Understanding a viral polymerase complex: a multienzyme 'molecular motor' that drives virus replication

Lead Research Organisation: London Sch of Hygiene and Trop Medicine
Department Name: Infectious and Tropical Diseases

Abstract

Viruses can only successfully infect animals by hijacking normal cellular functions and using these to make many more copies of the virus. However, cells do not contain all of the machinery necessary to copy the virus genes, and so the virus has to carry some of this with it when it infects the animal. As this part of the virus has no cellular equivalent, but is necessary for the virus to reproduce, it is a very good target for medicine that interferes with virus infection. Bluetongue virus causes a devastating disease in sheep where up to 70% if infected animals die. For this virus we know all of the genes in the virus, understand the overall structure of the pathogen, and have demonstrated the function of some of the parts of the machinery that copies the virus at a test tube level. What we do not know is precisely how these individual pieces of the machinery work together and that is the focus of this research study. The approach taken will involve a study of the structure of the machinery at a very small scale, as well as experiments that test how well each part of the overall machine is working. Outputs from this research will provide opportunities for the design of new medicine that interferes with the virus machinery and prevents it from copying itself.

Technical Summary

The structure of the virus core of dsRNA viruses within the family Reoviridae has revealed a functional molecular machine dedicated to the efficient transcription of RNA upon cell entry, so initiating the infectious cycle. The present level of understanding has required a merging of structural and biophysical studies to map the protein-protein interactions within the core with biochemistry of individual reactions within the protein microenvironment and represents a novel and holistic approach to uncovering the action of the virion core. It is still the case however that the described activity of each individual core protein is incomplete and that a complete understanding of how the core proteins act in concert is lacking. This proposal will address these issues with a comprehensive analysis of each of the viral enzymes associated with transcription from the virus core. The proposed work will reveal how these proteins come together to provide the complete viral transcription function and, in an important and unique objective, we will reconstitute a functional transcriptase its component parts in vitro / a first in the field. The output from the work will contribute to the growing area of the structural basis of transcription and pave the way for the rational design of viral transcriptase inhibitors with the potential to block orbivirus replication and provide lead compounds of relevance to many related viruses of pathogenic significance to man and animals.

Publications

10 25 50
 
Description Viruses establish infection in human and animals by generating multiple copies of the virus through replication. The process of replication depends on the structure of the viral genetic material and the host cells that the virus is infecting. The purpose of this study was to understand how Bluetongue virus which possesses unique type of genetic materials (10 pieces of duplex nucleic acids, dsRNAs) accomplishes efficiently this process during infection of host cells. Bluetongue virus was selected since it is an important pathogen of livestock and much is known on viral protein structure and molecular biology. The extensive background knowledge should form the basis for understanding the replication mechanism. The main aim of the project was to reveal how these proteins come together to provide the genome replication function and, in an important and unique objective, was to reconstitute a functional replicase complex from its component parts in vitro, a first in the field. In addition, gaining this understanding for Bluetongue virus should serve as a highly useful model for other viruses that infect humans and animals. This project has been undertaken using a multi-disciplinary approach including genetics, biochemical and biophysical methods.

The output from this work should contribute to the growing research area of the structural basis of virus replication and pave the way for the rational design of anti-viral agents with the potential to block virus replication and provide a foundationof agents that will be of relevance to the control of many related viruses that infect man and animals.
Exploitation Route N/a
Sectors Other

 
Title Defective BTV strains 
Description A series of replication defective virus with site specific mutations/truncations or insertions in either VP6, VP7 or NS2 were generated using the reverse genetics system 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact Analysis of the effect of mutations in different virus genes has a huge impact on the development of new generation of vaccines for BTV 
 
Title In vitro polymerase 
Description Using in vitro polymerase assay system, we demonstrated that VP1 could synthesize the 10 dsRNAs simultaneously from BTV ssRNA templates in a single in vitro reaction as well as genomic dsRNA segments from rotavirus ssRNA templates that possess no sequence similarity with BTV. Further, we showed that synthesis of dsRNAs from capped ssRNA templates were significantly higher than that from uncapped ssRNA templates and the addition of dinucleotides enhanced activity as long as the last nucleotide of ssRNA template was complemented by the dinucleotide. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Understanding BTV replication mechanisms has an impact on the development of antiviral strategies 
 
Title Modified S7 exact copy constructs 
Description A series of constructs were produced that have specific mutations, trunctions and insertions of GFP into the coding region of VP7. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact These range of mutations were designed in order to test genetic stability of disabled viruses that can be potentially used as vaccine. 
 
