Picornaviruses encompass a large variety of medically important human and animal viruses which include those inducing common cold (rhinovirus), poliomyelitis, infectious hepatitis (hepatitis A), Coxsackie virus, and foot-and-mouth disease virus (FMDV) among others. While capped cellular mRNAs are translated by a ribosomal 'scanning' mechanism, naturally uncapped picornaviral mRNAs are translated by a distinct mechanism involving internal entry of ribosomes within the 5'untranslated region of viral RNA, While trying to express infectious poliovirus cDNA in the yeast S.cerevisiae, it was discovered serendipitously that yeast cells were unable to translate poliovirus RNA. This was due to a transacting small RNA (called inhibitor RNA, I-RNA) present in yeast cell lysates that was able to inhibit translation of PV RNA in HeLa cell extracts. The I-RNA has been purified and a synthetic clone has been prepared. I-RNA synthesized from the clone by transcription with T7 RNA polymerase, blocks internal entry of ribosomes and thus inhibits picornaviral translation but not cellular RNA translation both in vivo and in vitro. Initial studies suggest that I-RNA interacts with a cellular protein (p52) involved in internal initiation of translation. Both biochemical and genetic approaches will be used to determine the mechanism by which I-RNA selectively inhibits internal initiation of translation. We will investigate whether viral processes other than translation (such as RNA synthesis or protein processing) is also affected by the I-RNA. Identity and normal function of I-RNA in the yeast S.cerevisiae will be addressed by cloning and characterizing the gene encoding the I-RNA sequence. I-RNA gene """"""""knock out"""""""" will be performed to better understand the role of I-RNA in yeast cell growth. We will also determine whether yeast proteins which bind to I-RNA plays a role in translation. Whether I-RNA has potential to inhibit different viruses (including hepatitis C) that use internal initiation of translation for protein synthesis will be determined. Finally, a stable HeLa cell line expressing I-RNA will be prepared to determine the effect of long term expression of I-RNA in animal cells and whether cells expressing I-RNA will be resistant to picornavirus infection. It is hoped that studies proposed here will not only provide a rational basis for antiviral drug design but also further our understanding of the mechanism of internal initiation of translation in eukaryotic cells.
