Our understanding of the structure and mechanism of action of the RNA-dependent RNA polymerase (RdRP) of Paramyxoviruses is rudimentary. We have recently determined the structure of a C-terminal fragment of Nipah virus L polymerase. The aim of the project is to characterise the biochemical function of the C-terminal domain of NiV L, using structure based approaches, with the aim of solving the structures of complexes of this fragment with ligands.
Studies will be initiated to determinine the function of the structure of the NiV L domain, using a structure based mutagenesis approach combined with in vivo and in vitro assays, along with co-crystallisation experiments.
At present we do not know whether the fragment solved has any functional activity. Evidence exists suggesting that in VSV the corresponding region is involved in controlling MTase activity. Although the sequence conservation in this region between VSV and NiV L is rather poor, it is intriguing to note that the corresponding NiV L region is surface exposed, which suggests it could be functionally relevant. A number of structure based mutations in this region will be introduced into the full length protein and their effect assessed by cell-based and in vitro methods as described below.
Cell-based assays for L functionality: For NiV L we will use an established plasmid-driven minigenome system obtained from Ramon Flick, BioProtection Systems Corporation, Ames, USA. Briefly, we will reconstitute NiV RNPs by co-expressing viral L, P, and N in transiently transfected 293T cells using standard protein expression vectors. A minigenome with leader and trailer sequences flanking a reporter gene, e.g. CAT, will be expressed from an RNA Pol I-driven construct (Pol I is endogenous and gives cleaner transcripts than T7). Polymerase activity will be monitored by assaying for CAT-activity. Alternatively, we will establish a primer extension-based assay, similar to that developed for influenza virus RNAs, to monitor the accumulation of NiV mRNA, genomic and antigenomic RNA. This assay will provide information on the functionality of L mutants; it will also distinguish between mutants deficient only in transcription (mRNA synthesis) or genome replication.
In vitro activity assays for L: If a particular mutation leads to a reduction in minigenome transcription and/or replication, we will investigate further the effect of these mutations in vitro with the aim of determining the step in transcription/replication affected by the mutation.
Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Project reference number: 347
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Jonathan M Grimes | Structural Biology | Oxford University | UK | jonathan@strubi.ox.ac.uk |
| Dr Ervin Fodor | Dunn School of Pathology | University of Oxford | UK | ervin.fodor@path.ox.ac.uk |
2005. A single amino acid change in the L-polymerase protein of vesicular stomatitis virus completely abolishes viral mRNA cap methylation. J. Virol., 79 (12), pp. 7327-37. Read abstract | Read more
The vesicular stomatitis virus (VSV) RNA polymerase synthesizes viral mRNAs with 5'-cap structures methylated at the guanine-N7 and 2'-O-adenosine positions (7mGpppA(m)). Previously, our laboratory showed that a VSV host range (hr) and temperature-sensitive (ts) mutant, hr1, had a complete defect in mRNA cap methylation and that the wild-type L protein could complement the hr1 defect in vitro. Here, we sequenced the L, P, and N genes of mutant hr1 and found only two amino acid substitutions, both residing in the L-polymerase protein, which differentiate hr1 from its wild-type parent. These mutations (N505D and D1671V) were introduced separately and together into the L gene, and their effects on VSV in vitro transcription and in vivo chloramphenicol acetyltransferase minigenome replication were studied under conditions that are permissive and nonpermissive for hr1. Neither L mutation significantly affected viral RNA synthesis at 34 degrees C in permissive (BHK) and nonpermissive (HEp-2) cells, but D1671V reduced in vitro transcription and genome replication by about 50% at 40 degrees C in both cell lines. Recombinant VSV bearing each mutation were isolated, and the hr and ts phenotypes in infected cells were the result of a single D1671V substitution in the L protein. While the mutations did not significantly affect mRNA synthesis by purified viruses, 5'-cap analyses of product mRNAs clearly demonstrated that the D1671V mutation abrogated all methyltransferase activity. Sequence analysis suggests that an aspartic acid at amino acid 1671 is a critical residue within a putative conserved S-adenosyl-l-methionine-binding domain of the L protein. Hide abstract
2008. Establishment and characterization of plasmid-driven minigenome rescue systems for Nipah virus: RNA polymerase I- and T7-catalyzed generation of functional paramyxoviral RNA. Virology, 370 (1), pp. 33-44. Read abstract | Read more
In this study we report the development and optimization of two minigenome rescue systems for Nipah virus, a member of the Paramyxoviridae family. One is mediated by the T7 RNA polymerase supplied either by a constitutively expressing cell line or by transfection of expression plasmids and is thus independent from infection with a helper virus. The other approach is based on RNA polymerase I-driven transcription, a unique approach for paramyxovirus reverse genetics technology. Minigenome rescue was evaluated by reporter gene activities of (i) the two different minigenome transcription systems, (ii) genomic versus antigenomic-oriented minigenomes, (iii) different ratios of the viral protein expression plasmids, and (iv) time course experiments. The high efficiency and reliability of the established systems allowed for downscaling to 96-well plates. This served as a basis for the development of a high-throughput screening system for potential antivirals that target replication and transcription of Nipah virus without the need of high bio-containment. Using this system we were able to identify two compounds that reduced minigenome activity. Hide abstract
2002. A single amino acid mutation in the PA subunit of the influenza virus RNA polymerase inhibits endonucleolytic cleavage of capped RNAs. J. Virol., 76 (18), pp. 8989-9001. Read abstract | Read more
The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA-->cRNA-->vRNA), but its transcriptional activity (vRNA-->mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNA-binding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity. Hide abstract