The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.Herpes simplex virus type 1 (HSV-1) can enter into cells via the fusion of its viral envelope with cellular membranes. Also, the virus can spread from infected to uninfected cells by causing virus-induced cell fusion, allowing virions to enter into uninfected cells without being exposed to extracellular spaces. These membrane fusion phenomena are known to be mediated by viral glycoproteins and other viral proteins (reviewed in reference 36). Although wild-type viruses cause a limited amount of virus-induced cell fusion, certain mutations cause extensive virus-induced cell-to-cell fusion (syncytial, or syn, mutations). These syncytial mutations are located predominantly within the UL20 gene (5, 27, 28); the UL24 gene (25, 38); the UL27 gene, encoding glycoprotein gB (7, 15, 18, 32); and the UL53 gene, coding for gK (6, 11, 24, 34, 35, 37).The presence of syncytial mutations within different viral genes, as well as other accumulating evidence, suggests that virus-induced cell fusion is mediated by the concerted action and interactions of the viral glycoproteins gD, gB, and gH/gL as well as gK and the membrane protein UL20p. Specifically, recent studies have shown that gD interacts with both gB and gH/gL (1, 2, 21). However, gB and gH/gL can also interact with each other even in the absence of gD (3). In this membrane fusion model, the binding of gD to its cognate receptors, including nectin-1, herpesvirus entry mediator (HVEM), and other receptors (8, 19, 30, 39-42), is thought to trigger sequential conformational changes in gH/gL and gB causing the fusion of the viral envelope with cellular membranes during virus entry as well as fusion among cellular membranes (22, 23). The transient coexpression of gB, gD, and gH/gL causes cell-to-cell fusion (31, 43), suggesting that these four viral glycoproteins are necessary and sufficient for membrane fusion. However, this transient fusion system does not accurately depict virus-induced cell fusion. Specifically, viral glycoprotein K (gK) and the UL20 membrane protein (UL20p) have been shown to be strictly required for virus-induced cell fusion (10, 27, 29). Moreover, syncytial mutations within gK (6, 11, 24, 34, 35, 37) or UL20 (5, 27, 28) promote extensive virus-induced cell fusion, and viruses lacking gK enter more slowly than the wild-type virus into susceptible cells (17). In contrast, the transient coexpression of gK carrying a syncytial mutation with gB, gD, and gH/gL did not enhance cell fusion, while the coexpression of wild-type gK with gB, gD, and gH/gL was reported previously to inhibit cell fusion in certain cell lines (4). To date, there is no direct evidence that either gK or UL20p interacts with gB, gD, gH, or gL.The X-ray structure of the ectodomain of HSV-1 gB has been determined and was predicted to assume at least two major conformations, one of which may be necessary for the fusogenic properties of gB (23). Single-amino-acid changes within the carboxyl terminus of gB located intracellularly as well as the deletion of the terminal 28 amino acids (aa) of gB cause extensive virus-induced cell fusion, presumably because they alter the extracellular conformation of gB (15, 31, 43). We have previously shown that HSV-1 gK and UL20p functionally and physically interact and that these interactions are absolutely necessary for their coordinate intracellular transport, cell surface expression, and functions in the HSV-1 life cycle (13, 16). In contrast to gB, syncytial mutations in gK map predominantly within extracellular domains of gK and particularly within the amino-terminal portion of gK (domain I) (12), while syncytial mutations of UL20 are located within the amino terminus of UL20p shown to be located intracellularly (27).Recently, we showed that the a peptide composed of the amino-terminal 82 amino acids of gK (gKa) can complement in trans for gB-mediated cell fusion caused by the deletion of the carboxyl-terminal 28 amino acids of gB, suggesting that the gKa peptide interacted with gB or other viral glycoproteins involved in virus-induced cell fusion (10). In this work, we demonstrate that UL20p and the amino terminus of gKa physically interact with gB in infected cells, while the gKa peptide is also capable of binding to the extracellular portion of gH, suggesting that gK/UL20p modulates virus-induced cell fusion via direct interactions with gB and gH.