Double-stranded RNA binding by the human cytomegalovirus PKR antagonist TRS1

•TRS1 specifically binds dsRNA as short as 20 base pairs.•TRS1 binds dsRNA with a weaker affinity than PKR.•TRS1 is more abundant than PKR when dsRNA is present during infection.•TRS1 must bind to dsRNA at least weakly to antagonize PKR.•dsRNA and PKR binding are separable functions of TRS1.

Protein Kinase R (PKR) inhibits translation initiation following double-stranded RNA (dsRNA) binding and thereby represses viral replication. Human cytomegalovirus (HCMV) encodes two noncanonical dsRNA binding proteins, IRS1 and TRS1, and the expression of at least one of these PKR antagonists is essential for HCMV replication. In this study, we investigated the role of dsRNA binding by TRS1 in PKR inhibition. We found that purified TRS1 binds specifically to dsRNA with an affinity lower than that of PKR. Point mutants in the TRS1 dsRNA binding domain that were deficient in rescuing the replication of vaccinia virus lacking its PKR antagonist E3L were unable to bind to dsRNA but retained the ability bind to PKR. Thus TRS1 binding to dsRNA and to PKR are separable. Overall, our results are most consistent with a model in which TRS1 binds simultaneously to both dsRNA and PKR to inhibit PKR activation.