Centromeric protein CENP-B proteasomal degradation induced by the viral protein ICP0

The ICP0 protein of herpes simplex virus type 1 (HSV-1) is a nuclear protein that possesses a well-characterized E3 ubiquitin ligase activity. This activity is responsible for the proteasomal-dependent degradation of several cellular proteins. This study shows that ICP0 induces the proteasomal-dependent degradation of the centromeric protein CENP-B in infected as well as ICP0-expressing cells. It is also shown that the ICP0-induced CENP-B degradation occurs as efficiently in human and mouse cells. CENP-B is one of the major proteins of centromeres and its degradation is likely to contribute to the severe damage induced to centromeres by ICP0.     

Herpes Simplex Virus Type 1 ICP27 Protein： Its Expression, Purification and Specific Antiserum Production

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. Coli and purified by nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin column,finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.     

Analysis of the Cellular Localization of Herpes Simplex Virus 1 Immediate-early Protein ICP22

Nuclear proteins often form punctiform structures, but the precise mechanism for this process is unknown. As a preliminary study, we investigated the aggregation of an HSV-1 immediate-early protein, infected-cell protein 22 (ICP22), in the nucleus by observing the localization of ICP22-EGFP fusion protein. Results showed that, in high-level expression conditions, ICP22-EGFP gradually concentrates in the nucleus, persists throughout the cell cycle without disaggregation even in the cell division phase, and i...     

Herpes simplex virus tegument protein VP22 contains an internal VP16 interaction domain and a C-terminal domain that are both required for VP22 assembly into the virus particle

Many steps along the herpesvirus assembly and maturation pathway remain unclear. In particular, the acquisition of the virus tegument is a poorly understood process, and the molecular interactions involved in tegument assembly have not yet been defined. Previously we have shown that the two major herpes simplex virus tegument proteins VP22 and VP16 are able to interact, although the relevance of this to virus assembly is not clear. Here we have constructed a number of recombinant viruses expressing N- and C-terminal truncations of VP22 and have used them to identify regions of the protein involved in its assembly into the virus structure. Analysis of the packaging of these VP22 variants into extracellular virions revealed that the C terminus of VP22 is absolutely required for this process, with removal of the C-terminal 89 residues abrogating its incorporation. However, while these 89 residues alone were sufficient for specific incorporation of small amounts of VP22 into the tegument, efficient packaging of VP22 to the levels of full-length protein required an additional 52 residues of the protein. Coimmunoprecipitation assays indicated that these 52 residues also contained the interaction domain for VP16. Furthermore, analysis of the subcellular localization of the mutant forms of VP22 revealed that only those truncations that were efficiently assembled formed characteristic cytoplasmic trafficking complexes, suggesting that these complexes may represent the cellular location for VP22 assembly into the virus. Taken together, these results suggest that there are two determinants involved in the packaging of VP22-a C-terminal domain and an internal VP16 interaction domain, both of which are required for the efficient recruitment of VP22 to sites of virus assembly.     

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

Herpes simplex virus VP22 is a major tegument protein of unknown function. Very recently, we reported that the predominant effect of deleting the VP22 gene was on the expression, localization, and virion incorporation of ICP0. In addition, the Delta22 virus replicated poorly in epithelial MDBK cells. We have also previously shown that VP22 interacts with the tegument protein VP16 and the cellular microtubule network. While the majority of VP22 in infected cells is highly phosphorylated, the nonphosphorylated form of VP22 is the predominant species in the virion, suggesting a differential requirement for phosphorylation through virus replication. Hence, to study the significance of VP22 phosphorylation, we have now constructed two recombinant viruses expressing green fluorescent protein-VP22 (G22) in which the previously identified serine phosphorylation sites have been mutated either to alanine to abolish the phosphorylation status of VP22 (G22P-) or to glutamic acid to mimic permanent phosphorylation (G22P+). Localization studies indicated that the G22P- protein associated tightly with microtubules in some infected cells, suggesting that VP22 phosphorylation may control its interaction with the microtubule network. By contrast, VP22 phosphorylation had no effect on its ability to interact with VP16 and, importantly, had no effect on the relative packaging of VP22. Intriguingly, virion packaging of ICP0 was reduced in the G22P+ virus while ICP0 expression was reduced in the G22P- virus, suggesting that these two ICP0 defects, previously observed in the Delta22 virus, were attributable to different forms of VP22. Furthermore, the Delta22 virus replication defect in MDBK cells correlated with the expression of constitutively charged VP22 in the G22P+ virus. Taken together, these results suggest an important role for VP22 phosphorylation in its relationship with ICP0.     

