To understand the mechanisms of rubella virus (RV) RNA replication, analysis of the function of conserved sequence (cis-acting) elements of genomic RNA thought to be involved in virus replication, is essential for viral pathogenicity. The cis- acting elements are present at the 5' and 3' end of RV RNA and can potentially form stem-loop (SL) structures. We have analyzed the function of these putative SL structures in RNA translation and negative-strand RNA synthesis by constructing chimeric chloramphenicol acetyl transferase (CAT) RNAs, flanked either by both 5' and 3' terminal sequence domains or several deletions derivatives of the same sequences. Both in vivo and in vitro translation activities of the chimeric RNAs revealed that the presence of 5' and 3' SL sequences of RV RNA, in correct orientation and context, was necessary for efficient translation of chimeric RV/CAT RNAs. Further, the presence of RV 5' (+) SL sequences in negative-strand RNA synthesis, the chimeric RV/CAT constructs were cloned in an expression vector under the control of adenovirus major late promoter and transfected in Vero 76 cells. Cell lines expressing chimeric RNAs were either mock or RV infected. Negative strand RNAs, complimentary to chimeric CAT RNAs were detected from such cells. From such an analysis it was evident that both 5' and 3' SL sequences are essential for negative strand RNA synthesis. In addition, initiation of negative-strand RNA synthesis was dependent on RV infection. Currently, studies are in progress to define the functional domains of cis-acting elements of RV RNA and their interaction with host and viral encoded proteins in viral replication.
