Structural insight into RNA encapsidation by the severe fever with thrombocytopenia syndrome virus nucleocapsid protein

ABSTRACT Severe fever with thrombocytopenia syndrome virus (SFTSV), an emerging highly pathogenic bunyavirus, poses a significant public health threat with a case fatality rate of up to 30%. The viral nucleocapsid protein (NP) encapsidates the genomic RNA to form a ribonucleoprotein (RNP) complex, which is critical for transcription and replication. However, the molecular mechanism underlying SFTSV RNA encapsidation remains poorly understood, largely due to the lack of structural information on the NP-RNA complex. Here, we report a cryo-electron microscopy structure of the SFTSV NP in complex with single-stranded RNA. The structure reveals a pentameric NP assembly that sequesters RNA along the inner surface of the oligomeric ring in a sequence-independent manner. Strikingly, all the RNA bases face the protein, rendering them inaccessible for transcription and replication. Each NP subunit accommodates four nucleotides within an evolutionarily conserved hydrophobic cleft, with an additional two to three nucleotides bound at the inter-subunit interface. The functional importance of the NP-RNA interactions is further corroborated by a minigenome-based assay. This work provides structural insight into RNA encapsidation by SFTSV NP and offers a foundation for the rational design of antiviral therapeutics targeting this essential viral protein. IMPORTANCE Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic bunyavirus that causes severe hemorrhagic fever, leukopenia, thrombocytopenia, and multi-organ failure, with a case fatality rate of up to 30%. No licensed vaccines or specific antiviral therapies are currently available. The viral nucleocapsid protein (NP) is essential for viral transcription and replication, forming a ribonucleoprotein complex (RNP) by encapsidating viral genomic RNA. However, the structural basis of RNA recognition and encapsidation by SFTSV NP remains poorly understood. In this study, we determined a cryo-electron microscopy structure of the SFTSV NP-RNA complex. Structural comparisons and evolutionary conservation analysis of NPs across the family Phenuiviridae uncovered a conserved RNA-binding mode among phenuiviruses, suggesting a shared RNA encapsidation mechanism among related viruses. Our findings provide critical structural insights into SFTSV RNA encapsidation and will aid future efforts to develop antivirals against SFTSV and related pathogenic viruses.