Structural Characterization of the Full-length Hantaan Virus Polymerase

Abstract

Hantaviridae are a family of segmented negative-sense RNA viruses that contains important human and animal pathogens including Andes orthohantavirus and Hantaan orthohantavirus. Hantaviridae contain a viral RNA-dependent RNA polymerase that replicates and transcribes the viral genome in the cytoplasm of infected cells. Here we establish the expression and purification of the polymerase from the Old World Hantaan orthohantavirus and characterise the structure using single particle cryo electron microscopy. We determine a series of structures at resolutions between 2.7 and 3.3 Å of RNA free polymerase comprising the core, core and endonuclease, and a full-length polymerase. The full-length polymerase structure depicts the location of the cap binding and C-terminal domains which are arranged in a conformation that is incompatible with transcription and in a novel conformation not observed in previous conformations of cap-snatching viral polymerase. We further describe structures with 5′ vRNA promoter in the presence and absence of a nucleotide triphosphate. The nucleotide bound structure mimics a replication pre-initiation complex and the nucleotide stabilises the motif E in a conformation distinct from those previously observed. We observe motif E in four distinct conformations including β- sheet, two helical arrangements, and an ordered loop. The insights gained here form a starting point for future detailed mechanistic studies of both the transcription and replication activities of the hantavirus polymerase and for the development of therapeutic targets.