Mechanism of tripartite RNA genome packaging in Rift Valley fever virus

The Bunyaviridae family includes pathogens of medical and veterinary importance. Rift Valley fever virus (RVFV), a member in the Phlebovirus genus of the family Bunyaviridae, is endemic to sub-Saharan Africa and causes a mosquito-borne disease in ruminants and humans. Viruses in the family Bunyaviridae carry a tripartite, single-stranded, negative-sense RNA genome composed of L, M, and S RNAs. Little is known about how the three genomic RNA segments are copackaged to generate infectious bunyaviruses. We explored the mechanism that governs the copackaging of the three genomic RNAs into RVFV particles. The expression of viral structural proteins along with replicating S and M RNAs resulted in the copackaging of both RNAs into RVFV-like particles, while replacing M RNA with M1 RNA, lacking a part of the M RNA 5' UTR, abrogated the RNA copackaging. L RNA was efficiently packaged into virus particles released from cells supporting the replication of L, M, and S RNAs, and replacing M RNA with M1 RNA abolished the packaging of L RNA. Detailed analyses using various combinations of replicating viral RNAs suggest that M RNA alone or a coordinated function of M and S RNAs exerted efficient L RNA packaging either directly or indirectly. Collectively, these data are consistent with the possibility that specific intermolecular interactions among the three viral RNAs drive the copackaging of these RNAs to produce infectious RVFV.