Mj. Wentz et al., THE 3'-TERMINAL CONSENSUS SEQUENCE OF ROTAVIRUS MESSENGER-RNA IS THE MINIMAL PROMOTER OF NEGATIVE-STRAND RNA-SYNTHESIS, Journal of virology, 70(11), 1996, pp. 7833-7841
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.