Our results demonstrated that WNV inhibits one of the first steps of the IFN signal transduction pathway and, hence, provided an explanation for previous observations made with WNV-infected mice and tissue culture cells (5
). Interactions between viral proteins and components of the innate immune response are major determinants in viral pathogenesis. This fact is best exemplified with genetically modified mice that are deficient in their response to IFN-α due to the lack of functional IFN-α/β receptors or STAT proteins and, as a consequence, rapidly succumb to viral infections (9
). On the other hand, most, if not all, viruses depend on mechanisms to attenuate the IFN response for their survival (22
). For example, certain parainfluenza viruses can inhibit the IFN pathway in human cells with the help of the accessory protein V that induces the proteasome-dependent degradation of STAT proteins (8
The exact mechanism by which WNV inhibits the IFN response is not yet known. Our results showed that the structural proteins are not required and, hence, by inference, have invoked a role for one or several NS proteins in blocking the activation of the innate immune response. However, it is also possible that the mechanism of inhibition was indirect. For example, it could have been caused by changes in the cytoplasm of infected cells that are known to occur as a consequence of viral replication. These changes include the induction and rearrangement of intracellular membranes (25
). Because HCV replication induces similar changes (10
) and yet is very sensitive to IFNs (Fig. ), we do not favor this possibility. Instead, we propose the following scenarios to explain our results. First, NS proteins may directly bind to the IFN receptor and block the activation of the Janus kinases. This model would imply that the NS viral proteins bind to both IFN receptors, because both IFN responses were inhibited albeit with slightly different efficiencies (Fig. , , and ). Alternatively, NS proteins could directly interact with JAK1 and Tyk2 and inhibit their tyrosine kinase activities, perhaps similar to the V proteins of human parainfluenza viruses as described above. In fact, we observed a more profound inhibition of Tyk2 than of JAK1, which could be caused by differences in the affinity of one or more NS proteins for the two kinases. Second, WNV replication may lead to the activation of negative feedback loops that could inhibit or reverse JAK1 and Tyk2 phosphorylation. The suppressor of cytokine signaling-3 (SOCS3) or the protein tyrosine phophatases, such as the Src homology 2 (SH2) domain-containing protein tyrosine phophatases (SHP-2), are known members of such pathways (18
). For example, herpes simplex virus type I (HSV1) was recently shown to induce SOCS3 expression leading to the inhibition of the IFN response (37
). Finally, inhibition may occur through a reduction in the surface expression of IFN receptors. Although a comparison of IFNAR2 present on the surface of parental HeLa and KUNCD20 cells by flow cytometry analysis revealed a slightly lower expression in KUNCD20 cells, similar comparison with normal and WNV-infected Vero cells did not reveal any substantial differences (results not shown). Moreover, a Western blot analysis of IFNRA1 expression did not reveal any significant alteration in KUNCD20 and WNV-infected Vero cells compared with normal HeLa and Vero cells (Fig. and results not shown). Hence, based on these results, we consider this possibility unlikely.
While this study was in progress, Muñoz-Jordán et al. (29
) reported that the Dengue virus replication could inhibit the phosphorylation of STAT1 and, hence, explain the previously observed inhibition of the IFN response. Moreover, these authors provided evidence for a role of the NS protein 4B in the modulation of Stat1 phosphorylation. However, this activity was relatively weak compared with that of other known inhibitors of the IFN response and was amplified when NS4B was coexpressed with other NS proteins. To identify the WNV protein(s) responsible for the observed inhibition of the IFN signaling pathway, we used a similar approach and transfected HeLa cells with plasmids encoding individual NS proteins. Unfortunately, our efforts have so far not yielded any positive results (results not shown). It is possible that under our selected conditions, the expression levels of individual NS proteins were lower than those obtained with Dengue NS proteins and those present in KUNCD20 or WNV-infected cells. Alternatively, it is conceivable that during natural replication viral proteins are sequestered in a manner that was not reproduced under our experimental conditions.
The physiological relevance of the well-documented resistance of WNV to IFN treatment can so far only be explained by inference based on other systems, because viral mutants that have lost the ability to block the JAK-STAT pathway are not yet available. One of the best examples is herpes simplex virus, where a functional relationship between the viral infected cell protein 34.5 (ICP34.5) and the IFN-inducible double-stranded RNA-dependent protein kinase R exists that controls viral replication (20
). Interestingly, Gale and colleagues have just reported that WNV could delay the expression of IFN-β (11
). The expression of this gene is controlled by a pathway that depends on its activation by proteins that can sense virus particles or products of replication, such as certain viral RNA structures or viral proteins. An example of a sensor would be the recently identified retinoic acid inducible gene 1 (RIG-1) (38
). Hence, under physiological conditions, WNV might inhibit more than one cellular pathway of the innate immune response.
In summary, our study revealed the mechanism by which WNV inhibits the cellular innate immune response elicited by IFN-α and IFN-γ and provided an explanation for the previously observed resistance of this virus to IFN treatment in animals and cell cultures. These findings set the stage for the identification of the viral proteins and cellular factors that control the innate immune reaction and, hence, will provide opportunities to better understand novel virus-host interactions.