Phosphorylation of the Herpes Simplex Virus Tegument Protein VP22 Has No Effect on Incorporation of VP22 into the Virus but Is Involved in Optimal Expression and Virion Packaging of ICP0

UL16 is a tegument protein of herpes simplex virus (HSV) that is conserved among all members of the Herpesviridae, but its function is poorly understood. Previous studies revealed that UL16 is associated with capsids in the cytoplasm and interacts with the membrane protein UL11, which suggested a "bridging" function during cytoplasmic envelopment, but this conjecture has not been tested. To gain further insight, cells infected with UL16-null mutants were examined by electron microscopy. No defects in the transport of capsids to cytoplasmic membranes were observed, but the wrapping of capsids with membranes was delayed. Moreover, clusters of cytoplasmic capsids were often observed, but only near membranes, where they were wrapped to produce multiple capsids within a single envelope. Normal virion production was restored when UL16 was expressed either by complementing cells or from a novel position in the HSV genome. When the composition of the UL16-null viruses was analyzed, a reduction in the packaging of glycoprotein E (gE) was observed, which was not surprising, since it has been reported that UL16 interacts with this glycoprotein. However, levels of the tegument protein VP22 were also dramatically reduced in virions, even though this gE-binding protein has been shown not to depend on its membrane partner for packaging. Cotransfection experiments revealed that UL16 and VP22 can interact in the absence of other viral proteins. These results extend the UL16 interaction network beyond its previously identified binding partners to include VP22 and provide evidence that UL16 plays an important function at the membrane during virion production. Infectious herpesviruses contain approximately 40 viral proteins and are produced when their DNA-containing capsids are wrapped with a cell-derived membrane in the cytoplasm (1). This envelopment process is driven by complex interactions that are still poorly understood but is known to involve bridging interactions provided by a growing list of tegument proteins, which provide linkages between the capsid and viral membrane proteins (1-3). The UL16 tegument protein of herpes simplex virus (HSV) is remarkable for its numerous interactions with several other viral proteins, namely, tegument protein UL21 (4, 5), membrane protein UL11 (4, 6-8), membrane glycoprotein E (gE) (4, 9), and an unidentified protein(s) that is associated with the capsid (10-12). UL16 is conserved among all the alpha-, beta-, and gammaherpesvirinae (2, 13, 14), but its actual function remains unknown.There are several reasons for suggesting a role for UL16 in HSV envelopment. The earliest study showed that a U L 16-null mutant (here named the ⌬U L 16B mutant) produces infectious virions at a level only one-tenth that of the wild-type virus (15). Also, UL16 has been shown to be bound in some manner to cytoplasmic capsids (10, 16, 17), and thus, its direct interactions with membrane proteins UL11 (8) and gE (9) suggest that UL16 might provide bridging functions that help drive virion production, as first pro...

Section: Ultrastructural Characterization Of Ul16-null Mutantsmentioning

confidence: 49%

Section: Ultrastructural Characterization Of Ul16-null Mutantsmentioning

confidence: 63%

Elucidation of the Block to Herpes Simplex Virus Egress in the Absence of Tegument Protein UL16 Reveals a Novel Interaction with VP22

Starkey

Han

Chadha

et al. 2014

“…We have also shown that this defect can be rescued by both WT VP22 and a variant of VP22 in which all of the phosphorylation sites have been mutated (12,36). Therefore, we next wished to determine if introduction of the gEbind mutant form of VP22 into the ⌬22 virus was also capable of rescuing this replication defect.…”

Section: Resultsmentioning

confidence: 99%

Virion Incorporation of the Herpes Simplex Virus Type 1 Tegument Protein VP22 Occurs via Glycoprotein E-Specific Recruitment to the Late Secretory Pathway