Title S9 exact copy constructs with mutations and insertions 
Description A series of constructs were produced that have specific mutations, trunctions and insertions of GFP into the coding region of VP6. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact These range of mutations were designed in order to test genetic stability of disabled viruses that can be potentially used as vaccine. 
 
Title Stoichiometry of VP6 
Description A method was established to study the stoichiometry of VP6 when interacting with RNA. We modified glass cover-slips with amino groups modified with silane and attached fluorescently labelled protein by cross-linking amino groups from the protein with amino groups on the glass surface. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact No major impact, however, it may be used in future to understand specific RNA packaging motif in VP6. 
 
Title Genome packaging 
Description The genome packaging signals are present in 155 nucleotides at 5' end and 135 nucleotides at 3' end. 
Type Of Material Database/Collection of data 
Provided To Others? No  
 
Title Immunoprecipitation data 
Description The guk kinase domain (KL domain; 1-108 aa) of VP4, the capping enzyme of BTV, has been produced in baculovirus with a His-tag and Flag-tag. Immunoprecipitation data with the 3 functional domains of VP1 indicate that KL binds to the C terminal domain (CTD) of VP1 consistent with structural data. 
Type Of Material Database/Collection of data 
Provided To Others? No  
 
Title In vitro CFA method 
Description We have established a cell-free system to reconstitute subcore and core structures with the entire set of proteins and 10 ssRNAs and demonstrated that reconstituted cores are infectious in insect cells. Further, we showed that the 10 positive sense BTV ssRNAs are essential to drive the assembly reaction and that there is a distinct order of internal protein recruitment during the assembly process. 
Type Of Material Data analysis technique 
Year Produced 2011 
Provided To Others? Yes  
Impact Understand packaging signals and requirements for BTV. 
 
Title NMR structure of VP6 
Description To determine the NMR structure of VP6, both full-length and truncated versions of VP6 were cloned and expressed with an N-terminal His-tag in E. coli. VP6 was expressed in presence of 15NH4Cl and 13C6-glucose in D2O and labelled VP6 was purified by Ni-NTA resin followed by size exclusion chromatography. Protein samples were concentrated and NMR assignment spectra collected at 298K which showed an excellent, high dispersed peak of 2D 1H-15N HSQC spectrum, indicative of well-folded protein. Several parts of VP6 NMR structure have subsequently been determined. Our data clearly indicate that both N terminal and C terminal regions are well structured; however there is a large flexible region in the centre, due to glycine-rich amino acid sequences. Further there are two additional regions aa34 to aa134 and aa183 to aa220 were too unstable to analyze the structure. This region prevents to obtain high dispersed peak of spectrum. To resolve this problem, we constructed several truncated versions of VP6 that lack these flexible regions and expect this may allow us to obtain better spectrum of the protein. The truncated complexes may be more suitable for crystallograpy. 
Type Of Material Database/Collection of data 
Provided To Others? No  
 
Title Replication 
Description BTV uses a 2-stage replication cycle during infection. The primary replication synthesizes the first set of proteins that form polymerase complex and the subcore layer VP3 as well as a non-structural protein, NS2 which forms the virus inclusion bodies (the core assembly site). The secondary replication involves genome packaging. 
Type Of Material Database/Collection of data 
Provided To Others? No  
 
Title Replication 
Description Nonstructural protein NS1 is necessary only for enhancing translation of BTV proteins in early replication. 
Type Of Material Database/Collection of data 
Provided To Others? No