Characterization of Rift Valley fever virus transcriptional terminations

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome and causes a mosquito-borne disease among humans and livestock in sub-Saharan African and Arabian Peninsula countries. Phlebovirus L, M, and N mRNAs are synthesized from the virus-sense RNA segments, while NSs mRNA is transcribed from the anti-virus-sense S segment. The present study determined the 3′ termini of all RVFV mRNAs. The 3′ termini of N and NSs mRNAs were mapped within the S-segment intergenic region and were complementary to each other by 30 to 60 nucleotides. The termini of M and L mRNAs were mapped within 122 to 107 nucleotides and 16 to 41 nucleotides, respectively, from the 5′ ends of their templates. Viral RNA elements that control phlebovirus transcriptional terminations are largely unknown. Our studies suggested the importance of a pentanucleotide sequence, CGUCG, for N, NSs, and M mRNA transcription terminations. Homopolymeric tracts of C sequences, which were located upstream of the pentanucleotide sequence, promoted N and M mRNA terminations. Likewise, the homopolymeric tracts of G sequences that are found upstream of the pentanucleotide sequence promoted NSs mRNA termination. The L-segment 5′-untranslated region (L-5′ UTR) had neither the pentanucleotide sequence nor homopolymeric sequences, yet replacement of the S-segment intergenic region with the L-5′ UTR exerted N mRNA termination in an infectious virus. The L-5′ UTR contained two 13-nucleotide-long complete complementary sequences, and their sequence complementarities were important for L mRNA termination. A computer-mediated RNA secondary structure analysis further suggested that RNA secondary structures formed by the sections of the two 13-nucleotide-long sequences and by the sequence between them may have a role in L mRNA termination. Our data demonstrated that viral RNA elements that govern L mRNA termination differed from those that regulate mRNA terminations in the M and S segments.