5' SEQUENCES OF RUBELLA-VIRUS RNA STIMULATE TRANSLATION OF CHIMERIC RNAS AND SPECIFICALLY INTERACT WITH 2 HOST-ENCODED PROTEINS

Sequences at the 5' and 3' ends of the rubella virus (RV) genomic RNA can potentially form stable stem-loop (SL) structures that are postula ted to be involved in virus replication. We have analyzed the function of these putative SL structures in RNA translation by constructing ch imeric chloramphenicol acetyltransferase (CAT) RNAs, flanked either by both 5'- and 3'-terminal sequence domains from the RV genome or sever al deletion derivatives of the same sequences. After in vitro transcri ption of chimeric RNAs, the translational efficiencies of these RNAs w ere compared by the rabbit reticulocyte lysate translation system. For in vivo translation studies, the level of CAT activity was measured f or chimeric RV/CAT RNAs expressed in transfected cells by the adenovir us major late promoter. Both in vivo and in vitro translation activiti es of the chimeric RNAs revealed that the presence of 5' and 3' SL seq uences of RV RNA, in correct (+) orientation and context [5'(+)SL and 3'(+)SL, respectively] was necessary for efficient translation of chim eric RV/CAT RNAs. The presence of the RV 5'(+)SL sequence had the prim ary enhancing effect on translation. To identify host proteins which i nteract with the 5'(+)SL which may be involved in RV RNA translation, RNA gel-shift and UV cross-linking assays were employed. Two host prot eins 59 and 52 kDa in size, present in cytosolic extracts from both un infected and RV-infected cells, specifically interacted with the RV 5' (+)SL RNA. Direct binding comparisons between wild-type and mutant 5'( +)SL RNAs demonstrated that sequences in and around the bulge region o f the terminal stem domain of this structure constituted a protein bin ding determinant. Human serum, qualified for anti-Ro/SS-A antigen spec ificity, immunoprecipitated 59- and 52-kDa protein-RNA complexes conta ining the RV 5'(+)SL RNA. However, poly- and monoclonal antisera raise d against the recombinant 60- and 52-kDa Ro proteins failed to precipi tate complexes containing the 5'(+)SL RNA. The identity of the protein s binding this RV cis-acting element remains to be determined; however , their role in RV translation is discussed.