This study identifies VZV-encoded modulation of the NF-κB signaling pathway within infected DCs and identifies the viral gene product of ORF61 as functioning to inhibit this important signal transduction pathway. Given that DCs are proposed as the first immune cells to encounter VZV following inoculation, these results indicate that ORF61 plays an important role in immune evasion of the NF-κB signal transduction pathway. This pathway controls transcription of many immune molecules required to initiate an immune response to foreign pathogens, and so disruption of this pathway is likely to suppress critical immune effector capacity of the host cell.
Other herpesviruses modulate the NF-κB pathway in a cell type-dependent manner (3
). Our work indicates VZV modulates the NF-κB pathway within DCs. The cytoplasmic retention of NF-κB protein subunits p50 and p65 indicates that the pathway is modulated prior to their translocation into the nucleus, as shown by immunofluorescence and confocal microscopy. These results are distinct from those reported for HSV-1 infection, where NF-κB proteins are translocated into the nucleus of HSV-1-infected C33-A cells (24
). The NF-κB pathway in VZV-infected DCs was not modulated by downregulation of TLR3, TLR8, TLR9, TNFR-1, or TNFR-2, which signal the activation of the NF-κB pathway, since these were unaffected by VZV infection. This finding is consistent with VZV interfering with NF-κB signaling at a point downstream of cell surface (TNFR-1 and TNFR-2) or intracellular receptors which trigger this pathway (TLR3, TLR8, and TLR9).
A critical component of activation of the NF-κB pathway is phosphorylation of IκBα (5
). We therefore explored the phosphorylation state of IκBα in infected DCs. The increase in phosphorylated IκBα within VZV-infected DCs indicated that the NF-κB pathway was able to be activated at this level following infection. In addition, IκBα protein levels within DCs did not decrease following infection with VZV. Thus, IκBα can be phosphorylated and is not necessarily degraded following VZV infection of DCs. The NF-κB pathway is therefore apparently inhibited following phosphorylation of IκBα but prior to translocation of NF-κB p50 and p65 into the nucleus of VZV-infected DCs. The identification of this step is under further consideration.
We identified VZV ORF61 as a gene responsible for inhibiting TNF-α-stimulated NF-κB reporter expression. VZV ORF61 is transcribed within 1 h of infection; however, it is not a component of the virion, supporting reports of UV-inactivated VZV being unable to inhibit the NF-κB pathway within fibroblasts and that de novo
gene synthesis is required (17
). ORF61 has also been implicated in the modulation of other cellular pathways, such as the mitogen-activated protein kinase (MAPK) pathways where a significant increase in the phosphorylation of JNK/SAPK and a decrease in p38/MAPK phosphorylation were observed in MeWo cells transfected with ORF61 (25
). ORF61 also downmodulates the IRF3-mediated IFN-β pathway by its direct binding to and degradation of IRF3 (35
). We demonstrate that when ORF61 is expressed alone, it is sufficient to inhibit TNF-α-induced IκBα degradation, further defining the role of ORF61 in NF-κB pathway inhibition. We also showed that the E3 ubiquitin ligase domain of ORF61 is essential for NF-κB pathway inhibition, since mutations within this domain rendered ORF61 unable to inhibit NF-κB reporter activity. HSV-1 ICP0 and VZV ORF61 share homology within their RING finger domains, which are involved in E3 ubiquitin ligase activity (21
). ICP0 modulates the NF-κB pathway in a cell type-dependent manner, as shown by transfection of ICP0 expression constructs into SHSY-5Y and HEK293 cells. Within HEK293 cells stably expressing either TLR2 or TLR4, ICP0 causes inhibition of TLR-mediated NF-κB signaling (9
). ICP0 inhibits the NF-κB pathway within these cells by binding and transporting the host cell protein USP7, a ubiquitin-specific protease, to the cytoplasm, where it deubiquitinates TRAF6 and IKKγ. The USP7 binding site on ICP0 is within the C-terminal region; due to the homology of ICP0 and ORF61 being restricted to the RING finger domain in the N terminus, it is unlikely that ORF61 encodes this USP7 binding region and therefore inhibits the NF-κB pathway in a manner different from that of its HSV-1 homologous protein in HEK293/TLR cells. Expression of ICP0 within SHSY-5Y cells stimulated NF-κB reporter activity through the E3 ubiquitin ligase action of ICP0, resulting in ubiquitination and subsequent degradation of IκBα (10
). Our demonstration that the E3 ubiquitin ligase domain of ORF61 is responsible for the inhibition of NF-κB reporter activity indicates that it may be the process of ubiquitination causing the effect; however, protein expression analysis of IκBα within VZV-infected DCs suggests that IκBα is not degraded. The Vpu protein of HIV, however, blocks proteasome-dependent degradation of IκBα by binding to βTRCP in the E3 ubiquitin ligase complex that is involved in the regulated degradation of IκBα (7
). This suggests that the E3 ubiquitin ligase activity of ORF61 may have an indirect effect on NF-κB proteins.
Although we have shown that ORF61 is a viral factor that downregulates NF-κB, we have not yet verified whether it is the only viral factor able to do this. This requires the construction of VZV that lacks the ORF61 protein, particularly in the amino-terminal ring finger domain. Such mutants have proven very difficult to develop. Using a VZV bacterial artificial chromosome (BAC) system, we found that VZV BAC constructs engineered to contain the amino acid substitution within the RING finger domain of ORF61 (C19G) or a stop codon inserted directly after the RING finger domain or a stop codon inserted directly following the E3 ubiquitin ligase domain all resulted in either failure to obtain any recombinant VZV or the reversion of the point mutation to the wild type (M. B. Yee and P. Kinchington, unpublished data). This mirrors previous reports of ORF61 playing an important role in virus replication (8
). Thus, although ORF61 has been identified as functioning to inhibit the NF-κB pathway, it remains possible that other VZV gene products may also encode a similar function. In addition, whether the E3 ubiquitin ligase activity of ORF61 exerts a direct effect on NF-κB proteins or whether it impacts on the cellular regulators of these transcription factors will be an important focus of future work to delineate the mode of action of ORF61 in suppressing NF-κB signaling. The continued presence of phosphorylated IκBα within VZV-infected DCs indicates that it is not being degraded. Therefore, it would be useful to also investigate ubiquitin/protease pathways that control the degradation of IκBα following NF-κB pathway activation.
In summary, we identify here inhibition of the NF-κB signal transduction pathway in VZV-infected human MDDCs, which occurs following phosphorylation of IκBα, but prior to the translocation of NF-κB p50 and p65 into the nucleus. We also demonstrate that the immediate-early gene ORF61 inhibits TNF-α-stimulated NF-κB reporter expression, indicating that this viral gene can inhibit NF-κB pathway activation. Furthermore, we show that the region of ORF61 important for its inhibitory affects on TNF-α-stimulated NF-κB reporter activity is the E3 ubiquitin ligase domain, indicating that it is the process of ubiquitination that causes this pathway inhibition. During the preparation of this manuscript, Wang et al. (32
) presented findings that ORF61 binds SUMO-1 via three SUMO-interacting motifs (SIMs) and that these SIMs were required for ORF61 binding to and disrupting of PML nuclear bodies (32
). That study also reported that ORF61 can act as an inhibitor of TNF-α-induced NF-κB reporter activity in a transient-transfection assay within a melanoma cell line, supporting the results presented here. VZV-encoded modulation of the NF-κB pathway may be the mechanistic basis for the observed downregulation of immune molecules in VZV-infected DCs, an important immune evasion strategy of VZV.