Nucleocapsids of the Rift Valley fever virus ambisense S segment contain an exposed RNA element in the center that overlaps with the intergenic region

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen. Its RNA genome consists of two negative-sense segments (L and M) with one gene each, and one ambisense segment (S) with two opposing genes separated by the noncoding “intergenic region” (IGR). These vRNAs and the complementary cRNAs are encapsidated by nucleoprotein (N). Using iCLIP2 (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to map all N-vRNA and N-cRNA interactions, we detect N coverage along the L and M segments. However, the S segment vRNA and cRNA each contain approximately 100 non-encapsidated nucleotides stretching from the IGR into the 5’-adjacent reading frame. These exposed regions are RNase-sensitive and predicted to form stem-loop structures with the mRNA transcription termination motif positioned near the top. Moreover, optimal S segment transcription and replication requires the entire exposed region rather than only the IGR. Thus, the RVFV S segment contains a central, non-encapsidated RNA region with a functional role. Rift Valley fever virus is a WHO-prioritized zoonotic pathogen. The authors identified an RNA element on its genome that is devoid of nucleoprotein, has the potential for a hairpin structure, and is required for optimal transcription and replication.