The cellular response to viral infection often includes activation of pathways that shut off protein synthesis and thereby inhibit viral replication. In order to enable efficient replication, many viruses carry genes such as the E3L gene of vaccinia virus that counteract these host antiviral pathways. Vaccinia virus from which the E3L gene has been deleted (VVDeltaE3L) is highly sensitive to interferon and exhibits a restricted host range, replicating very inefficiently in many cell types, including human fibroblast and U373MG cells. To determine whether human cytomegalovirus (CMV) has a mechanism for preventing translational shutoff, we evaluated the ability of CMV to complement the deficiencies in replication and protein synthesis associated with VVDeltaE3L. CMV, but not UV-inactivated CMV, rescued VVDeltaE3L late gene expression and replication. Thus, complementation of the VVDeltaE3L defect appears to depend on de novo CMV gene expression and is not likely a result of CMV binding to the cell receptor or of a virion structural protein. CMV rescued VVDeltaE3L late gene expression even in the presence of ganciclovir, indicating that CMV late gene expression is not required for complementation of VVDeltaE3L. The striking decrease in overall translation after infection with VVDeltaE3L was prevented by prior infection with CMV. Finally, CMV blocked both the induction of eukaryotic initiation factor 2alpha (eIF2alpha) phosphorylation and activation of RNase L by VVDeltaE3L. These results suggest that CMV has one or more immediate-early or early genes that ensure maintenance of a high protein synthetic capacity during infection by preventing activation of the PKR/eIF2alpha phosphorylation and 2-5A oligoadenylate synthetase/RNase L pathways.