| Viral infections often produce double-stranded RNA (dsRNA), which triggers potent antiviral responses, including repression of protein synthesis mediated by protein kinase R (PKR) and 2'--5' oligoA synthetase (OAS). Evasion of this response is vital for viral infection, and as a consequence many viruses have evolved genes that counteract these pathways. Human cytomegalovirus (HCMV) encodes two related proteins, pIRS1 and pTRS1, that bind dsRNA and prevent PKR and OAS activation. I have shown that an HCMV mutant lacking both pIRS1 and pTRS1 (HCMV[DeltaI/DeltaT]) has a severe replication defect due to its inability to prevent PKR activation, which results in the shutoff of protein synthesis and abrogates viral replication. These experiments demonstrated that expression of at least one of the HCMV dsRNA-binding proteins is absolutely required for viral replication. Interestingly, the OAS pathway is not activated during HCMV[DeltaI/DeltaT] infection, suggesting that the main role of pIRS1 and pTRS1 is in evading PKR. The mechanism of pIRS1 and pTRS1 function involves binding dsRNA via a domain in their conserved amino-terminal region and also binding PKR using a domain in their divergent carboxy-terminal region. Both of these interactions are required for pIRS1- or pTRS1-mediated rescue of vaccinia virus deleted for its dsRBP E3L (VVDeltaE3L). Our data support the hypothesis that pIRS1 and pTRS1 function by forming a trimolecular complex of one molecule of pIRS1 or pTRS1, one molecule of PKR, and dsRNA. This complex both sequesters dsRNA and prevents the PKR homodimerization that is required for activation. Additionally, pIRS1 and pTRS1 cause nuclear accumulation of PKR, suggesting that nuclear PKR might mediate the transcriptional activation function that has been ascribed to pIRS1 and pTRS1. Indeed, I established that the PKR-binding region of pIRS1 and pTRS1 is required for pIRS1/pTRS1-mediated transgene activation. However, when PKR expression is repressed, pIRS1 is not necessary for high levels of plasmid-derived transgene expression. Therefore, the transgene activation activity of IRS1 and TRS1 is most likely just a manifestation of the critical primary function of these proteins in repressing the PKR response. |
