Investigation of RNA packaging in Rift Valley fever virus

Rift Valley fever virus (RVFV), a bunyavirus, is transmitted by mosquitoes and has caused large outbreaks among humans and ruminants in many countries in Africa and the Arabian Peninsula. In most cases, patients recover from the disease, but some develop retinal or macular changes, which result in vision impairment, and severe diseases, characterized by hemorrhagic fever or encephalitis. Introduction of RVFV into non-endemic countries potentially occurs by the movement of infected travelers, animals, and mosquitoes. Moreover, naturally occurring mosquito populations in the U.S. support RVFV replication. RVFV outbreaks in the U.S. would cause serious public health, agricultural, and economic problems. RVFV is a select agent and belongs to the NIAID Category A list of pathogens and the CDC list of potential bioterrorism agents. RVFV is also listed among eight named pathogens that cause priority diseases for WHO research and development in emergency context. The lack of availability of licensed vaccines or anti-RVFV reagents for use in humans or domestic animals is of great concern. RVFV is an enveloped RNA virus, carrying a tripartite, single-stranded, negative-sense RNA genome composed of L, M and S RNAs. L RNA encodes L protein, the RNA-dependent RNA polymerase, and M RNA encodes the two major envelope glycoproteins, Gn and Gc, along with two accessory proteins. The S RNA uses an ambi-sense coding strategy to express N and NSs, the latter of which is the major viral virulence factor. Our long-term goal is to elucidate the mechanism of viral RNA packaging and co-packaging mechanisms of RVFV, as insight into the underlying rules and mechanisms that govern the packaging/co-packaging of the segmented RNA genome into RVFV particles is valuable for understanding the regulation of virus replication, virus evolution, and the pathogenic potential of the virus. This knowledge is also critical for the development of antiviral drugs that can inhibit infectious virus production and the design of strategies for live attenuated vaccines. The present application will test the central hypothesis that intersegmental intracellular viral RNA-RNA interactions drive packaging/co-packaging of viral RNA segments, leading to efficient production of infectious RVFV. Our previous study showed that effective packaging of LRNA relies on other viral RNAs. Specific aim 1 will test the hypothesis that intersegmental interaction between intracellular M RNA and intracellular L RNA facilitates the formation of a packaging-competent conformation of L RNA capable of efficient binding to Gn, leading to efficient packaging of L RNA into RVFV particles. We demonstrated that ntigenomic S RNA binds efficiently to Gn and undergoes efficient packaging. Specific aim 2 will test the hypothesis that efficient binding of antigenomic S RNA to Gn, along with the sequence of the antigenomic S RNA, facilitates its interaction with M and L RNAs, resulting in the co-packaging of these viral RNAs into RVFV particles.