Characterization of Punta Toro S mRNA species and identification of an inverted complementary sequence in the intergenic region of Punta Toro phlebovirus ambisense S RNA that is involved in mRNA transcription termination

Virology. 1987 Jan;156(1):1-11. doi: 10.1016/0042-6822(87)90430-2.

Abstract

The transcription termination sites for the subgenomic N and NS(S) mRNA species coded by the 1904 nucleotide, ambisense S RNA species of Punta Toro (PT) phlebovirus (Bunyaviridae, Ihara et al., 1984) have been identified by Northern analyses using a series of synthetic oligodeoxynucleotides. For the viral-complementary N mRNA species the oligonucleotides that were used represented viral RNA sequences; for the viral-sense NS(S) mRNA species they represented viral-complementary sequences. The results have been confirmed by employing the same oligodeoxynucleotides to backcopy purified mRNA preparations in the presence of appropriate chain-terminating dideoxynucleotides. The results obtained have allowed the 3' termini of both mRNA species to be mapped to a common region of the viral S RNA between RNA residues 977 and 1017. Based on these results and the presence of short non-viral sequences at the 5' ends of the PT mRNA species (Ihara et al., 1985), the estimated sizes of the PT N and NS(S) mRNA species are of the order of 1000 and 900 nucleotides, respectively. Computer analysis of DNA sequences representing the centrally located intergenic region (i.e., residues 768-1127) revealed a long inverted complementary sequence corresponding to nucleotides 886-1092. The peak of this potential hairpin structure, residue 996, correlates to the region of the genome identified by Northern analyses to be involved in the termination of mRNA transcription. Neither the N nor NS(S) mRNA species appeared to be polyadenylated to the extent that is characteristic of most eukaryotic mRNA species, although from the indicated sequences the viral mRNA species contain 3' proximal regions that are rich in short stretches of adenylic acid residues. This is particularly true for the N mRNA species, permitting its purification by selective oligo(dT) chromatography. In vitro translation of purified N and NSS mRNA species by rabbit reticulocyte lysates resulted in the synthesis of proteins that were identified as the viral N and NS(S) species, respectively, by comparison with proteins obtained from PT virus infected Vero cells.