Inhibition of the protein kinase R pathway by cytomegalovirus double-stranded RNA binding proteins
Bierle, Craig John
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Double-stranded RNA (dsRNA) that accumulates during many viral infections is recognized by the host cell and elicits an antiviral response. Protein kinase R (PKR) is activated by dsRNA binding, phosphorylates eIF2α, and inhibits translation initiation, potentially blocking the synthesis of viral proteins. Human cytomegalovirus (HCMV) expresses two noncanonical dsRNA binding proteins, TRS1 and IRS, that antagonize PKR. In this study, I investigated the dsRNA-binding properties of TRS1 and related proteins to help clarify their mechanisms of PKR inhibition and roles in viral replication. I found that purified TRS1 specifically bound to dsRNAs as short as 20 base pairs, albeit with a weaker affinity than PKR. However, TRS1 was over 10-fold more abundant than PKR at late times during infection when detectable levels of dsRNA are present. I mutagenized the TRS1 dsRNA binding domain and identified TRS1 variants that impaired dsRNA binding. The TRS1 mutants most deficient for dsRNA binding did not support the replication of vaccinia virus deleted of its PKR antagonist E3L (VVdeltaE3L) but did retain the ability to bind PKR. This indicates that while dsRNA and PKR binding are separate functions of TRS1, TRS1 must at least weakly bind dsRNA to antagonize PKR. These results are most consistent with a model in which TRS1 forms a trimolecular complex with dsRNA and PKR to prevent PKR activation. However, it remains possible that the abundance of TRS1 enables it to sequester dsRNA, thereby blocking PKR and other dsRNA-activated pathways. I also investigated whether guinea pig cytomegalovirus (GPCMV), a model of congenital CMV infection, encodes a PKR antagonist. Of the GPCMV dsRNA binding proteins that I identified in a proteomic screen, only gp145 rescued VVdeltaE3L replication. gp145 reversed the effects of PKR on transfected gene expression and reduced the levels of PKR and eIF2 α phosphorylation in VVdeltaE3L infected cells, demonstrating gp145's capacity to inhibit PKR. Mapping studies demonstrated that gp145 has a dsRNA binding domain homologous to that of TRS1, but also that dsRNA binding by gp145 is not sufficient for PKR inhibition. These studies have demonstrated that dsRNA-binding is a conserved and essential function of the CMV PKR antagonists.