Oxaliplatin br Results br Discussion PKR
Discussion PKR is activated by viral dsRNA and exerts its antiviral effect through phosphorylation of eIF2a. Because eIF2a phosphorylation leads to attenuation of protein synthesis, many viruses have evolved mechanisms to block or suppress PKR activation. Some viruses use alternative translational mechanisms. For example, caliciviruses encode viral proteins that act as cap-analogs while picornaviruses initiate translation from an internal ribosome entry site (IRES) in an eIF2a-independent manner (Lopez-Lastra et al., 2010). Other viruses produce a small RNA or a viral protein that either directly or indirectly inhibits PKR activity (Garcia et al., 2007). WNV genome RNA translation is cap-dependent and so would be susceptible to eIF2a-mediated translation inhibition. However, shut-down of host translation does not occur in flavivirus infected Oxaliplatin (Lindenbach et al., 2007). We previously reported only low levels of eIF2a phosphorylation in WNV-infected BHK cells (Emara and Brinton, 2007). Consistent with this observation, no increase in PKR phosphorylation in WNV-infected BHK cells and only a slight increase in infected wild type MEFs mediated by IFN was observed in the present study. The authors of a previous study done with WNV virus-like particles (VLPs) containing WNV replicons concluded that PKR activation provides antiviral protection against WNV (Gilfoy and Mason, 2007). However, a similar number of foci forming units (FFU) were detected in wild type and PKR−/− MEFs 48h after infection with WNV VLPs and IFN-treatment led to a reduction in FFUs in wild type but not in PKR−/− MEFs suggesting that the PKR activation observed was mediated by IFN and not by WNV dsRNA. The results of this previous study are consistent with those of the present study. An additional previous study, that utilized VLPs containing a WNV replicon with a C-terminal EMCV IRES driving translation of a neomycin gene ORF (Jiang et al., 2010), reported a reduction in virus yield from cells overexpressing PKR compared to control cells. The EMCV IRES was previously shown to activate PKR in vitro and in vivo (Arnaud et al., 2010, Shimoike et al., 2009) and may have contributed to the PKR activation observed. Suppression of PKR by known cellular inhibitors requires association with PKR (Garcia et al., 2007). Among the cellular inhibitors tested, only Nck colocalized with PKR that concentrated in the perinuclear region of WNV-infected cells. Since Nck interacts with inactive PKR, the detection of Nck colocalizing with PKR in the perinuclear region of infected cells suggested that the majority of the PKR in this region is in an inactive state. Co-immunoprecipitation of Nck and PKR in both infected and uninfected cells suggested that this interaction was not disrupted by WNV infection. It was previously reported that a minimum of 2μg/ml of poly(I:C) was sufficient to outcompete Nck binding to PKR (Cardin and Larose, 2008). However, even though the WNV genome RNA and the 3′sfRNA, which were shown to contain several structures that could activate PKR in vitro, as well as viral dsRNA replicative intermediates increase exponentially between 6 and 24h after infection, these viral RNAs did not compete with Nck for binding to PKR. The minimal activation of PKR detected in WNV-infected Nck1,2−/− MEFs provided additional evidence that PKR was not activated by viral dsRNA in infected cells and the observation that poly(I:C) induced significant PKR phosphorylation in WNV-infected cells suggested that PKR phosphorylation is not actively suppressed in WNV-infected cells. The similar virus yields produced by wildtype and PKR−/− MEFs infected with WNV provided additional confirmation that PKR does not mediate significant antiviral activity in WNV-infected cells. PKR was reported to associate with ER membranes (Garcia et al., 2007) and the perinuclear concentration of PKR observed in WNV-infected cells could be a by-stander effect of virus-directed ER membrane rearrangement rather than due to recruitment by a viral component.