Herpes simplex virus type 1 (HSV-1)-induced cell fusion is mediated by viral glycoproteins and other membrane proteins expressed on infected cell surfaces. Certain mutations in the carboxyl terminus of HSV-1 glycoprotein B (gB) and in the amino terminus of gK cause extensive virus-induced cell fusion. Although gB is known to be a fusogenic glycoprotein, the mechanism by which gK is involved in virus-induced cell fusion remains elusive. To delineate the amino-terminal domains of gK involved in virus-induced cell fusion, the recombinant viruses gK⌬31-47, gK⌬31-68, and gK⌬31-117, expressing gK carrying in-frame deletions spanning the amino terminus of gK immediately after the gK signal sequence (amino acids [aa] 1 to 30), were constructed. Mutant viruses gK⌬31-47 and gK⌬31-117 exhibited a gK-null (⌬gK) phenotype characterized by the formation of very small viral plaques and up to a 2-log reduction in the production of infectious virus in comparison to that for the parental HSV-1(F) wild-type virus. The gK⌬31-68 mutant virus formed substantially larger plaques and produced 1-log-higher titers than the gK⌬31-47 and gK⌬31-117 mutant virions at low multiplicities of infection. Deletion of 28 aa from the carboxyl terminus of gB (gB⌬28syn) caused extensive virus-induced cell fusion. However, the gB⌬28syn mutation was unable to cause virus-induced cell fusion in the presence of the gK⌬31-68 mutation. Transient expression of a peptide composed of the amino-terminal 82 aa of gK (gKa) produced a glycosylated peptide that was efficiently expressed on cell surfaces only after infection with the HSV-1(F), gK⌬31-68, ⌬gK, or UL20-null virus. The gKa peptide complemented the gK⌬31-47 and gK⌬31-68 mutant viruses for infectious-virus production and for gK⌬31-68/gB⌬28syn-mediated cell fusion. These data show that the amino terminus of gK modulates gB-mediated virus-induced cell fusion and virion egress.Herpes simplex virus type 1 (HSV-1) specifies at least 11 virally encoded glycoproteins, as well as several nonglycosylated and lipid-anchored membrane-associated proteins, which serve important functions in virion infectivity and virus spread. Although cell-free enveloped virions can efficiently spread viral infection, virions can also spread by causing cell fusion of adjacent cellular membranes. Virus-induced cell fusion, which is caused by viral glycoproteins expressed on infected cell surfaces, enables transmission of virions from one cell to another, avoiding extracellular spaces and exposure of free virions to neutralizing antibodies (reviewed in reference 56). Most mutations that cause extensive virus-induced cell-to-cell fusion (syncytial or syn mutations) have been mapped to at least four regions of the viral genome: the UL20 gene (5,42,44); the UL24 gene (37, 58); the UL27 gene, encoding glycoprotein B (gB) (9, 51); and the UL53 gene, coding for gK (7,15,35,53,54,57).Increasing evidence suggests that virus-induced cell fusion is mediated by the concerted action of glycoproteins gD, gB, and gH/gL. Recent studies have shown tha...

We have shown that glycoprotein K (gK) and its interacting partner, the UL20 protein, play crucial roles in virion envelopment. Specifically, virions lacking either gK or UL20 fail to acquire an envelope, thus causing accumulation of capsids in the cytoplasm of infected cells. The herpes simplex virus 1 (HSV-1) UL37 protein has also been implicated in cytoplasmic virion envelopment. To further investigate the role of UL37 in virion envelopment, the recombinant virus DC480 was constructed by insertion of a 12-amino-acid protein C (protC) epitope tag within the UL37 amino acid sequence immediately after amino acid 480. The DC480 mutant virus expressed full-size UL37 as detected by the anti-protC antibody in Western immunoblots, accumulated unenveloped capsids in the cytoplasm of infected cells, and produced very small plaques on African green monkey kidney (Vero) cells that were similar in size to those produced by the UL20-null and UL37-null viruses. The DC480 virus replicated nearly 4 log less efficiently than the parental wild-type virus when grown on Vero cells. However, DC480 mutant virus titers increased nearly 20-fold when the virus was grown on FRT cells engineered to express the UL20 gene in comparison to the titers on Vero cells, while the UL37-null virus replicated approximately 20-fold less efficiently than the DC480 virus on FRT cells. Coimmunoprecipitation experiments and proximity ligation assays showed that gK and UL20 interact with the UL37 protein in infected cells. Collectively, these results indicate that UL37 interacts with the gK-UL20 protein complex to facilitate cytoplasmic virion envelopment. IMPORTANCEHerpes simplex viruses acquire their final envelopes by budding into cytoplasmic membranes derived from the trans-Golgi network (TGN). The tegument proteins UL36 and UL37 are known to be transported to the TGN sites of virus envelopment and to function in virion envelopment, since mutants lacking UL37 accumulate capsids in the cytoplasm that are unable to bud into TGN membranes. Viral glycoprotein K (gK) also functions in cytoplasmic envelopment, in a protein complex with the membrane-associated protein UL20 (UL20mp). This work shows for the first time that the UL37 protein functionally interacts with gK and UL20 to facilitate cytoplasmic virion envelopment. This work may lead to the design of specific drugs that can interrupt UL37 interactions with the gK-UL20 protein complex, providing new ways to combat herpesviral infections.

