Rift Valley Fever Virus L Protein Forms a Biologically Active Oligomer

Abstract

Rift Valley fever virus (RVFV) (genus Rift Valley Fever virus (RVFV), which belongs to the genusPhlebovirus in the family Bunyaviridae, is endemic in subSaharan African countries and causes large outbreaks in endemic areas and countries outside of the endemic area, including Egypt, Saudi Arabia, and Yemen (2). RVFV is transmitted by mosquitoes, and the virus infection causes a high rate of abortions in pregnant ruminants and acute lethal hepatitis in newborn lambs (26). Most human patients show an acute febrile myalgic syndrome, with a small minority of patients, perhaps ϳ1%, experiencing severe hemorrhagic fever or encephalitis. Also, some patients show retinal vasculitis, which results in partial blindness for an undefined period (22).RVFV has three single-stranded genomic RNA segments, designated L, M, and S. The L and M RNA segments are of negative polarity. The L segment contains a 6,279-nucleotidelong open reading frame (ORF) encoding L protein and a viral RNA-dependent RNA polymerase (RdRp), while the M segment has a single ORF encoding NSm protein, a 78-kDa protein, and two major viral envelope proteins, Gn and Gc. The S segment is of ambisense polarity and encodes NSs protein and N protein; the former and the latter are encoded in anti-viralsense RNA and in viral-sense RNA, respectively. In bunyaviruses, both L protein and N protein are needed for viral RNA replication and viral mRNA synthesis, the latter of which uses a host mRNA-derived cap structure as a primer (3,16,29). N protein encapsidates viral RNAs to form helical nucleocapsids, which serve as a template for viral RNA synthesis (21, 23).RVFV L protein plays a central role in viral RNA synthesis, yet very little is known about how L protein exerts its functions; the properties of viral replication complexes, structures of viral RNA polymerases, and host factors needed for viral RNA synthesis have been characterized poorly for bunyaviruses. Past studies of other negative-stranded RNA viruses, including Sendai virus (36), parainfluenza virus 3 (37), and measles virus (4), all of which belong to the paramyxoviruses, and lymphocytic choriomeningitis virus (LCMV) (32), an arenavirus, suggested that their L proteins function as an oligomer to exert the RdRp function. In addition, the influenza A virus RdRp heterotrimer (18, 19) has been reported to form oligomers. Currently, it is unclear whether the L proteins of bunyaviruses also form an oligomer and exhibit RdRp activities in infected cells. We present here that RVFV L protein forms an oligomer. We further identified L protein regions that were involved in L oligomerization and explored the biological importance of the L protein oligomer for viral RNA synthesis. MATERIALS AND METHODSCells and viruses. BHK/T7-9 cells stably expressing T7 RNA polymerase (15) and Vero E6 cells were maintained as described previously (14). rMP12-rLuc (14), an RVFV MP-12 strain carrying the Renilla luciferase (rLuc) gene in place of the NSs gene in the S segment, was used.Plasmids. Plasmids encoding the mutant L protei...

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Recombinant Rift Valley fever vaccines induce protective levels of antibody in baboons and resistance to lethal challenge in mice

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