Modulation of viral genome 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. RVFV outbreaks in the U.S. are quite possible and would cause serious public health, agricultural, and economic problems. 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. Furthermore, antigenomic S RNA is also packaged efficiently in RVFV particles. L RNA encodes 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 suppresses host innate immune responses. One of the fundamental steps in a viral life cycle is the packaging of the viral genome into virus particles. The mechanism of genome packaging in bunyavirus differs from that of other envelope viruses, as most bunyaviruses, including RVFV, lack a matrix protein. We showed that Gn functions as a matrix protein surrogate and Gn-viral RNP interaction drives the packaging of RVFV RNAs. Although multiple regions of the RVFV RNAs directly bind to Gn in infected cells, noncoding regions of viral RNAs, which serve as cis-acting elements required for viral RNA replication, lack major Gn-binding sites, except for the presence of a prominent Gn-binding site, 6-25 region, at the 3’ end of the antigenomic S RNA. Notably, 6-25 region is important for efficient packaging of the antigenomic S RNA, leading to expression of NSs, which inhibits interferonβ mRNA transcription, early in RVFV infection. This indicates the contribution of 6-25 region to RVFV pathogenesis. Another notable feature of RVFV genome packaging is that L RNA undergoes inefficient packaging in the absence of other viral RNAs. We found that efficient L RNA packaging requires trans-acting functions of M RNA and N. Therefore, the overall goal of our proposal is to expand our understanding of the mechanism of viral RNA packaging in bunyaviruses, using RVFV as a model virus. Our central hypotheses are that presence of 6-25 region at the terminal region of the viral RNA is important for this region to function as a packaging signal, which promotes viral RNA packaging, and that adding 6-25 region to the terminal region of L RNA alters the L RNA packaging to be independent from the trans-acting functions of M RNA and N. Experiments testing these hypotheses and related studies proposed in the application will also provide new insights into development of a novel RVFV-based expression vector/vaccine platform and a safer live attenuated vaccine. Taken together, the data from the proposed studies will substantially advance our understanding of the mechanisms of bunyavirus RNA packaging, provide novel insight into RVFV pathogenicity at the molecular level, reveal the possible use of an attenuated strain of RVFV for wider scientific and medical applications, and open a new approach for improvement of live attenuated RVFV vaccine safety.
