Junji Xing

Varicella-Zoster Virus Immediate-Early Protein ORF61 Abrogates the IRF3-Mediated Innate Immune Response through Degradation of Activated IRF3

ABSTRACT Varicella-zoster virus (VZV) infection of differentiated cells within the host and establishment of latency likely requires evasion of innate immunity and limits secretion of antiviral cytokines. Here we report that its immediate-early protein ORF61 antagonizes the beta interferon (IFN-β) pathway. VZV infection down-modulated the Sendai virus (SeV)-activated IFN-β pathway, including mRNA of IFN-β and its downstream interferon-stimulated genes (ISGs), ISG54 and ISG56. Through a primary screening of VZV genes, we found that ORF61 inhibited SeV-mediated activation of IFN-β and ISRE (IFN-stimulated response element) promoter activities but only slightly affected NF-κB promoter activity, implying that the IFN-β pathway may be blocked in the IRF3 branch. An indirect immunofluorescence assay demonstrated that ectopic expression of ORF61 abrogated the detection of IRF3 in SeV-infected cells; however, it did not affect endogenous dormant IRF3 in noninfected cells. Additionally, ORF61 was shown to be partially colocalized with activated IRF3 in the nucleus upon treatment with MG132, an inhibitor of proteasomes, and the direct interaction between ORF61 and activated IRF3 was confirmed by a coimmunoprecipitation assay. Furthermore, Western blot analysis demonstrated that activated IRF3 was ubiquitinated in the presence of ORF61, suggesting that ORF61 degraded phosphorylated IRF3 via a ubiquitin-proteasome pathway. Semiquantitative reverse transcription-PCR (RT-PCR) analysis demonstrated that the level of ISG54 and ISG56 mRNAs was also downregulated by ORF61. Taken together, our results convincingly demonstrate that ORF61 down-modulates the IRF3-mediated IFN-β pathway by degradation of activated IRF3 via direct interaction, which may contribute to the pathogenesis of VZV infection.

Herpes Simplex Virus 1 Tegument Protein US11 Downmodulates the RLR Signaling Pathway via Direct Interaction with RIG-I and MDA-5

ABSTRACT 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.

Comprehensive Characterization of Interaction Complexes of Herpes Simplex Virus Type 1 ICP22, UL3, UL4, and UL20.5

ABSTRACT It has been reported that herpes simplex virus type 1 UL3, UL4, and UL20.5 proteins are localized to small, dense nuclear bodies together with ICP22 in infected cells. In the present study, we comprehensively characterized these interactions by subcellular colocalization, coimmunoprecipitation, and bimolecular fluorescence complementation assays. For the first time, it was demonstrated that both UL3 and UL20.5 are targeted to small, dense nuclear bodies by a direct interaction with ICP22, whereas UL4 colocalizes with ICP22 through its interaction with UL3 but not UL20.5 or ICP22. There was no detectable interaction between UL3 and UL20.5.

Molecular anatomy of subcellular localization of HSV-1 tegument protein US11 in living cells

Abstract The herpes simplex virus type I (HSV-1) US11 protein is an RNA-binding multifunctional regulator that specifically and stably associates with nucleoli. Although the C-terminal part of US11 was responsible for its nucleolar localization, the precise nucleolar localization signal (NoLS) and nuclear export signal (NES) of US11 and its nuclear import and export mechanisms are still elusive. In this study, fluorescence microscopy was employed to investigate the subcellular localization of US11 and characterize its transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein (EYFP), three novel NoLSs of US11 were for the first time mapped to amino acids 84–125, 126–152, and 89–146, respectively. Additionally, the NES was identified to locate between amino acids 89 and 119. Furthermore, the US11 protein was demonstrated to target to the cytoplasm through the NES by chromosomal region maintenance 1 (CRM1)-independent pathway, and to the nucleolus through Ran and importin β-dependent mechanism that does not require importin α5.

Characterization of the subcellular localization of herpes simplex virus type 1 proteins in living cells

In this study, we presented the construction of a library of expression clones for the herpes simplex virus type 1 (HSV-1) proteome and subcellular localization map of HSV-1 proteins in living cells using yellow fluorescent protein (YFP) fusion proteins. As a result, 21 proteins showed cytoplasmic or subcytoplasmic localization, 16 proteins showed nuclear or subnuclear localization, and others were present both in the nucleus and cytoplasm. Interestingly, most capsid proteins showed enriched or exclusive localization in the nucleus, and most of the envelope proteins showed cytoplasmic localization, suggesting that subcellular localization of the proteins correlated with their functions during virus replication. These results present a subcellular localization map of HSV-1 proteins in living cells, which provide useful information to further characterize the functions of these proteins.

