Protein Kinase R Is Responsible for the Phosphorylation of eIF2α in Rotavirus Infection

Abstract

The eukaryotic initiation translation factor 2 (eIF2) represents a key point in the regulation of protein synthesis. This factor delivers the initiator Met-tRNA to the ribosome, a process that is conserved in all eukaryotic cells. Many types of stress reduce global translation by triggering the phosphorylation of the ␣ subunit of eIF2, which reduces the formation of the preinitiation translation complexes. Early during rotavirus infection, eIF2␣ becomes phosphorylated, and even under these conditions viral protein synthesis is not affected, while most of the cell protein synthesis is blocked. Here, we found that the kinase responsible for the phosphorylation of eIF2␣ in rotavirus-infected cells is PKR, since in mouse embryonic fibroblasts deficient in the kinase domain of PKR, or in MA104 cells where the expression of PKR was knocked down by RNA interference, eIF2␣ was not phosphorylated upon rotavirus infection. The viral component responsible for the activation of PKR seems to be viral double-stranded RNA, which is found in the cytoplasm of infected cells, outside viroplasms. Taken together, these results suggest that rotaviruses induce the PKR branch of the interferon system and have evolved a mechanism to translate its proteins, surpassing the block imposed by eIF2␣ phosphorylation.Protein translation is the final step in the flow of genetic information, and unlike transcriptional control, regulation at this step allows for an immediate and rapid response to changes in physiological conditions. While every step of the translation process is amenable to regulation, under most circumstances mRNA translation is regulated primarily at the level of initiation (5). The translation of eukaryotic mRNAs involves the recognition and recruitment of mRNAs by the translation initiation machinery and the assembly of the 80S ribosome on the mRNA; this process is mediated by the eukaryotic initiation factors (eIFs). Translation initiation is a complex process that begins with the recognition of the cap nucleotide structure (m7GpppN) at the 5Ј end of mRNAs by the cap-binding protein eIF4E that is part of the cap-binding complex eIF4F. This complex is composed of eIF4E, eIF4A (an ATP-dependent RNA helicase), and the scaffolding protein eIF4G. The binding of Met-tRNA to the 40S ribosomal subunit is mediated by a ternary complex composed of eIF2-GTP-Met-tRNA. The binding of GTP to eIF2 is the ratelimiting step in the assembly of the ternary complex and is regulated by eIF2B. Once the 40S ribosomal subunit is bound to the mRNA, it is thought to scan the mRNA in the 5Ј to 3Ј direction (26). The joining of the 40S and 60S ribosomal subunits to form an 80S initiation complex then takes place. The release of eIFs is assisted by eIF5, which facilitates the hydrolysis of GTP carried out by eIF2. The GDP on eIF2 is exchanged for GTP by eIF2B in a regulated manner that is essential for ensuing rounds of initiation (25).Many types of stresses reduce global translation by triggering the phosphorylation of the ␣ subunit of eIF2 (eIF2␣) at ...

The Early Interferon Response to Rotavirus Is Regulated by PKR and Depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3

“…In support of this notion, UK rotavirus RNA synthesis increased significantly in IRF3 Ϫ/Ϫ MEFs compared to PKR Ϫ/Ϫ MEFs although both knockout cells supported much higher levels of viral RNA synthesis than WT MEFs. Interestingly in certain situations, rotavirus infection can lead to phosphorylation of eIF2␣ in a PKR-dependent manner (50), and it has been noted that triple-layered (i.e., intact) rotavirus particles replicate more efficiently in PKR Ϫ/Ϫ MEFs than WT MEFs (50). An earlier study found that PKR is critical for reovirus-induced IFN-␤ responses and influences virulence in a mouse model of myocarditis (56).…”

Alpha Interferon Restricts Human T-Lymphotropic Virus Type 1 and 2 De Novo Infection through PKR Activation

“…Active PKR is known to affect phosphorylation of eukaryotic initiation factor 2 (eIF2), which then suppresses mRNA translation (13). It would therefore be interesting to define whether HTLV-1/2 protein synthesis is affected through eIF2 phosphorylation, unlike a number of other viruses that evade this phenomenon (60,61,62).…”

Cooperative Roles of Fish Protein Kinase Containing Z-DNA Binding Domains and Double-Stranded RNA-Dependent Protein Kinase in Interferon-Mediated Antiviral Response

“…The latent PKR is activated by binding to dsRNA that occurs during virus replication, thus undergoing dimerization, autophosphorylation, and subsequently inhibition of viral protein synthesis via phosphorylating eIF2␣ (7). Consistent with its antiviral property, overexpression of PKR in many mammalian cell lines confers resistance to virus infection (17,23). PKRdeficient cells are more permissive for several RNA viruses (28,40) as well as DNA viruses (1), and PKR-deficient mice become highly susceptible to otherwise harmless infection of vesicular stomatitis virus (VSV) and influenza virus (2).…”