Structure of a rabies virus polymerase complex from electron cryo-microscopy

Abstract

Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication. rabies lyssavirus | NNS RNA viruses | vesicular stomatitis virus | transcription | replication

Nucleocapsid Structure of Negative Strand RNA Virus

“…In an NSV genome that encodes more than one viral protein, the promotor for transcription is located at the internal gene junction. The structure of the L-P complexes [55,[84][85][86] showed that the L protein is stabilized in the conformation prior to initiation by the bound P protein. The nascent RNA may exit to activate the capping and methyltransferase so that the elongation of mRNA will proceed [87].…”

Components and Architecture of the Rhabdovirus Ribonucleoprotein Complex

“…It can be divided into four domains. These are the N-terminal domain (P NTD, aa 1-90, RABV), which is disordered but adopts defined conformations upon interaction with N or L [37][38][39][40][41][42]; the central domain (P CED, aa 91-130, RABV); the C-terminal intrinsically disordered domain (P CID, aa 131-195, RABV); and the C-terminal domain (P CD, aa 195-296, RABV). The atomic structures of P CED (pdb: 2fqm [43], VSV; pdb: 3l32 [44], RABV), which mediates the dimerisation of the P-protein, and P CD (pdb: 2k47 [45], VSV; pdb: 1vyi [46], RABV), which is important for interacting with the N-RNA complex and parts of the VSV P NTD with N [37], have been resolved.…”

“…The P-protein is also the essential but catalytically inactive cofactor of L-protein. P NTD is the domain involved in the interaction with the L-protein [40,41,60,61]. This interaction is not essential for the enzymatic function of the L-protein, but rather for the generation of longer RNA molecules [62].…”

“…The RdRp and the capping domain form the core of the structure. In the absence of P, the remaining three domains-connector domain, methyltransferase domain and C-terminal domain-have no fixed position regarding the core of the structure [6,40,41,81]. The P-protein segments that bind to the L-protein that can be structurally resolved are aa 49-56 (interaction with the C-terminal domain), [82][83][84][85][86][87][88][89] (binding between the C-terminal domain and RdRp) and 94-105 (binding between the connector and C-terminal domains) for VSV [40].…”

“…The P-protein segments that bind to the L-protein that can be structurally resolved are aa 49-56 (interaction with the C-terminal domain), [82][83][84][85][86][87][88][89] (binding between the C-terminal domain and RdRp) and 94-105 (binding between the connector and C-terminal domains) for VSV [40]. For RABV, a 37 aa segment of P-protein, ending with Glu87, could be identified as being bound to the connector, the RdRp and the C-terminal domain of L-protein [41].…”