Identification of a novel NLS of herpes simplex virus type 1 (HSV-1) VP19C and its nuclear localization is required for efficient production of HSV-1.

Herpes simplex virus type 1 (HSV-1) triplex is a complex of three protein subunits, consisting of two copies of VP23 and one copy of VP19C. Here, we identified a non-classical NLS of VP19C between aa 50 and 61, and the nuclear import of VP19C was mediated by RanGTP and importin β1-, but not importin α5-, dependent pathway. Additionally, recombinant virus harbouring this NLS mutation (NLSm) replicates less efficiently as wild-type. These data strongly suggested that the nuclear import of VP19C is required for efficient HSV-1 production.

Characterization of the nuclear import and export signals, and subcellular transport mechanism of varicella-zoster virus ORF9

Varicella-zoster virus (VZV) open reading frame 9 (ORF9) mRNA is one of the most abundantly expressed messages during VZV infection. However, little is known concerning the function of ORF9 protein. Here, we found that transient expression of ORF9 fused to enhanced yellow fluorescent protein (EYFP) in COS-7 cells showed a predominantly cytoplasmic localization in the absence of other viral proteins. By constructing a series of ORF9 variants fused to EYFP, a bona fide bipartite nuclear localization signal of ORF9 was, for the first time, determined and mapped to aa 16-32 (RRKTTPSYSGQYRTARR). Additionally, the nuclear export signal (NES) was identified and found to be in a leucine-rich region at aa 103-117 (LRHELVEDAVYENPL). Finally, ORF9 was demonstrated to be targeted to the cytoplasm through the functional NES by Ran and the chromosomal region maintenance 1-dependent pathway, and to the nucleus via an importin β-dependent pathway that does not require importin α5.

Screening and identification of host factors interacting with UL14 of herpes simplex virus 1

The UL14 protein of herpes simplex virus type 1 (HSV-1) is highly conserved in herpesvirus family. However, its exact function during the HSV-1 replication cycle is little known. In the present study, a high throughput yeast two-hybrid system was employed to screen the cellular factors interacting with UL14, and five target candidates were yielded: (1) TSC22 domain family protein 3 (TSC22D3); (2) Mediator of RNA polymerase II transcription subunit 8 isoform 1(MED8); (3) Runt-related transcription factor 3 (RUNX3); (4) Arrestin beta-2 (ARRB2); (5) Cereblon (CRBN). Indirect immunofluorescent assay showed that both TSC22D3 and MED8 co-localized with UL14. Co-immunoprecipitation assay demonstrated that UL14 could be immunoprecipitated by TSC22D3, suggesting that UL14 interacted with TSC22D3 under physiological condition. In summary, this study opened up new avenues toward delineating the function and physiological significance of UL14 during the HSV-1 replication cycle.

Characterization of nuclear import and export signals determining the subcellular localization of WD repeat-containing protein 42A (WDR42A)

Karyopherin alpha‐1 physically interacts with WDR42A by anti tag co‐immunoprecipitation (View interaction) WDR42A physically interacts with Karyopherin alpha‐1 by pull down (View interaction) Karyopherin beta‐1 physically interacts with WDR42A by anti tag co‐immunoprecipitation (View interaction) WDR42A physically interacts with CRM1 by anti tag co‐immunoprecipitation (View interaction) WDR42A and RAN colocalize by fluorescence microscopy (View interaction)

A PY-nuclear localization signal is required for nuclear accumulation of HCMV UL79 protein.

Human cytomegalovirus UL79 protein is recently reported to be required for transcription or efficient accumulation of late viral mRNAs during viral infection. An absolute nuclear distribution of UL79 proteins has been observed with immunofluorescence assay, both during the infection of Flag-tagged UL79 recombinant virus and in the HFFs expressing HA-tagged UL79, with or without virus infection. However, little is known about the nuclear import mechanism of UL79 protein. Here, by utilizing living cells fluorescent microscopy, a predominant nuclear localization of UL79 protein in living cells was detected. Furthermore, the nuclear import of UL79 protein was demonstrated to be dependent on the transportin-1-mediated pathway. Finally, a hydrophobic PY-nuclear localization signal (PY-NLS) was delineated between the amino acids 66–92 of UL79 protein. Collectively, we provide evidence that a PY-NLS, firstly described in viral proteins, is responsible for the nuclear accumulation of UL79 protein.