Translational resistance of late alphavirus mRNA to eIF2α phosphorylation: a strategy to overcome the antiviral effect of protein kinase PKR

Abstract

The double-stranded RNA-dependent protein kinase (PKR) is one of the four mammalian kinases that phosphorylates the translation initiation factor 2␣ in response to virus infection. This kinase is induced by interferon and activated by double-stranded RNA (dsRNA). Phosphorylation of eukaryotic initiation factor 2␣ (eIF2␣) blocks translation initiation of both cellular and viral mRNA, inhibiting virus replication. To counteract this effect, most viruses express inhibitors that prevent PKR activation in infected cells. Here we report that PKR is highly activated following infection with alphaviruses Sindbis (SV) and Semliki Forest virus (SFV), leading to the almost complete phosphorylation of eIF2␣. Notably, subgenomic SV 26S mRNA is translated efficiently in the presence of phosphorylated eIF2␣. This modification of eIF2 does not restrict viral replication; SV 26S mRNA initiates translation with canonical methionine in the presence of high levels of phosphorylated eIF2␣. Genetic and biochemical data showed a highly stable RNA hairpin loop located downstream of the AUG initiator codon that is necessary to provide translational resistance to eIF2␣ phosphorylation. This structure can stall the ribosomes on the correct site to initiate translation of SV 26S mRNA, thus bypassing the requirement for a functional eIF2. Our findings show the existence of an alternative way to locate the ribosomes on the initiation codon of mRNA that is exploited by a family of viruses to counteract the antiviral effect of PKR.[Keywords: Translation; eIF2; eIF2A; PKR; alphaviruses; antiviral response] Supplemental material is available at http://www.genesdev.org.

Evaluation of cancer virotherapy with attenuated replicative Semliki forest virus in different rodent tumor models

“…Although the response in locally treated tumors was fast and strong, it was still insufficient to prevent virus replication and subsequent tumor cell destruction. Alphaviruses, including SFV, are known to resist IFN response, as they can slow down protein translation via formation of specific capsid RNA haipin structures, 29 which allows viral replication in the presence of active PKR. On the other hand, despite the slower onset of the IFN response in the i.v.-treated animals, the antitumor effect of the vector administered systemically was more modest.…”

Inhibition of host translation by virus infection in vivo

“…respectively, as compared with their counterparts that bear the viral DLP structure (SV-DLP Luc and SV-DLP EGFP). No differences in expression were detected in PKR o/o cells, showing that phosphorylation of eIF2 hampered translation of nonviral mRNAs as described before (22). Next, we quantified the extent of translational exclusion of EGFP mRNA in SV-infected cells compared with cellular mRNAs (e.g., β-actin).…”

“…A DLP structure included in the first 90 nts of the 26S mRNA coding sequence was also placed in the indicated reporter mRNAs. In SV-ΔDLP EGFP the secondary structure of DLP was disrupted by point mutations as described previously (22) …”

“…A remarkable exception to this are the members of alphavirus group (Sindbis and Semliki Forest virus). Thus, complete phosphorylation of eIF2 was found in cultured mouse fibroblasts infected with these viruses, so that only viral mRNA is translated under these circumstances (22,29). The presence of a secondary structure, termed downstream hairpin loop (DLP) and located 27 nts downstream from the initiator AUG in viral 26S transcripts, allows this mRNA to be translated by an eIF2-independent mechanism in infected cells (22,30).…”

“…Another important point of translation control in infected cells relies on the activity of eIF2 that brings the initiator Met-tRNA to 40S ribosomes (20)(21)(22)(23). In response to viral infection, host dsRNAactivated kinase (PKR) phosphorylates eIF2 in an attempt to block general translation (both cellular and viral) (24)(25)(26)(27).…”