Hantaan virus polymerase has an in vitro Terminal Nucleotidyl Transferase activity that is conserved across the Bunyaviricetes class

Abstract

Abstract section Bunyaviricetes is a class of segmented negative strand RNA viruses (sNSV) that includes causative agents of severe zoonotic diseases resulting in hemorrhagic fever in humans and livestock. Hantaan virus (HTNV), family Hantaviridae, is an example of life-threatening bunyavirus transmitted by rodents which leads to Hantavirus hemorrhagic fever with renal syndrome in humans. Infection relies on the replication and transcription of their tripartite genomes through multifunctional RNA-dependent RNA polymerases (RdRp) in the context of ribonucleoproteins (RNPs) made of one viral RNA fragment, one RdRp and multiple copies of viral nucleoproteins. The RdRp, also known as L protein, performs replication by de novo initiation through a prime-and-realign mechanism while transcription initiates through a cap snatching mechanism, that engages a cap-binding domain and an endonuclease (EN) domain present in the L protein C and N terminal regions, respectively. In this work we show that HTNV L protein has a terminal nucleotidyl transferase (TNTase) activity over double-stranded RNA substrates (dsRNA) in vitro. We show that the TNTase activity is also present in other bunyaviral L proteins by comparing HTNV L protein activity with La Crosse (LACV, family Orthobunyaviridae) and Crimean Congo Hemorrhagic Fever Virus (CCHFV, family Nairoviridae) L proteins. Our results show that TNTase activity appears to be a general feature of Bunyaviruses and report the differences on substrate and nucleotide specificity for each family of virus. Importance The L proteins are preferential antiviral targets for the treatment of bunyaviral emerging diseases. Our results provide new ground for the characterization of L protein enzymatic activity in vitro which should be considered for the development activity assays, paramount for the screening of antiviral compounds. Indeed, TNTase activity can be easily misinterpreted as replication activity. We indicate here the key experimental setup and controls that would need to be considered to distinguish both activities. Our in vitro observations pave the way for future investivation of the potential TNTase role(s) in the infected cells. Proposed hypotheses concern potential functions in the preservation of genome integrity, the prevention of RIG-I innate immune response activation, or modification of RNA metabolism in the cell cytoplasm. The TNTase activity assays we describe here finally provide a straightforward biochemical approach for screening of inhibitors targeting RdRp activity.