Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes

Abstract

18Bunyavirales is an order of segmented negative stranded RNA viruses comprising several life-19 threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus 20 (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific 21 treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales 22 genome constitute essential reactions of their viral cycle that are catalysed by the virally 23 encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-24 2 resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of 25 key C-terminal domains, notably the cap-binding domain that undergoes large movements 26 related to its role in transcription initiation and a zinc-binding domain that displays a fold not 27 previously observed. We capture the structure of LACV-L FL in two functionally relevant states, 28 pre-initiation and elongation, that reveal large conformational changes inherent to its 29 function. We uncover the coordinated movement of the polymerase priming loop, lid domain 30 and C-terminal region required for the establishment of a ten-base-pair template-product 31 RNA duplex before strand separation into respective exit tunnels. The revealed structural 32 details and dynamics of functional elements will be instrumental for structure-based 33 development of compounds that inhibit RNA synthesis by the polymerase. 34 35 INTRODUCTION 36Segmented negative stranded RNA viruses (sNSV) are divided into two major orders: 37Bunyavirales, that comprises more than 500 species classified in twelve families 1 , and 38Articulavirales, that include influenza virus from the Orthomyxoviridae family. Replication and 39 transcription of sNSV viral genomic segments are performed by the virally encoded RNA-40 dependent RNA polymerase or L protein 2 . These processes are performed in the cytoplasm of 41 infected cells for Bunyaviruses, whereas they occur in the nucleus for influenza virus 3,4 . 42Replication generates full-length genome or antigenome copies (vRNA and cRNA 43 respectively), whereas transcription produces capped viral mRNA that are recognized by the 44 cellular translation machinery to produce viral proteins. Transcription is initiated by a "cap-45 snatching" mechanism, whereby host 5ʹ capped RNAs are bound by the L cap-binding domain 46 (CBD), cleaved by the L endonuclease domain several nucleotides downstream, and then used 47 to prime synthesis of mRNA 2,5,6 . Although the overall mechanism of transcription initiation is 48 3 likely conserved between sNSVs, several elements suggest some divergences between viral 49 families. First, the source of capped RNA differs. Whereas influenza polymerase interacts 50 directly with the host polymerase II to snatch the caps of nascent transcripts in the nucleus 7 , 51 it is currently unclear which and in what context cytoplasmic capped RNAs are accessed by 52 bunyavirus polymerases and if the polymerase contains specific domains that w...