Characterization of the anti-viral effects inhibited by the vaccinia virus K1 protein

Vaccinia virus (VV), a member of the poxvirus family of double-stranded DNA viruses, is well-known as a highly effective vaccine against variola virus, the causative agent of smallpox. Poxviruses encode many strategies to evade the host immune and anti-viral response. One such host anti-viral response, activation of the double-stranded RNA activated protein kinase (PKR) pathway, is important in sensing the presence of intracellular viral genomes and their products. PKR is activated by dsRNA, a by-product of many virus infections, and exhibits its anti-viral effects in part via inhibition of protein synthesis and activation of the pro-inflammatory transcription factor NF-κB. I have identified the vaccinia virus K1 protein as an inhibitor of PKR activation. I determined that the C-terminal portion of the 2nd ankyrin repeat, a motif important for protein-protein interactions, is important for this inhibitory function. Further, K1 mediated PKR inhibition also blocks activation of downstream NF-κB in VV infected cells. Previous characterization of the K1 protein identified it as a host-range protein required for a productive infection. When the K1L gene is absent, replication is aborted due to shut-down in viral protein synthesis. I queried whether PKR was responsible for this effect. I found that replication when K1 was lacking could not be rescued by depletion of PKR protein levels. Hence K1 inhibition of PKR is not related to the host-range function. I next identified that the trigger for PKR activation is viral dsRNAs derived from early or intermediate viral transcription. As early dsRNAs have never been reported during a poxvirus infection, this finding was novel. Further, I found that there were higher levels of dsRNA present during a VV infection when K1 was absent or mutated. Finally, ectopic expression of K1 was able to inhibit PKR activation induced by viral dsRNAs. Together these data identifies a new function for the K1 protein and elucidates a strategy viruses utilize to inhibit detrimental host cell responses