncolytic HSV vectors.

Herpes Simplex Virus 1 Glycoprotein M and the Membrane-Associated Protein UL11 Are Required for Virus-Induced Cell Fusion and Efficient Virus Entry

Herpes simplex virus 1 (HSV-1) facilitates virus entry into cells and cell-to-cell spread by mediating fusion of the viral envelope with cellular membranes and fusion of adjacent cellular membranes. Although virus strains isolated from herpetic lesions cause limited cell fusion in cell culture, clinical herpetic lesions typically contain large syncytia, underscoring the importance of cell-to-cell fusion in virus spread in infected tissues. Certain mutations in glycoprotein B (gB), gK, UL20, and other viral genes drastically enhance virus-induced cell fusion in vitro and in vivo. Recent work has suggested that gB is the sole fusogenic glycoprotein, regulated by interactions with the viral glycoproteins gD, gH/gL, and gK, membrane protein UL20, and cellular receptors. Recombinant viruses were constructed to abolish either gM or UL11 expression in the presence of strong syncytial mutations in either gB or gK. Virus-induced cell fusion caused by deletion of the carboxyl-terminal 28 amino acids of gB or the dominant syncytial mutation in gK (Ala to Val at amino acid 40) was drastically reduced in the absence of gM. Similarly, syncytial mutations in either gB or gK did not cause cell fusion in the absence of UL11. Neither the gM nor UL11 gene deletion substantially affected gB, gC, gD, gE, and gH glycoprotein synthesis and expression on infected cell surfaces. Two-way immunoprecipitation experiments revealed that the membrane protein UL20, which is found as a protein complex with gK, interacted with gM while gM did not interact with other viral glycoproteins. Viruses produced in the absence of gM or UL11 entered into cells more slowly than their parental wild-type virus strain. Collectively, these results indicate that gM and UL11 are required for efficient membrane fusion events during virus entry and virus spread.

Herpes simplex virus type-1(HSV-1) oncolytic and highly fusogenic mutants carrying the NV1020 genomic deletion effectively inhibit primary and metastatic tumors in mice

BackgroundThe NV1020 oncolytic herpes simplex virus type-1 has shown significant promise for the treatment of many different types of tumors in experimental animal models and human trials. Previously, we described the construction and use of the NV1020-like virus OncSyn to treat human breast tumors implanted in nude mice. The syncytial mutation gKsyn1 (Ala-to-Val at position 40) was introduced into the OncSyn viral genome cloned into a bacterial artificial chromosome using double-red mutagenesis in E. coli to produce the OncdSyn virus carrying syncytial mutations in both gB(syn3) and gK(syn1).ResultsThe OncdSyn virus caused extensive virus-induced cell fusion in cell culture. The oncolytic potential of the OncSyn and OncdSyn viruses was tested in the highly metastatic syngeneic mouse model system, which utilizes 4T1 murine mammary cancer cells implanted within the interscapular region of Balb/c mice. Mice were given three consecutive intratumor injections of OncSyn, OncdSyn, or phosphate buffered saline four days apart. Both OncSyn and OncdSyn virus injections resulted in significant reduction of tumor sizes (p < 0.05) compared to control tumors. Virus treated mice but not controls showed a marked reduction of metastatic foci in lungs and internal organs. Mouse weights were not significantly impacted by any treatment during the course of the entire study (p = 0.296).ConclusionThese results show that the attenuated, but highly fusogenic OncSyn and OncdSyn viruses can effectively reduce primary and metastatic breast tumors in immuncompetent mice. The available bac-cloned OncSyn and OncdSyn viral genomes can be rapidly modified to express a number of different anti-tumor and immunomodulatory genes that can further enhance their anti-tumor potency.

551. Effective Treatment of Primary and Metastatic Tumors in an Aggressive Xenograft Breast Mouse Model System with the NV1020-Like Virus OncDSyn Specifying Syncytial Mutations in Glycoproteins B and K

551. Effective Treatment of Primary and Metastatic Tumors in an Aggressive Xenograft Breast Mouse Model System with the NV1020-Like Virus OncDSyn Specifying Syncytial Mutations in Glycoproteins B and K

Effective Treatment of Human Breast Tumor in a Mouse Xenograft Model with Herpes Simplex Virus Type 1 Specifying the NV1020 Genomic Deletion and the gBsyn3 Syncytial Mutation Enabling High Viral Replication and Spread in Breast Cancer Cells

