Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy

Abstract

The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a noncanonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains [RNA-dependent RNA polymerase (RdRp), polyribonucleotidyl transferase (PRNTase), and methyl transferase] and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.

A Point Mutation in the RNA-Binding Domain of Human Parainfluenza Virus Type 2 Nucleoprotein Elicits Abnormally Enhanced Polymerase Activity

“…In contrast, RdRps that initiate RNA synthesis at the NP gene start site and then cap the nascent NP mRNA become committed to transcription (23,24). Structural studies of both sNSV and nsNSV RdRps suggest that both can be found in a number of alternative states or conformations, each of which may be important during different steps of RNA synthesis (25)(26)(27)(28).…”

Physical‐Organic Chemistry: A Swiss Army Knife

“…Neutron diffraction is still a problem (and neutrons have the potential to be enormously useful in understanding the structures of water) because large crystals are required, and because neutrons are scarce. High-resolution electron cryomicroscopy is making astonishing advances in efforts to determine the structures of complex molecules [92,93] , but is extraordinarily expensive and complicated. Any consideration of mechanism, or thermodynamics, ultimately must rest on knowing the structures of the reactants and products.…”

An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus

“…This Cterminal component of L proteins of NNS RNA viruses, especially the region between domains V and VI, is highly variable, and considered as a domain of L which interacts with unique host cell transacting transcriptional cofactors (Dortmans et al, 2011;Poch et al, 1990;Sidhu et al, 1993). The mutations in guinea pig-lethal MARV L protein, however, affect the domain III close to the GDNQ/E motif, presumably directly modulating the processivity of viral RNA-dependent RNA polymerase (Liang et al, 2015). Hence, changes in the polymerase activity of L mutants are expected to be independent of direct interactions with host factors.…”

“…1a). The recently published structure of vesicular stomatitis virus polymerase L suggests that mCherry is located close to the methyltransferase domain of the enzyme (Liang et al, 2015). Insertion of mCherry-L in a homologous region did not impair the function of EBOV L protein (Hoenen et al, 2012).…”

“…L, together with the MARV protein VP35, forms the RNAdependent RNA polymerase complex which is essential for transcription and replication of the viral genome . Based on the sequence alignment of MARV L and polymerases of other NNS RNA viruses, six conserved sequence regions have been identified, designated domains I-VI (Mühlberger et al, 1992), which are believed to constitute various enzymatic activities of L protein (Liang et al, 2015;Poch et al, 1990;Sidhu et al, 1993;Stec et al, 1991). Domain III of MARV L is anticipated to represent the RNA polymerase active site.…”