Conformational changes in Lassa virus L protein associated with promoter binding and RNA synthesis activity

Abstract

Lassa virus, which causes annual outbreaks in West Africa with increasing case numbers in recent years, is recognized by the WHO R&D blueprint as a significant threat for public health with high epidemic potential and no effective countermeasures. The viral large (L) protein, which contains the RNA-dependent RNA polymerase, is a key player for transcription of viral mRNA and genome replication. Here we present nine cryo-EM structures of Lassa virus L protein in the apo-, promoter-bound pre-initiation and active RNA synthesis states. We characterize distinct binding pockets for the conserved genomic 3’ and 5’ promoter RNAs and show how full-promoter binding induces a distinct pre-initiation conformation. In the apo- and elongation states, the endonuclease is inhibited by the binding of two distinct L protein peptides in the active site, respectively, whereas in the pre-initiation state, the endonuclease is uninhibited. In the stalled, early elongation state, a template- product duplex is bound in the active site cavity together with an incoming non-hydrolysable nucleotide. In this configuration, the full C-terminal region of the L protein, including the putative cap-binding domain, is highly ordered. The structural data are complemented by in vitro and cell-based studies testing a broad range of L protein mutants to probe functional relevance. These data advance our mechanistic understanding of how this flexible and multifunctional molecular machine is activated and will underpin antiviral drug development targeting the arenavirus L protein.

Structural insights into viral genome replication by the severe fever with thrombocytopenia syndrome virus L protein

“…Overall, this analysis suggests that the 5′ RNA binding site is important for both genome replication and transcription as no selective defect in RNA synthesis could be detected. This matches previous observations for LASV L protein (27).…”

“…The first nt fit into the map at this site corresponds to U1 in the 3′ vRNA sequence and is found on the extremity of the L protein surrounded by several flexible sidechains (D383, K386 and E387), however, none of these form close interactions with the U1 base. This is not too surprising as we observed something similar for the LASV L protein (27). Nucleotide G2 sits in a pocket delineated by sidechains of Y408, E527, K544, L1254, P1256, I1344, and V1413.…”

“…This is possible because the 5′ and 3′ vRNA strands are shifted by 1 nt in respect to each other, meaning that G11 of the 5′ vRNA base-pairs with C12 of the 3′ vRNA, C12 of the 5′ vRNA base-pairs with G13 of the 3′ vRNA and so on (Figure 3B). In LACV, the 5′/3′ distal duplex is stabilised by 6 base-pair interactions (13), whereas, in LASV there are 8 base-pair interactions (27). This shift between the 3′ and 5′ vRNA strands presumably enables the SFTSV L protein to overcome the general lack of complementarity in the 3′/5′ duplex region.…”

“…Several bunyaviruses and influenza viruses are known to coordinate 3′ vRNA at secondary binding sites within the viral polymerase (13,27,28,52,54–56). In the late-stage elongation structure reported here, we find nts 1-5 of the 3′ vRNA bound in a tunnel immediately adjacent to the vRBL (Figure 5A).…”

Frequency of a nucleotide overhang at the 5’ end of hemorrhagic fever mammarenavirus genomes in public sequence data

“…In mammarenaviruses, the genome ends exhibit terminal complementarity of the 5' and 3' ends in each of the L and S segments to form a replication promoter. The interaction of each 5' and 3' end to L protein is essential for the initiation of RNA synthesis (7,8). The 5' end of some mammarenavirus genomes contains an additional nontemplated guanine residue (5'-G overhang) (9)(10)(11)(12), which has been considered to be generated by a prime-and-realign mechanism.…”