A new oncolytic and fusogenic herpes simplex virus type 1 (HSV-1) was constructed on the basis of the wildtype HSV-1(F) strain. To provide for safety and tumor selectivity, the virus carried a large deletion including one of the two alpha4, gamma(1)34.5, alpha0 genes and the latency-associated transcript region. The gamma(1)34.5 gene, a major neurovirulence factor, was replaced by a gene cassette constitutively expressing the red fluorescent protein gene. Homologous recombination was used to transfer the fusogenic gBsyn3 mutation to the viral genome to produce the OncSyn virus. OncSyn causes extensive virus-induced cell fusion (syncytia) and replicates to higher titers than the parental Onc and HSV-1(F) strains in breast cancer cells. Biochemical analysis revealed that the OncSyn virus retains a stable genome and expresses all major viral glycoproteins. A xenograft mouse model system using MDA-MB-435S-luc (MM4L) human breast cancer cells constitutively expressing the luciferase gene implanted within the interscapular region of animals was used to test the ability of the virus to inactivate breast tumor cells in vivo. Seventy-two mice bearing MM4L breast cancer xenografts were randomly divided into three groups and given two rounds of three consecutive intratumoral injections of OncSyn, inactivated OncSyn, or phosphate-buffered saline 3 days apart. A single round of virus injections resulted in a drastic reduction of tumor sizes (p <or= 0.0001) and diminution of chemiluminescence emitted by the cancer cells (p <or= 0.0002). This effect was enhanced by a second round of virus injections into the tumors 3 days after the first round (p <or= 0.0001). Systematic necropsy and pathological evaluation of the primary tumors revealed that the single round of injections resulted in extensive necrosis of tumor cells (p <or= 0.0001), which was enhanced by the second round of injections (p <or= 0.0002). Internal organs were not affected by virus inoculation. Mouse weights were not significantly impacted by any treatment during the course of the entire study (p = 0.46). These results show that the attenuated, fusogenic, and oncolytic HSV-1(F) virus strain OncSyn may effectively treat human breast tumors in vivo.

Identification of Functional Domains in Herpes Simplex Virus 2 Glycoprotein B

Glycoprotein B (gB) is one of four membrane proteins that are essential for the entry of herpes simplex viruses (HSV) into cells, and coexpression of the same combination of proteins in transfected cells results in cell fusion. The latter effect is reminiscent of the ability of virus infection to cause cell fusion, particularly since the degree of fusion is greatly increased by syncytial mutations in gB. Despite intensive efforts with the gB homologs of HSV and some other herpesviruses, information about functionally important regions in the 700-amino-acid ectodomain of this protein is very limited at present. This is largely due to the misfolding of the majority of the mutants examined. It was shown previously that the percentage of correctly folded mutants could be increased by targeting only predicted loop regions (i.e., not alpha-helix or beta-strand), and by using this approach new functional domains in HSV-2 gB have now been identified.

Pathogenic potentials of glycoprotein C-negative syncytial mutants from rabbit T cells infected persistently with herpes simplex virus type 1

Human T cell lymphotropic virus type I (HTLV-I)-transformed T cells of rabbits were infected persistently with Herpes simplex virus type 1 (HSV-1) strain KOS. These infected cells yielded syncytial mutants, either glycoprotein C (gC)-negative or -positive, which predominated over and replaced the wild-type virus in a long-term culture for 2 years. An alignment of nucleotide sequences showed multiple mutations in glycoprotein B (gB) and gC genes of these mutants, which are or may be responsible for the mutant phenotypes. One of four mutants analyzed produced extensively large syncytia and possessed point mutations within the cytoplasmic domain of gB. All four mutants possessed multiple point mutations in gC and two possessed single insertions which resulted in a frame shift, leading to the premature termination of the gC polypeptide chain. The supernatant of the 2-year culture of cells infected persistently, containing only gC-negative syncytial mutants, induced encephalitic symptoms in B/Jas inbred rabbits, when injected intravenously. One gC-negative syncytial isolate from an encephalitic lesion, together with those from the culture supernatant, were examined for pathogenic potential in vitro and in vivo. All these mutants were more cytotoxic and more susceptible to complement inactivation than the parental virus, and could infect and replicate in adrenal glands when injected intravenously into rabbits. Invasion into the central nervous system appeared to be blocked at the portal of entry, the adrenal gland, i.e., none exhibited neuroinvasive potential by itself. Syncytial gC-negative mutants could thus be pathogenic in rabbits.