Herpes simplex virus 1 (HSV-1) viral glycoproteins gD (carboxyl terminus), gE, gK, and gM, the membrane protein UL20, and membrane-associated protein UL11 play important roles in cytoplasmic virion envelopment and egress from infected cells. We showed previously that a recombinant virus carrying a deletion of the carboxyl-terminal 29 amino acids of gD (gD⌬ct) and the entire gE gene ( Overall, comparisons of single, double, and triple mutant viruses generated in the same HSV-1(F) genetic background indicated that lack of either UL20 or gK expression caused the most severe defects in cytoplasmic envelopment, egress, and infectious virus production, followed by the double deletion of UL11 and gM.

Oncolytic virotherapy is a promising new tool for cancer treatment, but direct lytic destruction of tumor cells is not sufficient and must be accompanied by strong immune activation to elicit anti-tumor immunity. We report here the creation of a novel replication-competent recombinant oncolytic herpes simplex virus type 1 (VG161) that carries genes coding for IL-12, IL-15, and IL-15 receptor alpha subunit, along with a peptide fusion protein capable of disrupting PD-1/PD-L1 interactions. The VG161 virus replicates efficiently and exhibits robust cytotoxicity in multiple tumor cell lines. Moreover, the encoded cytokines and the PD-L1 blocking peptide work cooperatively to boost immune cell function. In vivo testing in syngeneic CT26 and A20 tumor models reveals superior efficacy when compared to a backbone virus that does not express exogenous genes. Intratumoral injection of VG161 induces abscopal responses in non-injected distal tumors and grants resistance to tumor re-challenge. The robust anti-tumor effect of VG161 is associated with T cell and NK cell tumor infiltration, expression of Th1 associated genes in the injection site, and increased frequency of splenic tumor-specific T cells. VG161 also displayed a superb safety profile in GLP acute and repeated injection toxicity studies performed using cynomolgus monkeys. Overall, we demonstrate that VG161 can induce robust oncolysis and stimulate a robust anti-tumor immune response without sacrificing safety.

Herpes simplex virus type 1 (HSV-1) acquires its final envelope by budding into cytoplasmic vesicles thought to be derived from trans-Golgi network membranes. This process is facilitated by interactions among the carboxyl termini of viral glycoproteins and tegument proteins. To directly investigate the relative importance of the carboxyl terminus of glycoprotein D (gD) in the presence or absence of gE, a recombinant virus (gD⌬ct) was constructed to specify a truncated gD lacking the carboxy-terminal 29 amino acids. Furthermore, two additional recombinant viruses were constructed by mutating from ATG to CTG the initiation codons of gE (gEctg) or both gE and gM (gEctg؉gMctg), causing lack of expression of gE or both gE and gM, respectively. A fourth mutant virus was constructed to specify the gEctg؉gD⌬ct mutations. The replication properties of these viruses were compared to those of a newly constructed recombinant virus unable to express UL20 due to alteration of the two initiation codons of UL20 (UL20ctgctg). All recombinant viruses were constructed by using the double-Red, site-directed mutagenesis system implemented on the HSV-1(F) genome cloned into a bacterial artificial chromosome. The gEctg, gEctg؉gMctg, gD⌬ct, and gEctg؉gD⌬ct viruses produced viral plaques on African monkey kidney cells (Vero), as well as other cells, that were on average approximately 30 to 50% smaller than those produced by the wild-type virus HSV-1(F). In contrast, the UL20ctgctg virus produced very small plaques containing three to five cells, as reported previously for the ⌬UL20 virus lacking the entire UL20 gene. Viral replication kinetics of intracellular and extracellular viruses revealed that all recombinant viruses produced viral titers similar to those produced by the wild-type HSV-1(F) virus intracellularly and extracellularly at late times postinfection, with the exception of the UL20ctgctg and ⌬UL20 viruses, which replicated more than two-and-a-half logs less efficiently than HSV-1(F). Electron microscopy confirmed that all viruses, regardless of their different gene mutations, efficiently produced enveloped virions within infected cells, with the exception of the UL20ctgctg and ⌬UL20 viruses, which accumulated high levels of unenveloped virions in the cytoplasm. These results show that the carboxyl terminus of gD and the full-length gE, either alone or in a redundant manner, are not essential in cytoplasmic virion envelopment and egress from infected cells. Similarly, gM and gE do not function alone or in a redundant manner in cytoplasmic envelopment and virion egress, confirming previous findings.Herpes simplex virus type 1 (HSV-1) virion assembly initiates in the nucleus, where procapsid proteins assemble around scaffolding proteins (52, 60), followed by the digestion of these scaffolding proteins, creating empty capsids to be filled with DNA (31, 57, 58). The most-prevalent model for HSV-1 virion egress and viral envelope acquisition calls for primary envelopment of nuclear capsids as they cross the inner nuclear membran...