Mechanisms of SARS-CoV-2 Inactivation Using UVC Laser Radiation.

Severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) has had a tremendous impact on humanity. Prevention of transmission by disinfection of surfaces and aerosols through a chemical-free method is highly desirable. Ultraviolet C (UVC) light is uniquely positioned to achieve inactivation of pathogens. We report the inactivation of SARS-CoV-2 virus by UVC radiation and explore its mechanisms. A dose of 50 mJ/cm2 using a UVC laser at 266 nm achieved an inactivation efficiency of 99.89%, while infectious virions were undetectable at 75 mJ/cm2 indicating >99.99% inactivation. Infection by SARS-CoV-2 involves viral entry mediated by the spike glycoprotein (S), and viral reproduction, reliant on translation of its genome. We demonstrate that UVC radiation damages ribonucleic acid (RNA) and provide in-depth characterization of UVC-induced damage of the S protein. We find that UVC severely impacts SARS-CoV- 2 spike protein's ability to bind human angiotensin-converting enzyme 2 (hACE2) and this correlates with loss of native protein conformation and aromatic amino acid integrity. This report has important implications for the design and development of rapid and effective disinfection systems against the SARS-CoV-2 virus and other pathogens.

Investigating multi-material hydrogel three-dimensional printing for in vitro representation of the neo-vasculature of solid tumours: a comprehensive mechanical analysis and assessment of nitric oxide release from human umbilical vein endothelial cells.

Many solid tumours (e.g. sarcoma, carcinoma and lymphoma) form a disorganized neo-vasculature that initiates uncontrolled vessel formation to support tumour growth. The complexity of these environments poses a significant challenge for tumour medicine research. While animal models are commonly used to address some of these challenges, they are time-consuming and raise ethical concerns. In vitro microphysiological systems have been explored as an alternative, but their production typically requires multi-step lithographic processes that limit their production. In this work, a novel approach to rapidly develop multi-material tissue-mimicking, cell-compatible platforms able to represent the complexity of a solid tumour's neo-vasculature is investigated via stereolithography three-dimensional printing. To do so, a series of acrylate resins that yield covalently photo-cross-linked hydrogels with healthy and diseased mechano-acoustic tissue-mimicking properties are designed and characterized. The potential viability of these materials to displace animal testing in preclinical research is assessed by studying the morphology, actin expression, focal adhesions and nitric oxide release of human umbilical vein endothelial cells. These materials are exploited to produce a simplified multi-material three-dimensional printed model of the neo-vasculature of a solid tumour, demonstrating the potential of our approach to replicate the complexity of solid tumours in vitro without the need for animal testing.

Do we really understand how drug eluted from stents modulates arterial healing?

The advent of drug-eluting stents (DES) has revolutionised the treatment of coronary artery disease. These devices, coated with anti-proliferative drugs, are deployed into stenosed or occluded vessels, compressing the plaque to restore natural blood flow, whilst simultaneously combating the evolution of restenotic tissue. Since the development of the first stent, extensive research has investigated how further advancements in stent technology can improve patient outcome. Mathematical and computational modelling has featured heavily, with models focussing on structural mechanics, computational fluid dynamics, drug elution kinetics and subsequent binding within the arterial wall; often considered separately. Smooth Muscle Cell (SMC) proliferation and neointimal growth are key features of the healing process following stent deployment. However, models which depict the action of drug on these processes are lacking. In this article, we start by reviewing current models of cell growth, which predominantly emanate from cancer research, and available published data on SMC proliferation, before presenting a series of mathematical models of varying complexity to detail the action of drug on SMC growth in vitro. Our results highlight that, at least for Sodium Salicylate and Paclitaxel, the current state-of-the-art nonlinear saturable binding model is incapable of capturing the proliferative response of SMCs across a range of drug doses and exposure times. Our findings potentially have important implications on the interpretation of current computational models and their future use to optimise and control drug release from DES and drug-coated balloons.

A novel ACE2 isoform is expressed in human respiratory epithelia and is upregulated in response to interferons and RNA respiratory virus infection.

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma membrane, resulting in viral RNA genome delivery into the host. Despite ACE2's critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.

Rhinovirus 2A is the key protease responsible for instigating the early block to gene expression in infected cells.