Herpes simplex virus 1 tegument protein US11 downmodulates the RLR signaling pathway via direct interaction with RIG-I and MDA-5

The interferon (IFN)-mediated antiviral response is a major defense of the host immune system. In order to complete their life cycle, viruses must modulate host IFN-mediated immune responses. Herpes simplex virus 1 (HSV-1) is a large DNA virus containing more than 80 genes, many of which encode proteins that are involved in virus-host interactions and show immune modulatory capabilities. In this study, we demonstrate that the US11 protein, an RNA binding tegument protein of HSV-1, is a novel antagonist of the beta IFN (IFN-β) pathway. US11 significantly inhibited Sendai virus (SeV)-induced IFN-β production, and its double-stranded RNA (dsRNA) binding domain was indispensable for this inhibition activity. Additionally, wild-type HSV-1 coinfection showed stronger inhibition than US11 mutant HSV-1 in SeV-induced IFN-β production. Coimmunoprecipitation analysis demonstrated that the US11 protein in HSV-1-infected cells interacts with endogenous RIG-I and MDA-5 through its C-terminal RNA-binding domain, which was RNA independent. Expression of US11 in both transfected and HSV-1-infected cells interferes with the interaction between MAVS and RIG-I or MDA-5. Finally, US11 dampens SeV-mediated IRF3 activation. Taken together, the combined data indicate that HSV-1 US11 binds to RIG-I and MDA-5 and inhibits their downstream signaling pathway, preventing the production of IFN-β, which may contribute to the pathogenesis of HSV-1 infection.     

Autophagy is involved in anti-viral activity of pentagalloylglucose (PGG) against Herpes simplex virus type 1 infection in vitro

Pentagalloylglucose (PGG) is a natural polyphenolic compound with broad-spectrum anti-viral activity, however, the mechanisms underlying anti-viral activity remain undefined. In this study, we investigated the effects of PGG on anti-viral activity against Herpes simplex virus type 1 (HSV-1) associated with autophagy. We found that the PGG anti-HSV-1 activity was impaired significantly in MEF-atg7^–/– cells (autophagy-defective cells) derived from an atg7^–/– knockout mouse. Transmission electron microscopy revealed that PGG-induced autophagosomes engulfed HSV-1 virions. The mTOR signaling pathway, an essential pathway for the regulation of autophagy, was found to be suppressed following PGG treatment. Data presented in this report demonstrated for the first time that autophagy induced following PGG treatment contributed to its anti-HSV activity in vitro.     

Self-association of herpes simplex virus type 1 ICP35 is via coiled-coil interactions and promotes stable interaction with the major capsid protein.

