The Adenovirus E1B 55-Kilodalton and E4 Open Reading Frame 6 Proteins Limit Phosphorylation of eIF2α during the Late Phase of Infection

During a productive infection, species C adenovirus reprograms the host cell to promote viral translation at the expense of cellular translation. The E1B 55-kilodalton (E1B-55K) and E4 open reading frame 6 (E4orf6) proteins are important in this control of gene expression. As part of a ubiquitin-protein ligase, these viral proteins stimulate viral mRNA export, inhibit cellular mRNA export, promote viral gene expression, and direct the degradation of certain host proteins. We report here that the E1B-55K and E4orf6 proteins limited phosphorylation of eIF2 alpha and the activation of the eIF2 alpha kinase PKR. Phospho-eIF2 alpha levels were observed to rise and fall at least twice during infection. The E1B-55K and E4orf6 proteins prevented a third increase at late times of infection. PKR appeared to phosphorylate eIF2 alpha only in the absence of E1B-55K/ E4orf6 function. PKR activation and eIF2 alpha phosphorylation was unrelated to the cytoplasmic levels of the adenovirus inhibitor of PKR, VA-I RNA. Nonetheless, expression of a PKR inhibitor, the reovirus double-stranded RNA-binding protein sigma 3, prevented PKR activation and eIF2 alpha phosphorylation. The sigma 3 protein largely corrected the defect in viral late protein synthesis associated with the E1B-55K and E4orf6 mutant viruses without affecting cytoplasmic levels of the late viral mRNA. The ubiquitin-protein ligase activity associated with the E1B-55K/ E4orf6 complex was necessary to prevent activation of PKR and phosphorylation of eIF2 alpha. These findings reveal a new contribution of the E1B-55K/ E4orf6 complex to viral late protein synthesis and the existence of multiple layers of regulation imposed on eIF2 alpha phosphorylation and PKR activation in adenovirus-infected cells.