David Rowlands collection

Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part by the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for virus replication as well as locations that can tolerate small genetic insertions. Such insertion sites can subsequently be adapted by the incorporation of commonly used epitope tags, facilitating their detection with commercially available reagents. In this study, we used random transposon-mediated mutagenesis to produce a library of 15 nt insertions in the FMDV non-structural polyprotein. Using a replicon-based assay, we isolated multiple replication-competent as well as replication-defective insertions. We adapted the replication-competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins for use in a variety of downstream assays. Additionally, we showed that replication of some of the replication-defective insertion mutants could be rescued by co-transfection of a ‘helper’ replicon, demonstrating a novel use of random mutagenesis to identify intergenomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornavirus replication complexes.

Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

A characteristic of many positive-strand RNA viruses is that, whilst replication of the viral genome is dependent on the expression of the majority of non-structural proteins in cis, virus particle formation can occur when most or all of the structural proteins are co-expressed in trans. Making use of a recently identified hepatitis C virus (HCV) isolate (JFH1) that can be propagated in tissue culture, this study sought to establish whether this is also the case for hepaciviruses. Stable cell lines containing one of two bicistronic replicons derived from the JFH1 isolate were generated that expressed non-structural proteins NS3–5B or NS2–5B. Release and transmission of these replicons to naïve Huh7 cells could then be demonstrated when baculovirus transduction was used to express the HCV proteins absent from the subgenomic replicons. Transmission could be blocked by a neutralizing antibody targeted at the E2 envelope protein, consistent with this phenomenon occurring via trans-encapsidation of replicon RNA into virus-like particles. Transmission was also dependent on expression of NS2, which was most effective at promoting virus particle formation when expressed in cis on the replicon RNA compared with in trans via baculovirus delivery. Density gradient analysis of the particles revealed the presence of a broad infectious peak between 1.06 and 1.11 g ml−1, comparable to that seen when propagating full-length virus in tissue culture. In summary, the trans-encapsidation system described offers a complementary and safer approach to study HCV particle formation and transmission in tissue culture.

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

We have developed a baculovirus delivery system that enables tetracycline-regulated expression of polII-derived hepatitis C virus (HCV) transcripts in hepatocyte-derived cell lines ( McCormick et al., 2002 ). As part of a study to determine whether such transcripts are replication competent, the transcription start site of the tetracycline-regulable promoter was mapped and three baculovirus transfer vectors containing a neo R-expressing culture adapted replicon cDNA were generated. These vectors either had the first nucleotide of the 5′UTR positioned −2 (mkI) and +1 (mkII) with respect to the transcription start site, or included a hammerhead ribozyme at the 5′ end of the transcript (5′HH) that cleaves between the ribozyme–5′UTR boundary. Transfection of all of the culture-adapted replicon constructs into Huh7 cells resulted in the formation of more neomycin-resistant colonies than seen with a polymerase knock-out replicon construct, although this was less pronounced in the mkI group. Furthermore, both the positive- and negative-strands of the replicon could be detected in all neomycin-resistant polyclonal cell lines except for those derived from transfection of the polymerase knock-out construct. Transduction of Huh7 cells with recombinant baculoviruses carrying the same expression cassettes improved replicon delivery, but the relative efficiency of the constructs remained the same. The baculovirus vectors were also used to introduce the replicon transcript into HepG2 cells. Expression of the culture-adapted but not the polymerase knock-out construct induced transcription of the β-interferon gene, a response that may contribute to this cell line being unable to maintain the replicon over long-term culture.

Baculovirus vectors have been used as efficient delivery vehicles for constitutive gene expression in a variety of mammalian cells. We have further developed the system to allow for regulable expression by placing the gene of interest under the control of an inducible promoter, and complementing it with a second baculovirus vector providing the control elements necessary for promoter activity. We have used this system to express (a) the lacZ gene, (b) a ‘minigenome’ derived from hepatitis C virus (HCV) and carrying lacZ or (c) the full-length HCV viral genome, in human hepatocyte cell lines in an inducible fashion. Control systems that rely on either the absence of tetracycline or presence of ponasterone to induce gene expression were tested. Expression of lacZ was controlled by ponasterone, but β-galactosidase activity was limited to 10–20% of cells. In contrast, the tetracycline-controlled expression system gave a low basal activity and was highly inducible in almost 100% of cells. Inducible expression was also obtained in almost 100% of cells infected with baculoviruses in which an HCV minigenome was placed downstream of the tetracycline-inducible promoter and upstream of either a hammerhead or hepatitis δ virus ribozyme. Northern blot analysis was consistent with accurate cleavage of the minigenome transcript by the hepatitis δ virus ribozyme. Finally, regulable transcript production and viral polypeptide processing could be demonstrated in HepG2 cells infected with baculoviruses bearing the full-length HCV genome. This system thus provides a novel tool for the analysis of HCV replication and host–cell interactions.