Herpes Simplex Virus Type 1 gK Is Required for gB-Mediated Virus-Induced Cell Fusion, While neither gB and gK nor gB and UL20p Function Redundantly in Virion De-Envelopment

Multiple amino acid changes within herpes simplex virus type 1 (HSV-1) gB and gK cause extensive virus-induced cell fusion and the formation of multinucleated cells (syncytia). Early reports established that syncytial mutations in gK could not cause cell-to-cell fusion in the absence of gB. To investigate the interdependence of gB, gK, and UL20p in virus-induced cell fusion and virion de-envelopment from perinuclear spaces as well as to compare the ultrastructural phenotypes of the different mutant viruses in a syngeneic HSV-1 (F) genetic background, gB-null, gK-null, UL20-null, gB/gK double-null, and gB/UL20 double-null viruses were constructed with the HSV-1 (F) bacterial artificial chromosome pYEBac102. The gK/gB double-null virus YEbacDeltagBDeltagK was used to isolate the recombinant viruses gBsyn3DeltagK and gBamb1511DeltagK, which lack the gK gene and carry the gBsyn3 or gBamb1511 syncytial mutation, respectively. Both viruses formed small nonsyncytial plaques on noncomplementing Vero cells and large syncytial plaques on gK-complementing cells, indicating that gK expression was necessary for gBsyn3- and gBamb1511-induced cell fusion. Lack of virus-induced cell fusion was not due to defects in virion egress, since recombinant viruses specifying the gBsyn3 or gKsyn20 mutation in the UL19/UL20 double-null genetic background caused extensive cell fusion on UL20-complementing cells. As expected, the gB-null virus failed to produce infectious virus, but enveloped virion particles egressed efficiently out of infected cells. The gK-null and UL20-null viruses exhibited cytoplasmic defects in virion morphogenesis like those of the corresponding HSV-1 (KOS) mutant viruses. Similarly, the gB/gK double-null and gB/UL20 double-null viruses accumulated capsids in the cytoplasm, indicating that gB, gK, and UL20p do not function redundantly in membrane fusion during virion de-envelopment at the outer nuclear lamellae.

Identification of syncytial mutations in a clinical isolate of herpes simplex virus 2

Small polykaryocytes resulting from cell fusion are found in herpes simplex virus (HSV) lesions in patients, but their significance for viral spread and pathogenesis is unclear. Although syncytial variants causing extensive fusion in tissue culture can be readily isolated from laboratory strains, they are rarely found in clinical isolates, suggesting that extensive cell fusion may be deleterious in vivo. Syncytial mutations have previously been identified for several laboratory strains, but not for clinical isolates of HSV type 2. To address this deficiency, we studied a recent syncytial clinical isolate, finding it to be a mixture of two syncytial and one nonsyncytial strain. The two syncytial strains have novel mutations in glycoprotein B, and in vitro cell fusion assays confirmed that they are responsible for syncytium formation. This panel of clinical strains may be ideal for examining the effect of increased cell fusion on pathogenesis.

Genetic analysis of the herpes simplex virus type 1 UL20 protein domains involved in cytoplasmic virion envelopment and virus-induced cell fusion.

The herpes simplex virus type 1 UL20 protein (UL20p) is an important determinant for cytoplasmic virion morphogenesis and virus-induced cell fusion. To delineate the functional domains of the UL20 protein, we generated a panel of single and multiple (cluster) alanine substitutions as well as UL20p carboxyl-terminal truncations. The UL20 mutant genes could be broadly categorized into four main groups: Group I UL20 mutant genes complemented for both virus production and virus-induced cell fusion; Group II UL20 mutant genes did not complement for either virus-induced cell fusion or infectious virus production; Group III UL20 mutant genes complemented for virus-induced cell fusion to variable extents but exhibited substantially decreased ability to complement UL20-null infectious virus production; Group IV mutant genes complemented for infectious virus production but had variable effects on virus-induced cell fusion; this group included two mutants that efficiently complemented for gBsyn3, but not for gKsyn1, virus-induced cell fusion. In addition, certain recombinant viruses with mutations in either the amino or carboxyl termini of UL20p produced partially syncytial plaques on Vero cells in the absence of any other virally encoded syncytial mutations. These studies indicated that the amino and carboxyl termini of UL20p contained domains that functioned both in infectious virus production and virus-induced cell fusion. Moreover, the data suggested that the UL20p's role in virus-induced cell fusion can be functionally separated from its role in cytoplasmic virion morphogenesis and that certain UL20p domains that function in gB-syn3 virus-induced cell fusion are distinct from those functioning in gKsyn1 virus-induced cell fusion.

The herpes simplex virus type 1 UL20 protein modulates membrane fusion events during cytoplasmic virion morphogenesis and virus-induced cell fusion.

