THE 3'-TERMINAL CONSENSUS SEQUENCE OF ROTAVIRUS MESSENGER-RNA IS THE MINIMAL PROMOTER OF NEGATIVE-STRAND RNA-SYNTHESIS

We used an in vitro template-dependent replicase assay (D. Chen, C. Ze ng, M. Wentz, M. Gorziglia, M. Estes, and R. Ramig. J. Virol. 68:7030- 7039, 1994) to identify the cis-acting signals required for replicatio n of a genome segment 9 template from the group A rotavirus strain OSU . The replicase phenotypes for a panel of templates with internal dele tions or 3'-terminal truncations indicated that no essential replicati on signals were present within the open reading frame and that key ele ments were present in the 5' and 3' noncoding regions. Chimeric constr ucts containing portions of viral sequence ligated to a nonviral backb one were generated to further map the regions required for in vitro re plication of segment 9. The data from these constructs showed that the 3'-terminal seven nucleotides of the segment 9 mRNA provided the mini mum requirement for replication (minimal promoter). Analysis of additi onal chimeric templates demonstrated that sequences capable of enhanci ng replication from the minimal promoter were located immediately upst ream of the minimal promoter and at the extreme 5' terminus of the tem plate. Mutational analysis of the minimal promoter revealed that the 3 '-terminal -CC residues are required for efficient replication. Compar ison of the replication levels for templates with guanosines and uridi nes at nucleotides -4 to -6 from the 3' terminus compared with levels for templates containing neither of these residues at these positions indicated that either or both residues must be present in this region for efficient replication in vitro.