Possible Involvement of the Double-Stranded RNA-Binding Core Protein ςA in the Resistance of Avian Reovirus to Interferon

Abstract

Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein A, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein A antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins.The alpha/beta interferons (IFNs) are a family of multifunctional cytokines encoded by intronless genes, which are expressed and secreted by leukocytes and fibroblasts in response to viral infection and which have the same cell receptors (for recent reviews, see references 15, 22, 42, and 53). Extracellular IFNs bind to specific high-affinity cell surface receptors to trigger the activation of signal transduction pathways that, through a phosphorylation cascade, induce increased expression of the designated IFN-responsive genes (for reviews, see references 19, 43, 57, and 63). Three of the many alpha/beta IFN-inducible gene products have been shown to play an important role in fighting virus infection, namely, Mx proteins, the 2Ј,5Ј-oligoadenylate synthetase system (2-5A synthetase), and the double-stranded RNA (dsRNA)-activated protein kinase (PKR) (for reviews, see references 19, 28, 32, 39, 41, 45, 56, and 57). Mx proteins are a family of related GTPases that are thought to inhibit the viral polymerase activity of susceptible viruses (reviewed in reference 40). Both 2-5A synthetase and PKR antiviral pathways play a key role in the intracellular regulation of protein synthesis. Increased expression of these enzymes is induced by IFN, but they are latent until after activation by dsRNA (28, 45). The activated 2-5A synthetase catalyzes the synthesis of short oligonucleotides of the general structure ppp(A2Јp5Ј)nA. These oligonucleotides bind and stimulate a latent endoribonuclease, designated RNase L, to degrade both cellular and viral RNAs, thus preventing protein synthesis (for reviews, see references 44 and 54). Interaction of PKR with dsRNA results in autophosphorylation and dimerization of the...

The Second Open Reading Frame of the Avian Reovirus S1 Gene Encodes a Transcription-Dependent and CRM1-Independent Nucleocytoplasmic Shuttling Protein

“…Nuclear p17 may avoid triggering the host immune response by blocking nuclear signaling pathways. This strategy might disrupt the interferonmediated response and may be one of the reasons that avian reoviruses are so resistant to the antiviral effects of interferons (9,29). Another possibility is that nuclear p17 decreases cellular transcription to induce host cell shutoff or to divert biosynthetic resources from the nucleus to the cytoplasm, the site of avian reovirus replication.…”

“…The infection of CEF by avian reovirus S1133 and metabolic radiolabeling have been described previously (29). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed in 15% polyacrylamide gels as described previously (26).…”

Impact of Protein Kinase PKR in Cell Biology: from Antiviral to Antiproliferative Action

“…The resistance conferred by the capsid 3 protein is certainly incomplete, however, since mammalian reoviruses are not protected against high levels of PKR activity, as found in most untransformed cells and in IFN-treated cells. Avian reoviruses express the A protein, which has much stronger dsRNA-binding affinity than 3 and interferes efficiently with PKR function (130,242), corresponding with lower IFN sensitivity of avian in comparison with mammalian reoviruses (242,334,373).…”

Reverse genetics system for introduction of site-specific mutations into the double-stranded RNA genome of infectious rotavirus

“…Although significant cytopathic effect could not be observed in the first passage, MA104 cells in the second passage showed cytopathic effect, suggesting the generation of transfectant viruses with the rescued VP4 gene derived from cDNA. The rescued virus was biologically cloned by three successive plaqueto-plaque purifications in CV-1 cells to isolate clones and to exclude the possibility of sequestrated irrelevant dsRNAs by core particles as described for avian reovirus (23) and bluetongue virus (24). After amplification in MA104 cells, the virus particles of transfectant clones were purified by isopycnic CsCl gradient centrifugation and subsequent fractionation, followed by a single major band formation (data not shown).…”