Stylianou

Maringer

Cook

et al. 2009

Self Cite

The mechanism by which herpesviruses acquire their tegument is not yet clear. One model is that outer tegument proteins are recruited by the cytoplasmic tails of viral glycoproteins. In the case of herpes simplex virus tegument protein VP22, interactions with the glycoproteins gE and gD have been shown. We have previously shown that the C-terminal half of VP22 contains the necessary signal for assembly into the virus. Here, we show that during infection VP22 interacts with gE and gM, as well as its tegument partner VP16. However, by using a range of techniques we were unable to demonstrate VP22 binding to gD. By using pulldown assays, we show that while the cytoplasmic tails of both gE and gM interact with VP22, only gE interacts efficiently with the C-terminal packaging domain of VP22. Furthermore, gE but not gM can recruit VP22 to the Golgi/trans-Golgi network region of the cell in the absence of other virus proteins. To examine the role of the gE-VP22 interaction in infection, we constructed a recombinant virus expressing a mutant VP22 protein with a 14-residue deletion that is unable to bind gE (⌬gEbind). Coimmunoprecipitation assays confirmed that this variant of VP22 was unable to complex with gE. Moreover, VP22 was no longer recruited to its characteristic cytoplasmic trafficking complexes but exhibited a diffuse localization. Importantly, packaging of this variant into virions was abrogated. The mutant virus exhibited poor growth in epithelial cells, similar to the defect we have observed for a VP22 knockout virus. These results suggest that deletion of just 14 residues from the VP22 protein is sufficient to inhibit binding to gE and hence recruitment to the viral envelope and assembly into the virus, resulting in a growth phenotype equivalent to that produced by deleting the entire reading frame.The herpesvirus tegument is the virion compartment located between the DNA-containing capsid and the virus envelope (6). Although it is well defined that the viral capsid assembles in the nucleus (37,38) and the viral envelope is acquired from cellular membranes (3, 24), the mechanism of tegument protein acquisition is still to be established. At least 20 virusencoded components are recruited into the herpes simplex virus type 1 (HSV-1) tegument (32), and there is increasing evidence to suggest that subsets of these proteins may be added as assembly progresses along the maturation pathway (28). To ensure efficient incorporation, it is likely that individual tegument proteins are specifically targeted to their cellular site of recruitment. Such targeting could involve interaction with a viral partner, a cellular partner, or both. A clearer understanding of how individual tegument proteins are acquired by newly assembling virions will help to define the herpesvirus assembly pathway.A number of protein-protein interactions between individual tegument proteins (13,40,42), and between tegument proteins and glycoproteins (19,20,22,32), have been described that may provide useful insight into the assembly process. In p...

“…2C). Furthermore, it has been reported that loss of VP22 causes reduced incorporation of other viral proteins into virions, such as ICP0 (70,71). VP22 also exists in a virion protein interaction network which is important for viral assembly.…”

Section: Discussionmentioning

confidence: 99%

Kaposi's Sarcoma-Associated Herpesvirus Inhibitor of cGAS (KicGAS), Encoded by ORF52, Is an Abundant Tegument Protein and Is Required for Production of Infectious Progeny Viruses

Avey

et al. 2016

Although Kaposi's sarcoma-associated herpesvirus (KSHV) ORF52 (also known as KSHV inhibitor of cGAS [KicGAS]) has been detected in purified virions, the roles of this protein during KSHV replication have not been characterized. Using specific monoclonal antibodies, we revealed that ORF52 displays true late gene expression kinetics and confirmed its cytoplasmic localization in both transfected and KSHV-infected cells. We demonstrated that ORF52 comigrates with other known virion proteins following sucrose gradient centrifugation. We also determined that ORF52 resides inside the viral envelope and remains partially associated with capsid when extracellular virions are treated with various detergents and/or salts. There results indicate that ORF52 is a tegument protein abundantly present in extracellular virions. To characterize the roles of ORF52 in the KSHV life cycle, we engineered a recombinant KSHV ORF52-null mutant virus and found that loss of ORF52 results in reduced virion production and a further defect in infectivity. Upon analysis of the virion composition of ORF52-null viral particles, we observed a decrease in the incorporation of ORF45, as well as other tegument proteins, suggesting that ORF52 is important for the packaging of other virion proteins. In summary, our results indicate that, in addition to its immune evasion function, KSHV ORF52 is required for the optimal production of infectious virions, likely due to its roles in virion assembly as a tegument protein. IMPORTANCEThe tegument proteins of herpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV), play key roles in the viral life cycle. Each of the three subfamilies of herpesviruses (alpha, beta, and gamma) encode unique tegument proteins with specialized functions. We recently found that one such gammaherpesvirus-specific protein, ORF52, has an important role in immune evasion during KSHV primary infection, through inhibition of the host cytosolic DNA sensing pathway. In this report, we further characterize ORF52 as a tegument protein with vital roles during KSHV lytic replication. We found that ORF52 is important for the production of infectious viral particles, likely through its role in virus assembly, a critical process for KSHV replication and pathogenesis. More comprehensive investigation of the functions of tegument proteins and their roles in viral replication may reveal novel targets for therapeutic interventions against KSHV-associated diseases. K aposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is the etiologic agent of Kaposi's sarcoma (KS) (1) and, also, two lymphoproliferative disorders, primary effusion lymphoma (PEL) (2) and multicentric Castleman disease (MCD) (3). KSHV belongs to the Rhadinovirus genus in the Gammaherpesvirinae subfamily and is related to rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS), and murine gammaherpesvirus 68 (MHV-68). The closest relative of KSHV among the known human herpesviruses is Epstein-Barr virus (EBV), which belongs to t...