Varicella-zoster virus IE63 protein phosphorylation by roscovitine-sensitive cyclin-dependent kinases modulates its cellular localization and activity

During the first stage of Varicella-Zoster virus (VZV) infection, IE63 (immediate early 63 protein) is mostly expressed in the nucleus and also slightly in the cytoplasm, and during latency, IE63 localizes in the cytoplasm quite exclusively. Because phosphorylation is known to regulate various cellular mechanisms, we investigated the impact of phosphorylation by roscovitine-sensitive cyclin-dependent kinase (RSC) on the localization and functional properties of IE63. We demonstrated first that IE63 was phosphorylated on Ser-224 in vitro by CDK1 and CDK5 but not by CDK2, CDK7, or CDK9. Furthermore, by using roscovitine and CDK1 inhibitor III (CiIII), we showed that CDK1 phosphorylated IE63 on Ser-224 in vivo. By mutagenesis and the use of inhibitors, we demonstrated that phosphorylation on Ser-224 was important for the correct localization of the protein. Indeed, the substitution of these residues by alanine led to an exclusive nuclear localization of the protein, whereas mutations into glutamic acid did not modify its subcellular distribution. When transfected or VZV-infected cells were treated with roscovitine or CiIII, an exclusive nuclear localization of IE63 was also observed. By using a transfection assay, we also showed that phosphorylation on Ser-224 and Thr-222 was essential for the down-regulation of the basal activity of the VZV DNA polymerase gene promoter. Similarly, roscovitine and CiIII impaired these properties of the wild-type form of IE63. These observations clearly demonstrated the importance of CDK1-mediated IE63 phosphorylation for a correct distribution of IE63 between both cellular compartments and for its repressive activity toward the promoter tested.     

Varicella-zoster virus infection of human dendritic cells and transmission to T cells: implications for virus dissemination in the host

During primary varicella-zoster virus (VZV) infection, it is presumed that virus is transmitted from mucosal sites to regional lymph nodes, where T cells become infected. The cell type responsible for VZV transport from the mucosa to the lymph nodes has not been defined. In this study, we assessed the susceptibility of human monocyte-derived dendritic cells to infection with VZV. Dendritic cells were inoculated with the VZV strain Schenke and assessed by flow cytometry for VZV and dendritic cell (CD1a) antigen expression. In five replicate experiments, 34.4% +/- 6.6% (mean +/- SEM) of CD1a(+) cells were also VZV antigen positive. Dendritic cells were also shown to be susceptible to VZV infection by the detection of immediate-early (IE62), early (ORF29), and late (gC) gene products in CD1a(+) dendritic cells. Infectious virus was recovered from infected dendritic cells, and cell-to-cell contact was required for transmission of virus to permissive fibroblasts. VZV-infected dendritic cells showed no significant decrease in cell viability or evidence of apoptosis and did not exhibit altered cell surface levels of major histocompatibility complex (MHC) class I, MHC class II, CD86, CD40, or CD1a. Significantly, when autologous T lymphocytes were incubated with VZV-infected dendritic cells, VZV antigens were readily detected in CD3(+) T lymphocytes and infectious virus was recovered from these cells. These data provide the first evidence that dendritic cells are permissive to VZV and that dendritic cell infection can lead to transmission of virus to T lymphocytes. These findings have implications for our understanding of how virus may be disseminated during primary VZV infection.     

The immediate-early 63 protein of Varicella-Zoster virus: analysis of functional domains required for replication in vitro and for T-cell and skin tropism in the SCIDhu model in vivo

The immediate-early 63-kDa (IE63) protein of varicella-zoster virus (VZV) is a phosphoprotein encoded by open reading frame (ORF) ORF63/ORF70. To identify functional domains, 22 ORF63 mutations were evaluated for effects on IE63 binding to the major VZV transactivator, IE62, and on IE63 phosphorylation and nuclear localization in transient transfections, and after insertion into the viral genome with VZV cosmids. The IE62 binding site was mapped to IE63 amino acids 55 to 67, with R59/L60 being critical residues. Alanine substitutions within the IE63 center region showed that S165, S173, and S185 were phosphorylated by cellular kinases. Four mutations that changed two putative nuclear localization signal (NLS) sequences altered IE63 distribution to a cytoplasmic/nuclear pattern. Only three of 22 mutations in ORF63 were compatible with recovery of infectious VZV from our cosmids, but infectivity was restored by inserting intact ORF63 into each mutated cosmid. The viable IE63 mutants had a single alanine substitution, altering T171, S181, or S185. These mutants, rOKA/ORF63rev[T171], rOKA/ORF63rev[S181], and rOKA/ORF63rev[S185], produced less infectious virus and had a decreased plaque phenotype in vitro. ORF47 kinase protein and glycoprotein E (gE) synthesis was reduced, indicating that IE63 contributed to optimal expression of early and late gene products. The three IE63 mutants replicated in skin xenografts in the SCIDhu mouse model, but virulence was markedly attenuated. In contrast, infectivity in T-cell xenografts was not altered. Comparative analysis suggested that IE63 resembled the herpes simplex virus type 1 U(S)1.5 protein, which is expressed colinearly with ICP22 (U(S)1). In summary, most mutations of ORF63 made with our VZV cosmid system were lethal for infectivity. The few IE63 changes that were tolerated resulted in VZV mutants with an impaired capacity to replicate in vitro. However, the IE63 mutants were attenuated in skin but not T cells in vivo, indicating that the contribution of the IE63 tegument/regulatory protein to VZV pathogenesis depends upon the differentiated human cell type which is targeted for infection within the intact tissue microenvironment.     

