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The Initiation Factors eIF2, eIF2A, eIF2D, eIF4A, and eIF4G Are Not Involved in Translation Driven by Hepatitis C Virus IRES in Human Cells.

ABSTRACT: Animal viruses have evolved a variety of strategies to ensure the efficient translation of their mRNAs. One such strategy is the use of internal ribosome entry site (IRES) elements, which circumvent the requirement for some eukaryotic initiation factors (eIFs). Much effort has been directed to unravel the precise mechanism of translation initiation by hepatitis C virus (HCV) mRNA. In the present study, we examined the involvement of several eIFs in HCV IRES-driven translation in human cells in a comparative analysis with mRNAs bearing the encephalomyocarditis virus or the Cricket paralysis virus IRES element. Consistent with previous findings, several inhibitors of eIF2 activity, including sodium arsenite, thapsigargin, tunicamycin, and salubrinal, had no inhibitory effect on the translation of an mRNA bearing the HCV IRES, and all induced the phosphorylation of eIF2α. In addition, hippuristanol and pateamine A, two known inhibitors of eIF4A, failed to block HCV IRES-directed translation. To test the release of nuclear proteins to the cytoplasm and to analyze the formation of stress granules, the location of the nuclear protein TIA1 was tested by immunocytochemistry. Both arsenite and pateamine A could efficiently induce the formation of stress granules containing TIA1 and eIF4G, whereas eIF3 and eIF2 failed to localize to these cytoplasmic bodies. The finding of eIF4A and eIF4G in stress granules suggests that they do not participate in mRNA translation. Human HAP1 cells depleted for eIF2A, eIF2D, or both factors, were able to synthesize luciferase from an mRNA bearing the HCV IRES even when eIF2α was phosphorylated. Overall, these results demonstrate that neither eIF2A nor eIF2D does not participate in the translation directed by HCV IRES. We conclude that eIF2, eIF4A, eIF2A, and eIF2D do not participate in the initiation of translation of HCV mRNA.

SUBMITTER: Gonzalez-Almela E

PROVIDER: S-EPMC5816946 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Nucleolin (NCL), a stress-responsive RNA-binding protein, has been implicated in the translation of internal ribosome entry site (IRES)-containing mRNAs, which encode proteins involved in cell proliferation, carcinogenesis, and viral infection (type I IRESs). However, the details of the mechanisms by which NCL participates in IRES-driven translation have not hitherto been described. Here, we identified NCL as a protein that interacts with the IRES of foot-and-mouth disease virus (FMDV), which is a type II IRES. We also mapped the interactive regions within FMDV IRES and NCL in vitro. We found that NCL serves as a substantial regulator of FMDV IRES-driven translation but not of bulk cellular or vesicular stomatitis virus cap-dependent translation. NCL also modulates the translation of and infection by Seneca Valley virus (type III-like IRES) and classical swine fever virus (type III IRES), which suggests that its function is conserved in unrelated IRES-containing viruses. We also show that NCL affects viral replication by directly regulating the production of viral proteins and indirectly regulating FMDV RNA synthesis. Importantly, we observed that the cytoplasmic relocalization of NCL during FMDV infection is a substantial step for viral IRES-driven translation and that NCL specifically promotes the initiation phase of the translation process by recruiting translation initiation complexes to viral IRES. Finally, the functional importance of NCL in FMDV pathogenicity was confirmed in vivo. Taken together, our findings demonstrate a specific function for NCL in selective mRNA translation and identify a target for the development of a broad-spectrum class of antiviral interventions. IMPORTANCE FMDV usurps the cellular translation machinery to initiate viral protein synthesis via a mechanism driven by IRES elements. It allows the virus to shut down bulk cellular translation, while providing an advantage for its own gene expression. With limited coding capacity in its own genome, FMDV has evolved a mechanism to hijack host proteins to promote the recruitment of the host translation machinery, a process that is still not well understood. Here, we identified nucleolin (NCL) as a positive regulator of the IRES-driven translation of FMDV. Our study supports a model in which NCL relocalizes from the nucleus to the cytoplasm during the course of FMDV infection, where the cytoplasmic NCL promotes FMDV IRES-driven translation by bridging the translation initiation complexes with viral IRES. Our study demonstrates a previously uncharacterized role of NCL in the translation initiation of IRES-containing viruses, with important implications for the development of broad antiviral interventions.

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The Initiation Factors eIF2, eIF2A, eIF2D, eIF4A, and eIF4G Are Not Involved in Translation Driven by Hepatitis C Virus IRES in Human Cells.

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