Rift Valley Fever Virus NSs mRNA Is Transcribed from an Incoming Anti-Viral-Sense S RNA Segment

Abstract

Analysis of purified Rift Valley fever virus (RVFV) particles demonstrated the presence of three negativesense RNA genomes, plus three anti-viral-sense RNA segments. The virion-associated anti-viral-sense S segment served as a template for the synthesis of NSs mRNA immediately after infection. NSs protein synthesis also occurred early in infection, suggesting that NSs protein produced early in infection probably has biologically significant roles in virus replication and/or survival in the host. Translation inhibitor treatment of mammalian cells infected with viruses belonging to the Bunyaviridae family generally inhibits viral mRNA synthesis. However, RVFV NSs mRNA synthesis, but not N mRNA synthesis, was resistant to puromycin treatment during primary transcription, pointing to the uniqueness of RVFV NSs mRNA synthesis.Viruses belonging to the Bunyaviridae family carry three single-stranded RNAs, designated L, M, and S. Viral RNA synthesis occurs in the cytoplasm, and the host mRNA-derived cap structure is used as a primer for viral mRNA synthesis (16). Host protein synthesis is essential for viral RNA replication (2,5,6,15,18) and is also important for viral mRNA synthesis in mammalian cells; viral mRNA elongation, but not initiation, is suppressed in the presence of protein synthesis inhibitors (2,15,18). How the host protein synthesis machinery facilitates viral mRNA elongation is unknown; it has been suggested that ribosome binding to nascent viral mRNA prevents its annealing to the template RNA and allows mRNA elongation (2).Rift Valley fever virus (RVFV) (family Bunyaviridae, genus Phlebovirus) causes severe epidemics among ruminants in the sub-Saharan African and has spread to Egypt, Yemen, and Saudi Arabia. It is also an important human pathogen that causes a syndrome of fever and myalgia, a hemorrhagic syndrome, ocular disease, and encephalitis (1, 13). The anti-viralsense L segment encodes L protein, a viral RNA polymerase, and the anti-viral-sense M segment encodes two structural glycoproteins, G1 and G2, NSm protein, and a 78-kDa protein.As in other viruses of the genus Phlebovirus, RVFV S segment uses an ambisense strategy to express N and NSs proteins, and the viral-sense S and anti-viral-sense S segments serve as templates for N mRNA and NSs mRNA, respectively (16). It has been believed that NSs mRNA is produced only after viralsense RNA has been copied to anti-viral-sense RNA; therefore, the NSs protein would appear at a later stage in infection than the structural proteins (6, 16).RVFV NSs protein plays an important role in RVFV pathogenesis and replication. This protein inhibits host mRNA synthesis, including alpha/beta interferon (IFN-␣/␤) mRNAs (3, 10), hence suppressing host innate immune responses to viral invasion, and it is a major virus virulence factor (4). We showed that coexpression of RVFV NSs protein with N and L proteins enhances viral RNA accumulation in the RVFV minigenome system (7). Because this effect appears to be independent of the NSs-mediated inhibition of IFN-␣/␤ produc...

Characterization of Wild-Type and Alternate Transcription Termination Signals in the Rift Valley Fever Virus Genome

“…In these viruses, the stop codon of the N ORF was located at a short distance (i.e., 7 or 12 nucleotides) from the transcription termination motif, compared to the wt situation, where the distance was 69 nucleotides. This strongly suggested to us that, as described previously for Bunyamwera virus (4), with transcription being coupled to translation (4,6,21,36), the presence of ribosomes on the nascent mRNA interferes with transcription termination, and a minimal distance between the stop codon and the transcription termination signal might be required for the transcriptase to recognize the termination motif and detach from the template. For Bunyamwera virus, it was shown that the translocation of the ribosomes on the nascent mRNA abrogated transcription termination.…”

“…We observed a similar situation with mutant viruses in which the motif was present but close to the stop codon of the ORF contained in the transcribed mRNA. The failure of the transcriptase to recognize the wild-type (wt) motif allowed us to propose a model taking into account that transcription is coupled to translation in RVFV-and other bunyavirus-infected cells (4,6,21,36). In addition, we found that although the conserved motif 5Ј-GCUGC-3Ј plays a major role in transcription termination, in some circumstances induced by mutations in the IGR or naturally found in the L segment, a slightly variant sequence can also be recognized as a transcription termination signal.…”

Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

“…These data indicate two points. First, SFTSV, like UUKV and RVFV (42)(43)(44), packages both types of full-length RNA into virus particles, as a 3= RACE protocol was used on virion RNA (i.e., RNA extracted from supernatant virus) and sequences corresponding to both genomic and antigenomic RNA 3= ends were obtained. Second, they highlight the importance of obtaining cDNA clones that exactly match the correct viral sequences in order to establish reverse genetics systems, as has recently been described for Oropouche orthobunyavirus (52).…”

Bunyavirus mRNA synthesis is coupled to translation to prevent premature transcription termination

“…This phenomenon of obligatorily coupled transcription and translation was first described for Akabane virus and BUNV Pattnaik and Abraham 1983), but has since been shown to apply to LaCrosse virus (LACV) and Germiston virus within the same genus (Patterson and Kolakofsky 1984;Vialat and Bouloy 1992). More recently, transcription of Rift Valley Fever virus has been shown to require protein synthesis (Ikegami et al 2005), suggesting this phenomenon is widespread among bunyaviruses.…”