Sequences at the 5' and the 3' end of the rubella virus (RV) genomic RNA can form potentially stable stem-loop (SL) structures and have been implicated to be involved in viral replication. To analyze the function of SL structures in viral translation, we constructed chimeric chloramphenicol acetyl transferase (CAT) RNAs, flanked either by both 5' and 3' SL sequences of the wild type rubella virus genome RNA or several deletion derivatives of the same sequences. The in vitro translational efficiency of chimeric RNAs transcribed by SP6 RNA polymerase was compared in both wheat germ or rabbit reticulocyte lysate translational systems. For in vivo translational studies, the chimeric CAT RNAs were expressed under the control of adenovirus major late promoter in transfected cells and the level of CAT activity was measured. Both in vivo and in vitro translation assays revealed that the presence of 5' and 3' SL sequences of RV RNA, in correct orientation, was necessary for efficient translation of RV/CAT hybrid RNA. The synergism between the 5' and the 3' SL RNA sequences in the translation coincides with their ability to specifically interact with two cellular proteins, 60 kDa Ro/SS-A antigen and calreticulin. The lose of translational capability, by the chimeric RV/CAT RNA, which has altered 5' SL sequence, correlated with its altered Ro/SS-A antigen binding activity. There was a complete inhibition of translation of chimeric CAT RNA in the presence of antibody directed against the Ro/SS-A protein. The role of Ro/SS-a antigen and calreticulin in the translation of RV RNA through their interaction with the SL sequences and its implication in viral replication is currently being studied.
