Features of the 3'-consensus sequence of rotavirus mRNAs critical to minusstrand synthesis

The last seven nucleotides of the 3 ' -end of rotavirus mRNAs, 5 ' -UGUGACC -3 ', are highly conserved acid form a cis-acting signal that can promote t he synthesis of (-) strand RNA to produce the viral dsRNA genome in vitro. Previous studies have shown that the sequence, location, and strandedness ( single- versus double-stranded) of the 3 ' -consensus sequence of the mRNA affect the efficiency of (-) strand synthesis. In this study. we have used exhaustive mutagenesis of the SA11 gene 8 mRNA and an in vitro replication system to define the importance of each of the residues in the consensus se quence in (-) strand synthesis. The analysis showed that the CC of the cons ensus sequence was the most critical for (-) strand synthesis. Furthermore, the data revealed that other, but not all, residues of the consensus seque nce contributed to efficient(-) strand synthesis in vitro. Mutant gene 8 RN As supported an intermediate level of (-) strand synthesis when the 15 nt s equence upstream of the CC was replaced with long tracts of poly(A) or poly (U), but not with poly(G). Predictions of the secondary structure of the mu tant RNAs suggested that the poly(G)-RNA could not replicate because its 3 ' -terminus was largely basepaired, instead of extending as a single-strand ed tail as is the case for the 3 ' -termini of the poly(A)- and poly(U)-RNA s and wild-type gene 8 RNA. Subsequent experiments performed with complemen tary oligonucleotides indicated that efficient RNA replication occurs in vi tro only when the last four residues of the 3 ' -consensus sequence, and mo st importantly the two terminal C's, existed in a single-stranded form. A s ingle-stranded CC may be crucial for formation of an initiation complex for (-) strand synthesis consisting of viral RdRP, mRNA, and the dinucleotide pGpG.