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

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 Å. We identify how the L protein binds the 5′ viral RNA (vRNA) in a hook-like conformation and show how the distal 5′ and 3′ vRNA ends form a duplex positioning the 3′ vRNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early- and late-stages of elongation with the RdRp core accommodating a 9-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3′ secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.

The mechanism of genome replication and transcription in bunyaviruses

“…However, arguably the major breakthrough in recent years has been the visualization of these proteins in action by determining structures of L proteins stalled in key active states. Following the pioneering work on influenza virus polymerase [ 46 , 47 ], snapshots have now been obtained for LACV, LASV, and the SFTSV L proteins [ 48 – 50 ]. LACV L structures revealed the key conformational changes necessary for genome replication and transcription [ 49 ].…”

“…LACV L structures revealed the key conformational changes necessary for genome replication and transcription [ 49 ]. SFTSV and LASV L snapshots depicted the conformational changes associated with promoter binding and genome replication activities [ 48 , 50 ] ( Fig 1 ). In addition, LASV, Junin virus (JUNV), and MACV L have been determined in complex with the viral matrix protein Z, providing structural insights into the regulation of Arenaviridae polymerase activity [ 51 – 53 ], which had been detected previously in in vitro and cell-based assays [ 54 – 59 ].…”

“…Both the linker region and the PA-C-like region buttress the central PB1-like region, which contains the conserved palm, thumb, and finger domains, features that are present in all viral RNA-dependent RNA polymerases (RdRps) and form the RdRp active site. The PB2-like region contains (i) a thumb-ring that is homologous to PB2-N in influenza virus and which surrounds the thumb domain; (ii) a bridge that connects the thumb/thumb-ring to the fingers; (iii) a lid that plays a central role in separating the template from the product facilitating their exit as single-stranded RNAs; and (iv) a protruding, multidomain CTER that undergoes large rearrangements during activity and is not visible in all the experimental maps of full-length L proteins [ 39 , 48 – 50 , 65 ].…”

“… (A) The LASV late-elongation (PDB: 7OJN) [ 48 ], SFTSV late-elongation (PDB: 8ASD) [ 50 ], and LACV early-elongation (PDB: 7ORM) [ 49 ] L protein structures are shown side by side. The core of each L protein is shown in grey with key domains colored as follows: endonuclease (orange), pyramid domain (only in LASV, dark blue), CBD (cyan), mid-link domain (pink), and 627-like/zinc-binding domain (purple).…”

“…Additional specific motifs of segmented negative-strand viruses (sNSV) include motif G in the thumb and H in the fingers domain [ 38 , 40 , 48 , 66 ]. The central active site cavity is connected to the exterior by four tunnels that correspond to (i) template entry; (ii) nucleotide entry; (iii) template exit; and (iv) product exit channels [ 8 , 38 ].The ENDO and the CTER protrude from the CORE and undergo large movements during activity [ 39 , 48 – 50 ].…”