The adenovirus E1B-55K and E4orf6 proteins limit PKR-mediated elF2alpha phosphorylation during the late phase of infection

During a productive adenovirus infection, the E1B-55K and E4orf6 proteins promote efficient late viral gene expression. The results presented in this dissertation reveal that the E1B-55K and E4orf6 proteins facilitate late viral protein synthesis by limiting phosphorylation of eIF2alpha by the dsRNA-dependent protein kinase R (PKR) during the late phase of infection. The phosphorylation of eIF2alpha systematically fluctuated throughout the viral life cycle. These fluctuations most likely reflect cellular responses to discrete viral events. Viral DNA replication was identified as the key event leading to PKR-independent phosphorylation of eIF2alpha between 6 and 24 hours after infection. At later times, the E1B-55K and E4orf6 proteins limited eIF2alpha phosphorylation by preventing an increase in PKR activation, which was most likely induced by double stranded RNA (dsRNA). The ability of the E1B-55K and E4orf6 proteins to prevent PKR-mediated eIF2alpha phosphorylation was correlated with their ability to promote efficient late viral protein synthesis. In support of these findings, expression of the reovirus sigma3 dsRNA-binding protein prevented PKR activation, blocked eIF2alpha phosphorylation, and restored late viral protein synthesis in cells infected with an E1B-55K or E4orf6-mutant adenovirus. This newly described function of the E1B-55K and E4orf6 proteins required a cullin 5-mediated activity of the E1B-55K/E4orf6 complex, most likely its associated ubiquitin-ligase function. Surprisingly, cytoplasmic levels of the adenovirus VAI RNA, which has been shown to prevent activation of PKR by dsRNA, were equivalent in mutant and wild-type virus-infected cells. Previous results established that the E4orf1 protein contributes to the defect in late viral protein synthesis in the absence of E1B-55K function. The findings reported here indicate that the E4orf1 protein promotes PKR activation and eIF2alpha phosphorylation in E1B-55K-mutant virus-infected cells, thus providing a potential molecular basis by which the E4orf1 protein restricts late viral protein synthesis in the absence of E1B-55K protein function. Collectively, the work presented here describes a previously unknown interplay between the E4orf1, E1B-55K, and E4orf6 proteins during the late phase of infection. Moreover, it provides insight into how these viral regulators govern late viral protein synthesis and help establish a precisely coordinated, productive adenovirus infection.