Human rhinoviruses (HRVs) express 2 cysteine proteases, 2A and 3C, that are responsible for viral polyprotein processing. Both proteases also suppress host gene expression by inhibiting mRNA transcription, nuclear export and cap-dependent translation. However, the relative contribution that each makes in achieving this goal remains unclear. In this study, we have compared both the combined and individual ability of the two proteases to shut down cellular gene expression using a novel dynamic reporter system. Our findings show that 2A inhibits host gene expression much more rapidly than 3C. By comparing the activities of a representative set of proteases from the three different HRV species, we also find variation in the speed at which host gene expression is suppressed. Our work highlights the key role that 2A plays in early suppression of the infected host cell response and shows that this can be influenced by natural variation in the activity of this enzyme.

Modelling drug release from polymer-free coronary stents with microporous surfaces.

Traditional coronary drug-eluting stents (DES) are made from metal and are coated with a permanent polymer film containing an anti-proliferative drug. Subsequent to stent deployment in a diseased coronary artery, the drug releases into the artery wall and helps prevent restenosis by inhibiting the proliferation of smooth muscle cells. Although this technology has proven to be remarkably successful, there are ongoing concerns that the presence of a polymer in the artery can lead to deleterious medical complications, such as late stent thrombosis. Polymer-free DES may help overcome such shortcomings. However, the absence of a rate-controlling polymer layer makes optimisation of the drug release profile a particular challenge. The use of microporous stent surfaces to modulate the drug release rate is an approach that has recently shown particularly promising clinical results. In this study, we develop a mathematical model to describe drug release from such stents. In particular, we develop a mathematical model to describe drug release from microporous surfaces. The model predicts a two-stage release profile, with a relatively rapid initial release of most of the drug, followed by a slower release of the remaining drug. In the model, the slow release phase is accounted for by an adsorption/desorption mechanism close to the stent surface. The theoretical predictions are compared with experimental release data obtained in our laboratory, and good agreement is found. The valuable insights provided by our model will serve as a useful guide for designing the enhanced polymer-free stents of the future.

Polyprotein-Driven Formation of Two Interdependent Sets of Complexes Supporting Hepatitis C Virus Genome Replication.

UNLABELLED: Hepatitis C virus (HCV) requires proteins from the NS3-NS5B polyprotein to create a replicase unit for replication of its genome. The replicase proteins form membranous compartments in cells to facilitate replication, but little is known about their functional organization within these structures. We recently reported on intragenomic replicons, bicistronic viral transcripts expressing an authentic replicase from open reading frame 2 (ORF2) and a second duplicate nonstructural (NS) polyprotein from ORF1. Using these constructs and other methods, we have assessed the polyprotein requirements for rescue of different lethal point mutations across NS3-5B. Mutations readily tractable to rescue broadly fell into two groupings: those requiring expression of a minimum NS3-5A and those requiring expression of a minimum NS3-5B polyprotein. A cis-acting mutation that blocked NS3 helicase activity, T1299A, was tolerated when introduced into either ORF within the intragenomic replicon, but unlike many other mutations required the other ORF to express a functional NS3-5B. Three mutations were identified as more refractile to rescue: one that blocked cleavage of the NS4B5A boundary (S1977P), another in the NS3 helicase (K1240N), and a third in NS4A (V1665G). Introduced into ORF1, these exhibited a dominant negative phenotype, but with K1240N inhibiting replication as a minimum NS3-5A polyprotein whereas V1665G and S1977P only impaired replication as a NS3-5B polyprotein. Furthermore, an S1977P-mutated NS3-5A polyprotein complemented other defects shown to be dependent on NS3-5A for rescue. Overall, our findings suggest the existence of two interdependent sets of protein complexes supporting RNA replication, distinguishable by the minimum polyprotein requirement needed for their formation. IMPORTANCE: Positive-strand RNA viruses reshape the intracellular membranes of cells to form a compartment within which to replicate their genome, but little is known about the functional organization of viral proteins within this structure. We have complemented protein-encoded defects in HCV by constructing subgenomic HCV transcripts capable of simultaneously expressing both a mutated and functional polyprotein precursor needed for RNA genome replication (intragenomic replicons). Our results reveal that HCV relies on two interdependent sets of protein complexes to support viral replication. They also show that the intragenomic replicon offers a unique way to study replication complex assembly, as it enables improved composite polyprotein complex formation compared to traditional trans-complementation systems. Finally, the differential behavior of distinct NS3 helicase knockout mutations hints that certain conformations of this enzyme might be particularly deleterious for replication.

NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system.

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.