Nuclear import of the varicella-zoster virus latency-associated protein ORF63 in primary neurons requires expression of the lytic protein ORF61 and occurs in a proteasome-dependent manner

Varicella-zoster virus (VZV) open reading frame (ORF) 63 protein (ORF63p) is one of six VZV ORFs shown to be transcribed and translated in latently infected human dorsal root ganglia. ORF63p accumulates exclusively in the cytoplasm of latently infected sensory neurons, whereas it is both nuclear and cytoplasmic during lytic infection and following reactivation from latency. Here, we demonstrate that infection of primary guinea pig enteric neurons (EN) with an adenovirus expressing ORF63p results in the exclusive cytoplasmic localization of the protein reminiscent of its distribution during latent VZV infection in humans. We show that the addition of the simian virus 40 large-T-antigen nuclear localization signal (NLS) results in the nuclear import of ORF63p in EN and that the ORF63p endogenous NLSs are functional in EN when fused to a heterologous protein. These data suggest that the cytoplasmic localization of ORF63p in EN results from the masking of the NLSs, thus blocking nuclear import. However, the coexpression of ORF61p, a strictly lytic VZV protein, and ORF63p in EN results in the nuclear import of ORF63p in a proteasome-dependent manner, and both ORF63p NLSs are required for this event. We propose that the cytoplasmic localization of ORF63p in neurons results from NLS masking and that the expression of ORF61p removes this block, allowing nuclear import to proceed.     

Varicella-zoster virus IE62 protein utilizes the human mediator complex in promoter activation

The varicella-zoster virus (VZV) major transactivator, IE62, is involved in the expression of all kinetic classes of VZV genes and can also activate cellular promoters, promoters from heterologous viruses, and artificial promoters containing only TATA elements. A key component of the mechanism of IE62 transactivation is an acidic activation domain comprising the N-terminal 86 amino acids of IE62. However, the cellular target of this N-terminal acidic activation is unknown. In the work presented here, we show that the IE62 activation domain targets the human Mediator complex via the Med25 (ARC92) subunit and that this interaction appears to be fundamental for transactivation by the IE62 activation domain. In contrast, the Med23 subunit (Sur2/TRAP150beta/DRIP130/CRSP130) of the Mediator complex is not essential for IE62-mediated activation. Further, the IE62 activation domain appears to selectively interact with a form of the Mediator complex lacking CDK8. Chromatin immunoprecipitation experiments showed that IE62 stimulates recruitment of Mediator to an IE62-responsive model promoter. Finally, immunofluorescence microscopy of VZV-infected cells demonstrated intranuclear translocation of the Mediator complex to viral replication compartments. These studies suggest that Mediator is an essential component for efficient VZV gene expression.     

Recombinant monoclonal antibody recognizes a unique epitope on varicella-zoster virus immediate-early 63 protein

We previously constructed a recombinant monoclonal antibody (rec-MAb 63P4) that detects immediate-early protein IE63 encoded by varicella-zoster virus (VZV) in the cytoplasm of productively infected cells. Here, we used ORF63 truncation mutants to map the rec-MAb 63P4 binding epitope to amino acids 141 to 150 of VZV IE63, a region not shared with other widely used anti-IE63 antibodies, and found that the recombinant antibody does not bind to the simian IE63 counterpart.     

Infection by human varicella-zoster virus confers norepinephrine sensitivity to sensory neurons from rat dorsal root ganglia.

Varicella-zoster virus (VZV) is a widespread human herpes virus causing chicken pox on primary infection and persisting in sensory neurons. Reactivation causes shingles, which are characterized by severe pain and often lead to postherpetic neuralgia. To elucidate the mechanisms of VZV-associated hyperalgesia, we elaborated an in vitro model for the VZV infection of sensory neurons from rat dorsal root ganglia. Between 35 and 50% of the neurons showed strong expression of the immediate-early virus antigens IE62 and IE63 and the late glycoprotein gE. When the intracellular calcium concentration was monitored microfluorometrically for individual cells after infection, the sensitivity to GABA or capsaicin was similar in controls and in VZV-infected neurons. However, the baseline calcium concentration was increased. Neurons became de novo sensitive to adrenergic stimulation after VZV infection. Norepinephrine-responsive neurons were more frequent and calcium responses to norepinephrine were significantly higher after infection with wild-type isolates than with the attenuated vaccine strain OKA. The adrenergic agonists phenylephrine and isoproterenol had similar efficacy. We suggest that the infection with wild-type VZV isolates confers norepinephrine sensitivity to sensory neurons by using alpha(1)- and/or beta(1)-adrenergic receptors providing a model for the pathophysiology of the severe pain associated with the acute reactivation of VZV.     

Characterization of regulatory functions of the varicella-zoster virus gene 63-encoded protein.

Varicella-zoster virus (VZV) gene 63 encodes a protein (IE63) with a predicted molecular mass of 30.5 kDa which has amino acid similarities to the immediate-early (IE) protein 22 (ICP22) of herpes simplex virus type 1. ICP22 is a polypeptide synthesized in herpes simplex virus type 1-infected cells, and as is the case for its VZV counterpart, its regulatory functions are unknown. On the basis of the VZV DNA sequence, it has been shown that IE63 exhibits hydrophilic and acidic properties, suggesting that this protein could play a regulatory role during the infectious cycle. We report in this article cotransfection experiments which demonstrate that the VZV gene 63 protein strongly represses, in a dose-dependent manner, the expression of VZV gene 62. On the other hand, transient expression of the VZV gene 63 protein can promote activation of the thymidine kinase gene but cannot affect the expression of the genes encoding glycoproteins I and II. The results of transient expression experiments strongly suggest that the VZV gene 63 protein could play a pivotal role in the repression of IE gene expression as well as in the activation of early gene expression.