Rift Valley fever virus nucleocapsids contain an exposed RNA sequence on the ambisense S segment that is not identical with the intergenic region

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen endemic in Africa. Its RNA genome consists of two negative-sense segments (L and M) with one gene each, and an ambisense segment (S) with two opposing genes separated by the noncoding “intergenic region” (IGR). These viral RNAs (vRNAs) as well as the complementary cRNAs are encapsidated by nucleoprotein (N). Using iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to map all N-vRNA and N-cRNA interactions, we detected consistent N coverage along the L and M segments. However, the S segment vRNA and cRNA each contained approximately 100 non-encapsidated nucleotides stretching from the downstream half of IGR into the terminus of the opposing gene. These exposed regions (EvSR and EcSR) have the potential to form stable stem-loop structures with the RVFV transcription termination motif positioned near the top. They are RNase-sensitive, and targeting with antisense oligomers decreases viral replication. The RVFV ambisense S segment thus contains a central non-encapsidated RNA region with a functional role.