Investigating the mechanism of RNA packaging in Rift Valley Fever phlebovirus

Rift Valley fever phlebovirus (RVFV) is a trisegmented, single-stranded, negative-sense RNA genome, consisting of L, M and S segments. The virion carries two envelope glycoproteins, Gn and Gc, along with ribonucleoprotein complexes (RNPs), composed of encapsidated genomes carrying the nucleocapsid protein, N, and the RNA-dependent RNA polymerase, L protein. Understanding the mechanisms that govern the recognition and recruitment of viral RNPs for packaging into RVFV particles is valuable for understanding the regulation of virus replication, virus evolution, and genetic reassortment. However, the mechanisms of viral RNA packaging in RVFV and other bunyaviruses are largely unknown. To study the accumulation kinetics and packaging profile of RVFV RNAs into virions, we established a novel strand-specific RT-qPCR assay that selectively quantifies the genomic and antigenomic RNAs of each of the three RVFV RNA segments. Using this assay, we were able to determine the accumulation kinetics of genomic and antigenomic viral RNAs in RVFV-infected cells and conduct a quantitative analysis of RNA packaged inside purified RVFV particles. Our analysis revealed similar packaging abilities among genome segments but among antigenomic segments, antigenomic S displayed a significantly higher packaging ability. Our data suggests the preferential incorporation of antigenomic S RNA compared to antigenomic L and M RNA. To delineate the factor(s) governing the differential packaging abilities of RVFV RNA segments, we characterized the molecular interactions between Gn and viral RNPs in RVFV-infected cells. Co-immunoprecipitation analysis demonstrated an interaction of Gn with viral RNPs in infected cells. Furthermore, UV-crosslinking and immunoprecipitation analysis showed that Gn also directly binds to genomic and antigenomic viral RNAs, revealing the presence of a direct interaction between Gn and viral RNAs in infected cells for the first time in bunyaviruses. Strikingly, among the antigenomic RNAs, Gn exhibited a significantly higher binding ability to antigenomic S RNA, which correlated with its higher packaging ability, suggesting the presence of a mechanism for the preferential packaging of antigenomic S RNA. Collectively, our study strongly suggests that a direct interaction between Gn and specific RNA elements in viral RNAs could be the primary factor that governs the packaging efficiency of RVFV RNA segments into virus particles.