Viral infections often produce double-stranded RNA (dsRNA), which in turn triggers potent antiviral responses, including the global repression of protein synthesis mediated by protein kinase R (PKR) and 2′-5′ oligoadenylate synthetase (OAS). As a consequence, many viruses have evolved genes, such as those encoding dsRNA-binding proteins, which counteract these pathways. Human cytomegalovirus (HCMV) encodes two related proteins, pTRS1 and pIRS1, which bind dsRNA and can prevent activation of the PKR and OAS pathways. HCMV mutants lacking either IRS1 or TRS1 replicate at least moderately well in cell culture. However, as we demonstrate in the present study, an HCMV mutant lacking both IRS1 and TRS1 (HCMV[ΔI/ΔT]) has a severe replication defect. Infection with HCMV[ΔI/ΔT] results in a profound inhibition of overall and viral protein synthesis, as well as increased phosphorylation of eukaryotic initiation factor 2α (eIF2α). The vaccinia virus E3L gene can substitute for IRS1 or TRS1, enabling HCMV replication. Despite the accumulation of dsRNA in HCMV-infected cells, the OAS pathway remains inactive, even in HCMV[ΔI/ΔT]-infected cells. These results suggest that PKR-mediated phosphorylation of eIF2α is the dominant dsRNA-activated pathway responsible for inhibition of protein synthesis and HCMV replication in the absence of both IRS1 and TRS1 and that the requirement for evasion of the PKR pathway likely explains the necessity for IRS1 or TRS1 for productive infection.