The herpes simplex virus type 1 (HSV-1) UL20 protein is an important determinant for virion morphogenesis and virus-induced cell fusion. A precise deletion of the UL20 gene in the HSV-1 KOS strain was constructed without affecting the adjacent UL20.5 gene. The resultant KOS/UL20-null virus produced small plaques of 8 to 15 cells in Vero cells while it produced wild-type plaques on the complementing cell line G5. Electron microscopic examination of infected cells revealed that the KOS/UL20-null virions predominantly accumulated capsids in the cytoplasm while a small percentage of virions were found as enveloped virions within cytoplasmic vacuoles. Recently, it was shown that UL20 expression was necessary and sufficient for cell surface expression of gK (T. P. Foster, X. Alvarez, and K. G. Kousoulas, J. Virol. 77:499-510, 2003). Therefore, we investigated the effect of UL20 on virus-induced cell fusion caused by syncytial mutations in gB and gK by constructing recombinant viruses containing the gBsyn3 or gKsyn1 mutations in a UL20-null genetic background. Both recombinant viruses failed to cause virus-induced cell fusion in Vero cells while they readily caused fusion of UL20-null complementing G5 cells. Ultrastructural examination of UL20-null viruses carrying the gBsyn3 or gKsyn1 mutation revealed a similar distribution of virions as the KOS/UL20-null virus. However, cytoplasmic vacuoles contained aberrant virions having multiple capsids within a single envelope. These multicapsid virions may have been formed either by fusion of viral envelopes or by the concurrent reenvelopment of multiple capsids. These results suggest that the UL20 protein regulates membrane fusion phenomena involved in virion morphogenesis and virus-induced cell fusion.

Overexpression of gK in gK-transformed cells collapses the Golgi apparatus into the endoplasmic reticulum inhibiting virion egress, glycoprotein transport, and virus-induced cell fusion

Intracellular transport and egress of alphaherpesviruses require the coordinate function of multiple proteins and glycoproteins. Recently, we showed that gK is expressed on infected cell surfaces and that gK cell-surface expression required the presence of the UL20 protein [J. Virol. 77 (2003), 499]. Overexpression of gK by gK-transformed cells blocked transport of enveloped virions from perinuclear spaces and inhibited virus-induced cell fusion caused by gK syncytial mutants [J. Virol. 69 (1995), 5401]. Therefore, we investigated whether altered synthesis and transport of gK was responsible for the observed gK-mediated interference phenomena. HSV-1 infection of the gK-transformed cell line Vero (gK9) caused a profound entrapment of gK in the endoplasmic reticulum and total inhibition of gK cell surface expression. In addition, gK drastically inhibited intracellular transport and maturation of gD and caused substantial defects in Golgi-dependent glycosylation of gB. Visualization of intracellular organelles via confocal microscopy revealed a profound collapse of the Golgi apparatus into the endoplasmic reticulum. These results were analogous to those observed in the presence of brefeldin A, a known Golgi disruptor. Therefore, virion entrapment within perinuclear spaces and inhibition of glycoprotein transport are due to gK-mediated collapse of the Golgi apparatus.

A single amino acid substitution in the cytoplasmic tail of the glycoprotein B of herpes simplex virus 1 affects both syncytium formation and binding to intracellular heparan sulfate

Herpes simplex virus 1 (HSV-1) (S) is a spontaneous syncytial mutant derived from the prototype HSV-1(F) after extensive plaque purification, and produces large syncytial plaques on Vero cells. Marker transfer experiments and DNA sequence analysis mapped the syncytial phenotype to a T–C base substitution at codon 787 of the cytoplasmic domain of mature gB, that results in Leu to Pro substitution and consequently belongs to the syn 3 locus. Both the cytoplasmic and the extracellular domains of gB are active in the fusion event since the addition of anti-gB monoclonal antibodies that recognize the extracellular domain of gB prevent HSV-1(S) induced cell fusion. Similarly, gD also participates in cell fusion since addition of anti-gD monoclonal antibodies also prevent HSV-1(S) induced cell fusion. Furthermore the glycoproteins B and D formed complexes in cells infected with mutant or wild type viruses. The amount of gB bound to total heparan sulfate is lower in the mutant than in the wild type strain. This difference becomes particularly profound when gB is associated with a portion of heparan sulfate intercalated to the membranes. The discrepancy in the binding of the mutant and wild type gB to heparan sulfate may be related to the mechanism of cell fusion induced by HSV-1(S).

