Epitope mapping of HSV-1 glycoprotein K (gK) reveals a T cell epitope located within the signal domain of gK

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To investigate the role of the signal sequences of herpes simplex virus 1 (HSV-1) gK on virus replication and viral pathogenesis, we constructed recombinant viruses with or without mutations within the signal sequences of gK. These recombinant viruses expressed two additional copies of the mutated (MgK) or native (NgK) form of the gK gene in place of the latency-associated transcript with a myc epitope tag to facilitate detection at their 3= ends. The replication of MgK virus was similar to that of NgK both in vitro and in vivo, as well as in the trigeminal ganglia (TG) of latently infected mice. The levels of gB and gK transcripts in the corneas, TG, and brains of infected mice on days 3 and 5 postinfection were markedly virus and time dependent, as well as tissue specific. Mutation in the signal sequence of gK in MgK virus blocked cell surface expression of gK-myc in rabbit skin cells, increased 50% lethal dose, and decreased corneal scarring in ocularly infected mice compared to the NgK or revertant (RgK) virus. MgK and NgK viruses, and not the RgK virus, showed a reduced extent of explant reactivation at the lower dose of ocular infection but not at the higher dose. However, the time of reactivation was not affected by overexpression of the different forms of gK. Taken together, these results strongly suggest that the 8mer peptide (ITAYGLVL) within the signal sequence of gK promotes cell surface expression of gK in infected cells and ocular pathogenesis in infected mice. IMPORTANCEIn this study, we show for the first time that mutations within the signal sequence of gK blocked cell surface expression of inserted recombinant gK in vitro. Furthermore, this blockage in cell surface expression was correlated with higher 50% lethal dose and less corneal scarring in vivo. Thus, these studies point to a key role for the 8mer within the signal sequence of gK in HSV-1-induced pathogenicity. Herpes simplex virus 1 (HSV-1) infections are among the most frequent serious viral eye infections in the United States and are a major cause of virus-induced blindness (1-3). It is estimated that 70 to 90% of American adults have antibodies to HSV-1 and/or HSV-2 and ca. 25% of these individuals have clinical symptoms upon routine clinical inquiry. HSV-1 is responsible for Ͼ90% of ocular HSV infections (1, 2, 4-7). HSV-1-induced corneal scarring (CS), also broadly referred to as herpes stromal keratitis (HSK), can lead to blindness. Furthermore, HSV-1 is the leading cause of corneal blindness due to an infectious agent in developed countries (1, 2, 4-7). In addition to necrotizing HSK, ocular infection with HSV-1 can cause eye disease ranging in severity from blepharitis, conjunctivitis, and dendritic keratitis to disciform stromal edema (4, 7-11). In the United States, approximately 30,000 people per year suffer recurrent ocular HSV episodes, requiring doctor visits, medication, and in severe cases, corneal transplants.Although it is well established that HSV-1-induced CS is due to immune responses to the virus, the exact...

Section: Discussionsupporting

confidence: 89%

Section: Discussionmentioning

confidence: 94%

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confidence: 94%

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Mutations within the Pathogenic Region of Herpes Simplex Virus 1 gK Signal Sequences Alter Cell Surface Expression and Neurovirulence

Matundan

Mott

Akhtar

et al. 2015

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“…The different characteristics of T CM and T EM cells suggest that they play different roles in recall responses and containment of herpes infection and disease (26,(28)(29)(30). We hypothesize that ASYMP individuals develop more of the protective HSV-specific CD8 ϩ T EM cells, while SYMP patients develop more of the nonprotective (or maybe pathogenic) HSV-specific CD8 ϩ T CM cells (31,32). In this study, we found two distinct phenotypic and functional patterns of protective and nonprotective HSV-1 gB-specific CD8 ϩ T cells that are associated with ASYMP versus SYMP ocular herpes, respectively.…”

Section: Importancementioning

confidence: 96%

Phenotypic and Functional Characterization of Herpes Simplex Virus Glycoprotein B Epitope-Specific Effector and Memory CD8⁺T Cells from Symptomatic and Asymptomatic Individuals with Ocular Herpes

