Structural and functional characterization of the Sin Nombre virus L protein

The Bunyavirales order is a large and diverse group of segmented negative-strand RNA viruses. Several virus families within this order contain important human pathogens, including Sin Nombre virus (SNV) of the Hantaviridae. Despite the high epidemic potential of bunyaviruses, specific medical countermeasures such as vaccines or antivirals are missing. The multifunctional similar to 250 kDa L protein of hantaviruses, amongst other functional domains, harbors the RNA-dependent RNA polymerase (RdRp) and an endonuclease and catalyzes transcription as well as replication of the viral RNA genome, making it a promising therapeutic target. The development of inhibitors targeting these key processes requires a profound understanding of the catalytic mechanisms. Here, we established expression and purification protocols of the full-length SNV L protein bearing the endonuclease mutation K124A. We applied different biochemical in vitro assays to provide an extensive characterization of the different enzymatic functions as well as the capacity of the hantavirus L protein to interact with the viral RNA. By using single-particle cryo-EM, we obtained a 3D model including the L protein core region containing the RdRp, in complex with the 50 promoter RNA. This first high-resolution model of a New World hantavirus L protein shows striking similarity to related bunyavirus L proteins. The interaction of the L protein with the 50 RNA observed in the structural model confirms our hypothesis of protein-RNA binding based on our biochemical data. Taken together, this study provides an excellent basis for future structural and functional studies on the hantavirus L protein and for the development of antiviral compounds. Author summary Hantaviruses are globally distributed zoonotic viruses and can cause severe disease in humans. New World hantaviruses, including Sin Nombre virus (SNV), are endemic to the Americas and cause cardiopulmonary syndromes with fatality rates of up to 35%. There are currently no effective vaccines or specific FDA-approved treatments available. A promising drug target would be the viral multifunctional large (L) protein, which plays a key role in viral genome transcription and replication. We succeeded in expressing and purifying the full-length L protein of SNV and present the first high-resolution structural model of the SNV L protein core region, including the RNA-dependent RNA polymerase, in complex with viral RNA. Additionally, we developed different biochemical assays to investigate protein-RNA interaction, RNA polymerase activity and endonuclease function of the full-length L protein. Based on these assays, we developed a model for viral promoter binding, which is supported by our structural data. The developed protocols and assays for the structural and functional characterization of the hantavirus L protein will facilitate further mechanistic studies and support the development of antiviral strategies.