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

Abstract

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␣ and the activation of the eIF2␣ kinase PKR. Phospho-eIF2␣ 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␣ only in the absence of E1B-55K/E4orf6 function. PKR activation and eIF2␣ 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␣ 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␣. 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␣ phosphorylation and PKR activation in adenovirus-infected cells.

Increased Susceptibility to DNA Virus Infection in Mice with a GCN2 Mutation

“…MCMV proteins m142 and m143 bind to PKR, possibly in conjunction with dsRNA, to prevent PKR activation (5). The noncoding virusassociated RNA molecule I (VAI RNA) of adenovirus binds to PKR and blocks its activation (26), while E1B-55K and E4orf6 proteins prevent PKR activation through a ubiquitin ligasedependent mechanism (33). Our findings clearly demonstrate that GCN2 deficiency increases susceptibility to MCMV infection in vitro and in vivo, albeit only slightly, and raise the possibility that viral mechanisms also have evolved to oppose eIF2␣ phosphorylation specifically by GCN2.…”

“…The existence of an eIF2␣ kinase specialized to sense and respond to viral infection underscores the effectiveness of this antiviral strategy. Indeed, many viruses have evolved countermeasures to PKR signaling (6,15,24,29,33,37).A few reports also have implicated eIF2␣ phosphorylation by PERK and GCN2 in antiviral responses. PERK is activated during herpes simplex virus 1 infection, likely as a result of accumulated viral proteins in the ER (7) and during vesicular stomatitis virus infections through an unknown mechanism (3).…”

“…The existence of an eIF2␣ kinase specialized to sense and respond to viral infection underscores the effectiveness of this antiviral strategy. Indeed, many viruses have evolved countermeasures to PKR signaling (6,15,24,29,33,37).…”

Complementation of the human adenovirus type 5 VA RNAI defect by the Vaccinia virus E3L protein and serotype-specific VA RNAIs

“…A recent report showed that the HAdV-5 E1B-55K and E4orf6 proteins block activation of the PKR protein and maintain low levels of eIF2α Ser51 phosphorylation in virus-infected cells (Spurgeon and Ornelles, 2009). The Cul5-based ubiquitin ligase activity associated with the E1B-55 K/E4orf6 protein complex appeared to be necessary to prevent activation of PKR and phosphorylation of eIF2α during the late phase of HAdV-5 infection.…”

Adenovirus E1B 55-Kilodalton Protein Is a p53-SUMO1 E3 Ligase That Represses p53 and Stimulates Its Nuclear Export through Interactions with Promyelocytic Leukemia Nuclear Bodies

“…The viral ubiquitin ligase also targets the integrin alpha 3 subunit, whose proteasomal degradation may promote release and spread of progeny virions (15). The viral ubiquitin ligase also stimulates late viral gene expression by stimulating the nuclear export of late viral mRNAs (80) and by preventing an increase in PKR activity and subsequent eIF2␣ phosphorylation that occurs in cells infected with E1B-55K or E4orf6 mutants (72), but the potential targets of proteasomal degradation involved in these processes are unknown.…”