Genetic complementation of hepatitis C virus nonstructural protein functions associated with replication exhibits requirements that differ from those for virion assembly.

UNLABELLED: Within the polyprotein encoded by hepatitis C virus (HCV), the minimum components required for viral RNA replication lie in the NS3-5B region, while virion assembly requires expression of all virus components. Here, we have employed complementation systems to examine the role that HCV polyprotein precursors play in RNA replication and virion assembly. In a trans-complementation assay, an HCV NS3-5A polyprotein precursor was required to facilitate efficient complementation of a replication-defective mutation in NS5A. However, this requirement for precursor expression was partially alleviated when a second functional copy of NS5A was expressed from an additional upstream cistron within the RNA to be rescued. In contrast, rescue of a virion assembly mutation in NS5A was more limited but exhibited little or no requirement for expression of functional NS5A as a precursor, even when produced in the context of a second replicating helper RNA. Furthermore, expression of NS5A alone from an additional cistron within a replicon construct gave greater rescue of virion assembly in cis than in trans. Combined with the findings of confocal microscope analysis examining the extent to which the two copies of NS5A from the various expression systems colocalize, the results point to NS3-5A playing a role in facilitating the integration of nonstructural (NS) proteins into viral membrane-associated foci, with this representing an early stage in the steps leading to replication complex formation. The data further imply that HCV employs a minor virion assembly pathway that is independent of replication. IMPORTANCE: In hepatitis C virus-infected cells, replication is generally considered an absolute prerequisite for virus particle formation. Here we investigated the role that the viral protein NS5A has in both replication and particle assembly using complementation assays and microscopy. We found that efficient rescue of replication required NS5A to be expressed as part of a larger polyprotein, and this correlated with detection of NS5A at sites where replication occurred. In contrast, rescue of particle assembly did not require expression of NS5A within the context of a polyprotein. Interestingly, although only partial restoration of particle assembly was possible by complementation, that proportion that could be rescued benefitted from expressing NS5A from the same RNA being packaged. Collectively, these findings provide new insight into aspects of polyprotein function. They also support the existence of a minor virion assembly pathway that bypasses replication.

Mutations in hepatitis C virus p7 reduce both the egress and infectivity of assembled particles via impaired proton channel function.

Hepatitis C virus (HCV) p7 protein is critical for the efficient production of infectious virions in culture. p7 undergoes genotype-specific protein-protein interactions as well as displaying channel-forming activity, making it unclear whether the phenotypes of deleterious p7 mutations result from the disruption of one or both of these functions. Here, we showed that proton channel activity alone, provided in trans by either influenza virus M2 or genotype 1b HCV p7, was both necessary and sufficient to restore infectious particle production to genotype 2a HCV (JFH-1 isolate) carrying deleterious p7 alanine substitutions within the p7 dibasic loop (R33A, R35A), and the N-terminal trans-membrane region (N15 : C16 : H17/AAA). Both mutations markedly reduced mature p7 abundance, with those in the dibasic loop also significantly reducing levels of mature E2 and NS2. Interestingly, whilst M2 and genotype 1b p7 restored the same level of intracellular infectivity as JFH-1 p7, supplementing with the isogenic protein led to a further increase in secreted infectivity, suggesting a late-acting role for genotype-specific p7 protein interactions. Finally, cells infected by viruses carrying p7 mutations contained non-infectious core-containing particles with densities equivalent to WT HCV, indicating a requirement for p7 proton channel activity in conferring an infectious phenotype to virions.

Increasing rate of cleavage at boundary between non-structural proteins 4B and 5A inhibits replication of hepatitis C virus.

In hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. Although proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However, it is not known whether slow cleavage is important for viral replication or a consequence of restrictions on sequences that can be tolerated at the cleaved ends of non-structural proteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary, and their effect on replication and polyprotein processing was examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codon degeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall, the data indicate that slow cleavage of the NS4B5A boundary is important and identify a previously unrecognized role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time.

A conserved proline between domains II and III of hepatitis C virus NS5A influences both RNA replication and virus assembly.

We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.

Expression of hepatitis C virus (HCV) structural proteins in trans facilitates encapsidation and transmission of HCV subgenomic RNA.

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.

Translation termination reinitiation between open reading frame 1 (ORF1) and ORF2 enables capsid expression in a bovine norovirus without the need for production of viral subgenomic RNA.

