We investigated the possible translational regulatory roles played by the interferon-induced, doublestranded-RNA-activated protein kinase (P68) and its natural substrate, eucaryotic initiation factor 2 (eIF-2), in poliovirus-infected cells. We demonstrated that protein kinase P68 was both highly autophosphorylated and activated during poliovirus infection. In accordance with these results, immunoprecipitation analysis revealed that phosphorylation of the endogenous eIF-2 alpha subunit also increased in poliovirus-infected cells. We found that double-stranded RNA synthesized during infection likely induced the high levels of P68 autophosphorylation. To determine whether the increase in kinase activity also could be attributed to induction of P68 synthesis, physical levels of protein kinase were measured. It was unexpectedly found that P68 protein levels did not increase but rather dramatically declined in poliovirus-infected cells. Pulse-chase experiments confirmed that the protein kinase was significantly degraded during virus infection. We corroborated our in vivo observations by developing an in vitro assay for P68 degradation using cell extracts. The possible consequences of P68 degradation and increased eIF-2 alpha phosphorylation for protein synthesis regulation in poliovirus-infected cells are discussed.

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Control of the interferon-induced double-stranded RNA (dsRNA) activated protein kinase (referred to as P68 because of its Mr of 68,000 in human cells) by animal viruses is essential to avoid decreases in protein synthetic rates during infection. We have previously demonstrated that poliovirus establishes a unique way of regulating the protein kinase, namely by inducing the specific degradation of P68 during infection (T. L.

We investigated the mechanisms by which influenza virus prevents shutoff of protein synthesis by a cellular protein kinase normally activated during infection. Earlier work has shown that influenza virus superinfection of cells previously infected by the adenovirus VAI RNA-negative mutant dl331 resulted in selective translation of influenza virus mRNAs and suppression of the elevated protein kinase levels normally found in cells infected by the mutant alone (M. G. Katze, B. M. Detjen, B. Safer, and R. M. Krug, Mol. Cell. Biol. 6:1741-1750, 1986). We elucidated the mechanisms of this kinase repression and can now report that influenza virus encodes a gene product which functions to directly block the autophosphorylation and activity of the interferon-induced, double-stranded-RNA-activated protein kinase, P68. Suppressed P68 activity was found not only in doubly infected cells but also in cells infected by influenza virus alone. Moreover, the decrease in P68 activity correlated with a decrease in the endogenous levels of phosphorylation of the alpha subunit of the eucaryotic initiation factor eIF-2, the natural substrate of the protein kinase. Suppression of P68 activity occurred as early as 2 h after influenza virus infection and required viral gene expression beyond the level of primary mRNA transcription to take place. We confirmed our in vivo observations with in vitro mixing experiments which showed that the influenza virus inhibitor can act in trans to block P68 activity. Combined repression of P68 function and eIF-2 alpha phosphorylation during influenza virus infection is essential for continued catalytic recycling of eIF-2 and efficient mRNA translation.