Binding and nuclear relocalization of protein kinase R by human cytomegalovirus TRS1

The human cytomegallovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2 alpha) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (W) deleted of the double-stranded RNA binding protein gene E3L (VV Delta E3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2 alpha kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VV Delta E3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type W or a WAE3L recombinant virus expressing EM. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VV Delta E3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VV Delta E3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2 alpha.