The non-structural protein NS3 of hepatitis C virus has been expressed in bacteria as a polyhistidine fusion protein which can be produced in a soluble form and easily purified by affinity chromatography. Using an in vitro transcription and translation system we have been able to demonstrate that this protein can proteolytically process substrate molecules derived from the non-structural region of the polyprotein. Using this assay system we have been able to optimize basic biochemical characteristics of the purified enzyme. Parallel experiments show that the full-length NS3 protein also possesses ATPase activity, indicating the bifunctional nature of the protein. In contrast, purified NS3 in which the predicted catalytic serine has been mutated loses protease but retains ATPase activity.

The protease activity of the hepatitis C virus (HCV) NS3 protein has been investigated using transient expression methods in mammalian cells, as well as in vitro transcription/translation systems. We confirmed that expression of the NS3−5 polyprotein in rabbit reticulocyte lysates results in efficient cis processing at the NS3/NS4 junction. However, processing at the other predicted sites of NS3-mediated cleavage varied markedly in efficiency, the site most susceptible being that between NS5A and NS5B. Time-course analysis of the proteolytic processing of the HCV non-structural precursor showed that the cis cleavage between NS3 and NS4 occurred extremely rapidly. However, efficient cleavage at this position was dependent on the prior removal of the NS2 protein. Furthermore, the presence of uncleaved NS2 sequences on the enzyme severely impeded NS3-mediated proteolysis at downstream sites in the polyprotein. This suggests therefore that efficient cleavage at the NS2/NS3 junction is a pivotal event in HCV replication. During the course of this study a proteolytically inactive mutant of NS3 was characterized carrying a previously unreported amino acid substitution near the proposed active site of the enzyme. Molecular modelling suggested that the amino acid present at this position may influence the conformation of the active site of the enzyme. Recently a number of reports have described a second protease activity, located in the NS2/NS3 region, which is responsible for cleavage at the NS2/NS3 junction. We have identified an isolate of HCV, obtained from a U.K. patient, which has a virtually inactive NS2/NS3 protease. The possible implications of this observation are discussed.

A total of 38 neutralization escape mutant viruses have been selected from a cloned stock of human rhinovirus serotype 2 (HRV-2), using either of two monoclonal antibodies (MAbs) which recognize overlapping epitopes as judged by competition binding. The mutant viruses were analysed for their sensitivity to a panel of antiviral MAbs by antibody binding and virus neutralization assays. The position and nature of the selected mutations was determined by sequencing of the virus RNAs, and the location of the substituted amino acids on the three-dimensional structure of the virus predicted from the co-ordinates determined for the closely related HRV-1A. Escape from neutralization could be attributed to single amino acid substitutions in all but one case, which had a deletion of four amino acids. In all cases in which the same mutation was found more than once, these mutations were transitions. The ratio of transition to transversion mutations was about 5:1 overall or about 1·7:1 if only unique substitutions are considered. Each antibody selected for a discrete cluster of mutations and the area of these clusters was considerably less than that determined to be in contact with antibodies from X-ray crystallographic analyses of antibody/protein complexes. One mutation did not occur within the cluster of others selected with the same antibody. This substitution occurred at the base of a small loop and may cause conformational changes at the virus surface.

Guinea-pigs were challenged with homologous or heterologous strains of foot-and-mouth disease virus (FMDV) following vaccination with baby hamster kidney (BHK) monolayer cell-adapted or BHK suspension cell-adapted strains of FMDV serotype A22 Iraq 24/64. The protection afforded by these vaccines was analysed as a function of antigen dose and the in vitro serum virus neutralization titres achieved. The results show that the level of neutralizing antibody induced that afforded 50% protection was similar for both vaccines in homologous or heterologous challenge situations. However, although the dose of antigen required to achieve this titre against homologous virus was similar for the two vaccines, approximately 20-fold more of the suspension cell-adapted virus was required to elicit a protective titre against heterologous challenge compared to the dose of monolayer cell-adapted virus required. A synthetic peptide representing the amino acid sequence 135 to 167 of VP1, which is identical in the A22 Iraq 24/64 variant viruses, was shown to induce protection against both homologous and heterologous virus challenge.