Functional and Physical Interactions of the Herpes Simplex Virus Type 1 UL20 Membrane Protein with Glycoprotein K

UL36p (VP1/2) is the largest protein encoded by herpes simplex virus 1 (HSV-1) and resides in the innermost layer of the viral tegument, lying between the capsid and the envelope. UL36p performs multiple functions in the HSV life cycle, including an essential role in cytoplasmic envelopment. We earlier described the isolation of a virion-associated cytoplasmic membrane fraction from HSV-infected cells. Biochemical and ultrastructural analyses showed that the organelles in this buoyant fraction contain enveloped infectious HSV particles in their lumens and naked capsids docked to their cytoplasmic surfaces. These organelles can also recruit molecular motors and transport their cargo virions along microtubules in vitro. Here we examine the properties of these HSV-associated organelles in the absence of UL36p. We find that while capsid envelopment is clearly defective, a subpopulation of capsids nevertheless still associate with the cytoplasmic faces of these organelles. The existence of these capsidmembrane structures was confirmed by subcellular fractionation, immunocytochemistry, lipophilic dye fluorescence microscopy, thin-section electron microscopy, and correlative light and electron microscopy. We conclude that capsid-membrane binding can occur in the absence of UL36p and propose that this association may precede the events of UL36p-driven envelopment. IMPORTANCEMembrane association and envelopment of the HSV capsid are essential for the assembly of an infectious virion. Envelopment involves the complex interplay of a large number of viral and cellular proteins; however, the function of most of them is unknown. One example of this is the viral protein UL36p, which is clearly essential for envelopment but plays a poorly understood role. Here we demonstrate that organelles utilized for HSV capsid envelopment still accumulate surface-bound capsids in the absence of UL36p. We propose that UL36p-independent binding of capsids to organelles occurs prior to the function of UL36p in capsid envelopment. Herpesviruses replicate their genomes and assemble DNApackaged capsids in the cell nucleus. It is then generally accepted that capsids bud into the inner nuclear membrane to generate primary enveloped perinuclear virions that subsequently fuse with the outer nuclear membrane, releasing mature nucleocapsids ("naked" capsids) into the cytoplasm (1-3). These naked cytoplasmic capsids subsequently undergo secondary envelopment at a postnuclear organelle to assemble the mature, infectious virion (4-9). Completion of cytoplasmic envelopment requires the coordinated interaction of multiple virally encoded proteins (8,(10)(11)(12)(13) and the participation of components of the cellular endosomal sorting complexes required for transport (ESCRT) machinery (14-19).UL36p (VP1/2) is the largest structural protein encoded by the herpesviridae (20, 21). It is a major constituent of the innermost layer of tegument, the complex protein layer between the capsid and the inner surface of the envelope, and connects the capsid to m...

Section: Discussionmentioning

confidence: 67%

mentioning

confidence: 99%

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confidence: 99%

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Herpes Simplex Virus Capsid-Organelle Association in the Absence of the Large Tegument Protein UL36p

Kharkwal

Furgiuele

Smith

et al. 2015

“…The gK expressed on virions forms a complex with the membrane-associated UL20 viral protein (52,56). HSV-1 mutants that lack gK fail to acquire a cytoplasmic envelope efficiently.…”

Section: Discussionmentioning

confidence: 99%

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

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...

“…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).…”

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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...