La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

Abstract

Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5′ untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

SUPPLEMENTAL MATERIAL

We thank Jerry Pelletier and Andrea Brueschke for critical reading of the manuscript, Karen Meerovitch for helpful discussions, Martin Holcik for providing the pcDNA-3-myc-La226-348 mutant, F. Lahser and E. Wimmer for the HCV-poliovirus chimera, and Colin Lister and Sandra Perrault for excellent assistance.

This work was supported by a grant from the Canadian Institute of Health Research (CIHR) to N.S., who is recipient of a CIHR Distinguished Scientific Award and a Howard Hughes Medical Institute International Scholarship. M.C.-M is a postdoctoral fellow supported by a CIHR fellowship.