Enterocin CRL35 inhibits late stages of HSV-1 and HSV-2 replication in vitro

The replication of herpes simplex virus (HSV) type 1 and 2 in Vero cells is inhibited in the presence of enterocin CRL35 (ECRL), a bacteriocin produced by Enterococcus faecium CRL35. Attempts to resolve the mode of action of ECRL indicate that virus adsorption and penetration are not affected. Instead, a late step of virus multiplication is hindered since the addition of 100 μg/ml of ECRL at 8 h post infection still causes a 90% inhibition of virus release. The effect of ECRL on HSV antigen expression was studied by immunofluorescence using a polyclonal serum and a monoclonal antibody against glycoprotein D (γ protein). These studies indicated that ECRL impeded the second round of infection, apparently as a consequence of the inhibition of glycoprotein D expression. The replication of syncytial mutants of HSV-1 was significantly inhibited at a ECRL concentration of 25 μg/ml. Both the percentage of fused cells and the polykaryocyte size were affected. Studies on the effect of ECRL on viral protein synthesis showed that in the presence of ECRL, HSV late γ proteins were not synthesized. From these findings, it is concluded that inhibition of HSV spreading by ECRL is due to the prevention of mainly late glycoprotein synthesis.

Truncation of herpes simplex virus type 2 glycoprotein B increases its cell surface expression and activity in cell-cell fusion, but these properties are unrelated.

Formation of small polykaryons by cell-cell fusion is characteristic of herpes simplex virus (HSV) lesions, but the great majority of viruses isolated from such lesions produce only limited cell fusion in tissue culture. Because of this, HSV laboratory strains that produce extensive cell fusion (syncytium formation) in culture are regarded as variants or mutants. Furthermore, the rarity of clinical isolates able to produce syncytia in culture suggests that extensive cell fusion is deleterious in vivo. Mutations that confer a syncytial phenotype can then be regarded as bypassing a mechanism that normally limits cell fusion. Determination of how these mutations, some of which are in the cytoplasmic tail of glycoprotein B (gB), lead to syncytium formation will likely reveal how fusion is controlled. Here we show the following. (i) Truncation of the cytoplasmic tail of HSV type 2 gB (gB-2) by a minimum of 25 residues or a maximum of 49 residues produces a syncytial phenotype. (ii) Truncation by 20 to 49 residues increases cell fusion when gB-2 is coexpressed with only gD-2, gH-2, and gL-2. (iii) Truncation by 25 or more residues removes a potential endocytosis motif and increases gB-2 cell surface expression. (iv) Mutation of this motif increases gB-2 cell surface expression but does not increase fusogenic activity, whereas mutation of another potential endocytosis motif does not increase surface expression but does increase fusogenic activity. Therefore, syncytial mutations in the cytoplasmic tail of gB-2 do not act by increasing cell surface levels of the protein.

Determination of the Transmembrane Topology of Herpes Simplex Virus Type 1 Glycoprotein K (gK)

Herpes simplex virus type 1 glycoprotein K (gK) plays an essential role in viral replication and cell fusion. gK is a very hydrophobic membrane protein that contains a signal sequence and several hydrophobic regions. It has been shown that mutations inducing cell fusion map to two distinct domains of gK, suggesting that these domains are functionally important. To understand the transmembrane topology of gK and the localization of these functional domains, we constructed a set of gK deletion, insertion, and truncation mutants and expressed these by in vitro translation in the presence of microsomal membranes. The transmembrane topology of gK was determined by examination of the post-translational processing and protease sensitivity of the mutant proteins. Our data demonstrate that gK contains three transmembrane domains (amino acids 125-139, 226-239, and 311-325). Another hydrophobic domain (amino acids 241-265), which is relatively less hydrophobic and much longer compared with the transmembrane sequences, is located in the extracellular loop. The analysis showed that the domains containing syncytial mutations are both ectodomains. They may interact with each other to form a complex tertiary structure that is critical for the biological function of gK.

Identification of the Fusion-from-without Determinants of Herpes Simplex Virus Type 1 Glycoprotein B

Fusion-from-without (FFWO) is the rapid induction of cell fusion at high multiplicities of infection and in the absence of viral protein synthesis. The ANG path strain and several other strains of herpes simplex virus type 1 (HSV-1) effectively cause FFWO of Vero cells. FFWO-inducing strains of HSV-1 contain syncytial mutations in the gB cytoplasmic domain; however, not all strains with such syncytial mutations cause FFWO. By characterization of recombinant viruses containing chimeric gB genes, it was shown that determinants in both the gB ectodomain and gB cytoplasmic domain control the FFWO phenotype of HSV-1. The complete nucleotide sequence of the ANG path gB gene was determined. Comparison of the predicted amino acid sequence of ANG path gB with other HSV-1 gB sequences showed that the gB genes of FFWO-inducing viruses must contain both syncytial mutations in the gB cytoplasmic domain and the fast rate-of-entry determinant at residue 553 in the gB ectodomain.