Varicella-Zoster Virus Inhibition of the NF-κB Pathway during Infection of Human Dendritic Cells: Role for Open Reading Frame 61 as a Modulator of NF-κB Activity

Abstract

V aricella-zoster virus (VZV) is an alphaherpesvirus causing chickenpox (varicella) during primary infection and shingles (herpes zoster) following reactivation from a latent infection. Following initial exposure to the virus, there is a 10-to 21-day incubation period before the appearance of the varicella rash. During this time it has been proposed that VZV actively evades immune recognition in this period, since the development of adaptive immunity is delayed (reviewed in reference 1). We have postulated that VZV infection of dendritic cells (DCs) and/or modulation of the immune function of these potent antigen-presenting cells would provide a strategy that would enhance the capacity of the virus to be transported from the site of inoculation to the draining lymph nodes to infect T cells while also evading immune detection.We have previously shown that VZV can productively infect human DCs in vitro and in vivo (2,16,22). These studies included demonstration that productively infected immature monocytederived DCs (MDDCs) are unable to upregulate the functionally important immune molecules CD80, CD83, CD86, major histocompatibility complex I, and CCR7, which are required for DC maturation and induction of an effective antiviral immune response (2). The expression of the immune molecules inhibited by VZV are largely regulated by the nuclear factor B (NF-B) signal transduction pathway (4,6,(12)(13)(14). The NF-B signal transduction pathway is an important regulator of innate immunity and inflammation that is triggered by a wide variety of stimuli, including virus infection, tumor necrosis factor alpha (TNF-␣), and other cytokines and pathogens (26,29). Activation of the NF-B pathway via pattern recognition receptors results in the phosphorylation of inhibitor of B kinase complex (IKK), which in turn phosphorylates IB, targeting it for ubiquitination and degradation, allowing NF-B proteins (p50 and p65) to translocate into the nucleus and bind to promoters containing NF-B response elements, initiating transcription of target genes (reviewed in references 26 and 29).Herpesviruses encode multiple proteins that function in immune evasion, and several herpesvirus proteins target and disrupt the NF-B pathway. Viral genes encoded by 27,28), cytomegalovirus (23,34), and herpes simplex virus 1 (HSV-1) (3, 9, 24) have been identified to regulate the NF-B pathway in a cell type-dependent manner. Jones and Arvin (17) reported that VZV inhibits the NF-B pathway in human fibroblasts in vitro and in vivo following the phosphorylation and ubiquitination of IB␣ but prior to the translocation of NF-B proteins into the nucleus.In the present study, we sought to extend these studies and examine the effect of VZV on the NF-B pathway within VZVinfected human MDDCs. Using flow cytometry, immunofluorescent staining, and Western blotting, we establish the point where VZV impacts the NF-B pathway in VZV antigen-positive DCs. In addition, using a transient-transfection approach and flow cytometry, we identified the E3 ubiquitin ligase domai...

The ORF61 Protein Encoded by Simian Varicella Virus and Varicella-Zoster Virus Inhibits NF-κB Signaling by Interfering with IκBα Degradation

“…In the present study, we demonstrate that, similar to VZV, infection with SVV leads to the stabilization of IB␣ (20,21). We further demonstrate that SVV ORF61, which shares 42.8% amino acid identity with VZV ORF61 (4) and regulates viral gene expression (32), also prevents IB␣ degradation.…”

“…Viral plaques appeared approximately 7 days posttransfection. (20,21). To determine whether SVV similarly interferes with NF-B signaling, we studied TNF-␣-induced NF-B activation in SVV-infected TRFs that stably express firefly luciferase under the control of an NF-B promoter and constitutively express Renilla luciferase (TRF NF-B).…”

“…The inhibition of NF-B activation observed in VZV-infected cells has been attributed to the ORF61 protein (21). VZV and SVV ORF61 proteins share 42.8% overall amino acid identity (4), and both proteins are implicated in the transactivation of expression of other viral genes (26,32).…”

“…The RING domain of VZV ORF61 has E3 ubiquitin ligase activity in vitro (30,31). This domain was found to be essential for the degradation of phosphorylated IRF3 (22) and for the inhibition of TNF-␣-induced NF-B signaling (21). Mutation of the cysteine at position 19 to a glycine residue (C19G) was shown to disrupt the RING domain of ORF61 and the protein's E3 ubiquitin ligase activity (28,31).…”

“…For VZV, it was shown that the virus interferes with TNF-␣-induced NF-B promoter activity by preventing the degradation of IB␣ (20). This interference seems to be at least partially mediated by ORF61, since Sloan et al showed that ORF61 expression led to the stabilization of IB␣ in TNF-␣-treated HEK 293T cells (21). However, Zhu et al reported that overexpression of ORF61 had only a minor effect on Sendai virus-induced NF-B signaling, indicating a pathway-specific countermechanism (22).…”