The ordered copolymerization of viral proteins to form the herpes simplex virus (HSV) capsid occurs within the nucleus of the infected cell and is a complex process involving the products of at least six viral genes. In common with capsid assembly in double-stranded DNA bacteriophages, HSV capsid assembly proceeds via the assembly of an outer capsid shell around an interior scaffold. This capsid intermediate matures through loss of the scaffold and packaging of the viral genomic DNA. The interior of the HSV capsid intermediate contains the viral protease and assembly protein which compose the scaffold. Proteolytic processing of these proteins is essential for and accompanies capsid maturation. The assembly protein (ICP35) is the primary component of the scaffold, and previous studies have demonstrated it to be capable of intermolecular association with itself and with the major capsid protein, VP5. We have defined structural elements within ICP35 which are responsible for intermolecular self-association and for interaction with VP5. Yeast (Saccharomyces cerevisiae) two-hybrid assays and far-Western studies with purified recombinant ICP35 mapped a core self-association domain between Ser165 and His219. Site-directed mutations in this domain implicate a putative coiled coil in ICP35 self-association. This coiled-coil motif is highly conserved within the assembly proteins of other alpha herpesviruses. In the two-hybrid assay the core self-association domain was sufficient to mediate stable self-association only in the presence of additional structural elements in either N- or C-terminal flanking regions. These regions also contain conserved sequences which exhibit a high propensity for alpha helicity and may contribute to self-association by forming additional short coiled coils. Our data supports a model in which ICP35 molecules have an extended conformation and associate in parallel orientation through homomeric coiled-coil interactions. In additional two-hybrid experiments we evaluated ICP35 mutants for association with VP5. We discovered that in addition to the C-terminal 25 amino acids of ICP35, previously shown to be required for VP5 binding, an additional upstream region was required. This region is between Ser165 and His234 and contains the core self-association domain. Site-directed mutations and construction of chimeric molecules in which the self-association domain of ICP35 was replaced by the GCN4 leucine zipper indicated that this region contributes to VP5 binding through mediating self-association of ICP35 and not through direct binding interactions. Our results suggest that self-association of ICP35 strongly promotes stable association with VP5 in vivo and are consistent with capsid formation proceeding via formation of stable subassemblies of ICP35 and VP5 which subsequently assemble into capsid intermediates in the nucleus.     

Microtubule reorganization during herpes simplex virus type 1 infection facilitates the nuclear localization of VP22, a major virion tegument protein

Full-length VP22 is necessary for efficient spread of herpes simplex virus type 1 (HSV-1) from cell to cell during the course of productive infection. VP22 is a virion phosphoprotein, and its nuclear localization initiates between 5 and 7 h postinfection (hpi) during the course of synchronized infection. The goal of this study was to determine which features of HSV-1 infection function to regulate the translocation of VP22 into the nucleus. We report the following. (i) HSV-1(F)-induced microtubule rearrangement occurred in infected Vero cells by 13 hpi and was characterized by the loss of obvious microtubule organizing centers (MtOCs). Reformed MtOCs were detected at 25 hpi. (ii) VP22 was observed in the cytoplasm of cells prior to microtubule rearrangement and localized in the nucleus following the process. (iii) Stabilization of microtubules by the addition of taxol increased the accumulation of VP22 in the cytoplasm either during infection or in cells expressing VP22 in the absence of other viral proteins. (iv) While VP22 localized to the nuclei of cells treated with the microtubule depolymerizing agent nocodazole, either taxol or nocodazole treatment prevented optimal HSV-1(F) replication in Vero cells. (v) VP22 migration to the nucleus occurred in the presence of phosphonoacetic acid, indicating that viral DNA and true late protein synthesis were not required for its translocation. Based on these results, we conclude that (iv) microtubule reorganization during HSV-1 infection facilitates the nuclear localization of VP22.     

Characterization of coliphage lambda hybrids carrying DNA fragments from Herpes simplex virus type 1 defective interfering particles

We describe the characterization of 34 hybrid lambda bacteriophages carrying EcoRI fragments obtained from DNA of defective interfering particles of the Patton strain of Herpes simplex virus type 1 (HSV-1). All cloned fragments contained S region terminal repeat sequences (TRs) fused to unique HSV-1 DNA. Several fragments contained deletions and rearrangements not described previously for DNA of HSV-1 defective interfering particles. A model describing the generation of defective interfering DNA based on recombination events involving the terminal "a" sequence as presented.     

Tyrosine phosphorylation of the herpes simplex virus type 1 regulatory protein ICP22 and a cellular protein which shares antigenic determinants with ICP22.

At least eight herpes simplex virus type 1 (HSV-1) and five HSV-2 proteins were tyrosine phosphorylated in infected cells. The first viral tyrosine phosphoprotein identified was the HSV-1 regulatory protein ICP22. Also, two novel phosphotyrosine proteins were bound by anti-ICP22 antibodies. H(R22) is a cellular protein, while the F(R10) protein is observed only in HSV-1-infected cells.