Poster Presentation 49th Lorne Conference on Protein Structure and Function 2024

Structural and Functional Characterization of the Hantaan Virus Polymerase Complex (#233)

Jeremy R. Keown 1 2 , Loic Carrique 1 , Ben Nilsson-Payant 3 , Ervin Fodor 4 , Jonathan M. Grimes 1
  1. Division of Structural Biology , University of Oxford, Oxford, Oxfordshire, United Kingdom
  2. School of Life Sciences, University of Warwick, Coventry, United Kingdom
  3. TWINCORE Centre for Experimental and Clinical Infection Research, Hannover, Germany
  4. Sir William Dunn School of Pathology, University of Oxford, Oxford, Oxfordshire, United Kingdom

Hantaviridae are a family of RNA viruses that contains important human and animal pathogens including Sin Nombre virus, Andes, and Hantaan orthohantavirus. Hantaviridae contain a viral RNA dependent RNA polymerase that replicates and transcribes the viral genome in the cytoplasm of infected cells. The polymerase is a large multifunctional enzyme that encompasses an RNA-dependent RNA polymerase domain, an N-terminal endonuclease domain, and a putative C-terminal cap-binding domain. Here we use structural biology complimented with in vitro assays to characterise the viral polymerase. We first developed expression and purification of an old world Hantaviridae polymerase from the Hantaan orthohantavirus. Functional characterisation show potent endonuclease activity that is further increased by the addition of MnCl2 and ablated with a single point mutation. We find the polymerase can efficiently initiate transcription using a capped RNA primer strongly suggesting the presence of a cap binding domain. Using single particle cryo electron microscopy we determined a series of polymerase conformations including structures of full length and RNA bound polymerase at resolutions between 2.8 and 3.8 Å. We observe a full-length polymerase in a conformation that is incompatible with transcription. We further observe movements and rearrangements within the core of the polymerase that open and close the product and template exit channels in response to nucleotide and RNA binding. The likely conservation of polymerase enzymatic functions across the Hantaviridae family makes them promising therapeutic targets. The insights provided here form a starting point for future detailed mechanistic studies of both transcription and replication of the viral polymerase and developed of therapeutic targets.