CIS-ACTING SIGNALS THAT PROMOTE GENOME REPLICATION IN ROTAVIRUS MESSENGER-RNA

A previous study has shown that rotavirus cores have an associated rep licase activity which can direct the synthesis of double-stranded RNA from viral mRNA in a cell-free system (D. Y. Chen, C. Q.-Y. Zeng, M. J . Wentz, M. Gorziglia, M. K. Estes, and R. F. Ramig, J. Virol, 68:7030 -7039, 1994), To define the cis-acting signals in rotavirus mRNA that are important for RNA replication, gene 8 transcripts which contained internal and terminal deletions and chimeric transcripts which linked gene 8-specific 3'-terminal sequences to the ends of nonviral sequence s were generated. Analysis of these RNAs in the cell-free system led t o the identification of a cis-acting signal in the gene 8 mRNA which i s essential for RNA replication and two cis-acting signals which, whil e not essential for replication, serve to enhance the process. The seq uence of the essential replication signal is located at the extreme 3' end of the gene 8 mRNA and, because of its highly conserved nature, i s probably a common feature of all 11 viral mRNAs. By site-specific mu tagenesis of the gene 8 mRNA, residues at positions -1, -2, -5, -6, an d -7 of the 3' essential signal were found to be particularly importan t for promoting RNA replication, One of the cis-acting signals shown t o enhance the replication in the cell-free system was located near the 5' end of the 3' untranslated region (UTR) of the gene 8 mRNA, while remarkably the other was located in the 5' UTR of the message, The exi stence of an enhancement signal in the 5' UTR raises the possibility t hat the 5' and 3' ends of the rotavirus mRNA may interact with each ot her and/or with the viral replicase during genome replication.