Khan

Srivastava

Spencer

et al. 2015

Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8؉ T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8؉ T cells play a key role in the "natural" protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8 O ver a billion individuals worldwide carry herpes simplex virus 1 (HSV-1), which causes a wide range of mild to life-threatening diseases (1-3). Although the virus reactivates from latency and is shed multiple times each year in body fluids (i.e., tears, saliva, and nasal and vaginal secretions), most reactivations are subclinical due to an efficient immune-mediated containment of the infection and disease (4-7). Thus, most infected individuals are asymptomatic (ASYMP) and do not present any apparent recurrent herpetic disease (e.g., cold sores, genital, or ocular herpetic disease). However, a small proportion of individuals experience endless recurrences of herpetic disease, usually multiple times a year, often necessitating continuous antiviral therapy (i.e., with acyclovir and derivatives) (8,9). In those symptomatic (SYMP) individuals, HSV-1 frequently reactivates from latency, reinfects the eyes, and may trigger recurrent and severe corneal herpetic disease, a leading cause of infectious corneal blindness in

“…HSV-1 gK has been shown to be involved in neurovirulence and immunomodulation (16,17). We have shown that HSV-1 gK and UL20 functionally and physically interact, and this interaction is mandatory for their coordinated intracellular transport, cell surface expression, and functions in virion egress, virus-induced cell fusion, and virus entry (18)(19)(20)(21).…”

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confidence: 99%

The Amino Terminus of Herpes Simplex Virus 1 Glycoprotein K Is Required for Virion Entry via the Paired Immunoglobulin-Like Type-2 Receptor Alpha

Chowdhury

Chouljenko

Naderi

et al. 2013

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The herpes simplex virus 1 (HSV-1) glycoprotein K (gK)/UL20 protein complex is incorporated into virion envelopes and cellular membranes and functions during virus entry and cell-to-cell spread. To investigate the role of gK/UL20 in the context of a highly neurovirulent virus strain, the HSV-1(McKrae) genome was cloned into a bacterial artificial chromosome plasmid T he herpes simplex virus 1 (HSV-1) entry mechanism is both complex and unique among enveloped viruses, involving multiple glycoproteins for attachment, binding, and membrane fusion (1). Viral glycoproteins interact with different cellular receptors to facilitate virus entry. Initial attachment of the virus to cellular membranes is mediated by interaction of glycoproteins gB and gC with glycosaminoglycan (GAG) moieties of cell surface proteoglycans (2, 3). Attachment of virions to cellular membranes facilitates subsequent binding of gD to cellular receptors, including the herpesvirus entry mediator (HVEM, also called HveA), nectin-1 (HveC), and 3-O-sulfated heparan sulfate (4-6). Apparently, gB can also bind to additional cellular receptors, including paired immunoglobulin-like type 2 receptor alpha (PILR␣), nonmuscle myosin heavy chain IIA (NMHC-IIA), and myelin-associated glycoprotein (MAG) (7-9). Sequential binding of gD and then gB to their respective cellular receptors during virus entry and virus-induced cell-to-cell fusion is thought to alter gB's conformation, resulting in gB-mediated membrane fusion (1, 10-12).HSV-1 can enter into cells by utilizing different cell-dependent pathways: (i) virus entry into Vero and HEp-2 cells is predominantly mediated via pH-independent fusion of the viral envelope with the host cell membrane (13); (ii) virus entry into HeLa and Chinese hamster ovary (CHO) cells expressing the nectin-1 gD receptor is predominantly achieved via receptor-mediated endocytosis, followed by pH-dependent fusion of the viral envelope with endocytic membranes (14); and (iii) virus entry into C10 murine melanoma cells predominantly occurs via pH-independent endocytosis (13). Recently, it has been suggested that gBspecific receptors, such as PILR␣, or other cellular plasma membrane factors determine whether virions enter predominantly via fusion at the plasma membrane or via receptor-mediated endocytosis (pH dependent or pH independent), followed by fusion of the viral envelope with endosomal membranes (15).HSV-1 gK has been shown to be involved in neurovirulence and immunomodulation (16,17). We have shown that HSV-1 gK and UL20 functionally and physically interact, and this interaction is mandatory for their coordinated intracellular transport, cell surface expression, and functions in virion egress, virus-induced cell fusion, and virus entry (18)(19)(20)(21). Recently, we showed that the amino terminus of gK interacts with gB and gH and can complement gB-mediated cell fusion (22,23). Virions lacking the entire gK or the amino terminus of gK (amino acids [aa] 31 to 68) enter susceptible cells substantially slower than the wild-type vi...