title: Viral double-stranded RNAs from vaccinia virus early or intermediate gene transcripts possess PKR activating function, resulting in NF-kappaB activation, when the K1 protein is absent or mutated.
abstract: PKR is a potent antiviral molecule that can terminate infection by inhibiting protein synthesis and stimulating NF-κB activation and apoptosis. Originally, it was thought that only intermediate and late gene transcription produced double-stranded (ds) RNA to activate PKR during vaccinia virus (VACV) infection. The VACV E3 or K3 proteins squelch this effect by binding to either dsRNA or PKR. However, in the absence of the K1 protein, VACV infection activates PKR at very early times post-infection and despite the presence of E3 and K3. These data suggest that VACV infection induces PKR activation by a currently unknown mechanism. To determine this mechanism, cells were infected with K1L-containing or -deficient VACVs. By using conditions that limited the progression of the poxvirus replication cycle, we observed that early gene transcripts activated PKR in RK13 cells, identifying a new PKR-activating mechanism of poxvirus infection. Using a similar approach for HeLa cells, intermediate gene transcription was sufficient to activate PKR. RNA isolated from infected RK13 or HeLa cells maintained PKR-activating properties only when dsRNA was present. Moreover, viral dsRNA was directly detected in infected cells either by RT-PCR or immunofluorescent microscopy. Interestingly, dsRNA levels were higher in infected cells in which the K1 protein was nonfunctional. Only K1 proteins with PKR inhibitory function prevented downstream NF-κB activation. These results reveal a new PKR activation pathway during VACV infection, in which the K1 protein reduces dsRNA levels early in VACV infection to directly inhibit PKR and several of its downstream antiviral effects, thereby enhancing virus survival.
authors.affiliation: From the Department of Microbiology, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
authors.forename: Kristen L
authors.initials: KL
authors.lastname: Willis
authors.affiliation: the Department of Naturopathic Research, Southwest College of Naturopathic Medicine, Tempe, Arizona 85282, and; the Biodesign Institute, Arizona State University, Tempe, Arizona 85287.
authors.forename: Jeffrey O
authors.initials: JO
authors.lastname: Langland
authors.affiliation: From the Department of Microbiology, College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,. Electronic address: jshisler@illinois.edu.
authors.forename: Joanna L
authors.initials: JL
authors.lastname: Shisler
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D014158:Transcription, Genetic / Q000235:genetics; D014615:Vaccinia / Q000235:genetics / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C083205:K1L protein, Vaccinia virus; D016328:NF-kappa B; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D014764:Viral Proteins; D051057:Proto-Oncogene Proteins c-akt