cis-Acting signals that promote genome replication in rotavirus mRNA

A previous study has shown that rotavirus cores have an associated replicase 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 sequences were generated. Analysis of these RNAs in the cell-free system led to the identification of a cis-acting signal in the gene 8 mRNA which is essential for RNA replication and two cis-acting signals which, while not essential for replication, serve to enhance the process. The sequence of the essential replication signal is located at the extreme 3' end of the gene 8 mRNA and, because of its highly conserved nature, is probably a common feature of all 11 viral mRNAs. By site-specific mutagenesis of the gene 8 mRNA, residues at positions -1, -2, -5, -6, and -7 of the 3' essential signal were found to be particularly important for promoting RNA replication. One of the cis-acting signals shown to 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 existence of an enhancement signal in the 5' UTR raises the possibility that the 5' and 3' ends of the rotavirus mRNA may interact with each other and/or with the viral replicase during genome replication.