A generally accepted view of norovirus replication is that capsid expression requires production of a subgenomic transcript, the presence of capsid often being used as a surrogate marker to indicate the occurrence of viral replication. Using a polymerase II-based baculovirus delivery system, we observed capsid expression following introduction of a full-length genogroup 3 norovirus genome into HepG2 cells. However, capsid expression occurred as a result of a novel translation termination/reinitiation event between the nonstructural-protein and capsid open reading frames, a feature that may be unique to genogroup 3 noroviruses.

Protection of hepatocytes from cytotoxic T cell mediated killing by interferon-alpha.

BACKGROUND: Cellular immunity plays a key role in determining the outcome of hepatitis C virus (HCV) infection, although the majority of infections become persistent. The mechanisms behind persistence are still not clear; however, the primary site of infection, the liver, may be critical. We investigated the ability of CD8+ T-cells (CTL) to recognise and kill hepatocytes under cytokine stimulation. METHODS/PRINCIPLE FINDINGS: Resting hepatocytes cell lines expressed low levels of MHC Class I, but remained susceptible to CTL cytotoxicity. IFN-alpha treatment, in vitro, markedly increased hepatocyte MHC Class I expression, however, reduced sensitivity to CTL cytotoxicity. IFN-alpha stimulated hepatocyte lines were still able to present antigen and induce IFN-gamma expression in interacting CTL. Resistance to killing was not due to the inhibition of the FASL/FAS- pathway, as stimulated hepatocytes were still susceptible to FAS-mediated apoptosis. In vitro stimulation with IFN-alpha, or the introduction of a subgenomic HCV replicon into the HepG2 line, upregulated the expression of the granzyme-B inhibitor-proteinase inhibitor 9 (PI-9). PI-9 expression was also observed in liver tissue biopsies from patients with chronic HCV infection. CONCLUSION/SIGNIFICANCE: IFN-alpha induces resistance in hepatocytes to perforin/granzyme mediate CTL killing pathways. One possible mechanism could be through the expression of the PI-9. Hindrance of CTL cytotoxicity could contribute to the chronicity of hepatic viral infections.

Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA.

Noroviruses are the major cause of nonbacterial gastroenteritis in humans. These viruses have remained refractory to detailed molecular studies because of the lack of a reverse genetics system coupled to a permissive cell line for targeted genetic manipulation. There is no permissive cell line in which to grow infectious human noroviruses nor an authentic animal model that supports their replication. In contrast, murine norovirus (MNV) offers a tractable system for the study of noroviruses with the recent discovery of permissive cells and a mouse model. The lack of a reverse genetic system for MNV has been a significant block to understanding the biology of noroviruses. We report recovery of infectious MNV after baculovirus delivery of viral cDNA to human hepatoma cells under the control of an inducible DNA polymerase (pol) II promoter. Recovered virus replicated in murine macrophage (RAW264.7) cells, and the recovery of MNV from DNA was confirmed through recovery of virus containing a marker mutation. This pol II promoter driven expression of viral cDNA also generated infectious virus after transfection of HEK293T cells, thus providing both transduction and transfection systems for norovirus reverse genetics. We used norovirus reverse genetics to demonstrate by mutagenesis of the protease-polymerase (pro-pol) cleavage site that processing of pro-pol is essential for the recovery of infectious MNV. This represents the first infectious reverse genetics system for a norovirus, and should provide approaches to address fundamental questions in norovirus molecular biology and replication.

Tagging of NS5A expressed from a functional hepatitis C virus replicon.

Knowledge of how hepatitis C virus (HCV) proteins associate with components of the host cell to form a functional replication complex is still limited. To address this issue, HCV replicon constructs were generated where either green fluorescent protein (GFP) or the Propionibacterium shermanii transcarboxylase domain (PSTCD) was introduced into the NS5A coding region. Insertion of both GFP and PSTCD was tolerated well, allowing formation of stable replicon-containing cell lines that contained viral protein and transcript levels that were comparable to those of an unmodified parental replicon. Cell lines generated from the GFP-tagged NS5A replicon allowed live-cell visualization of the location of NS5A. Cell lines generated from the PSTCD-tagged replicons allowed rapid and efficient precipitation of the PSTCD-tagged NS5A, as well as other HCV non-structural proteins, using streptavidin-coated magnetic beads. Both replicons represent useful tools that offer different but complementary ways of examining replication-complex formation in cells.

A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A.

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.

Hepatitis C virus NS5A-mediated activation of phosphoinositide 3-kinase results in stabilization of cellular beta-catenin and stimulation of beta-catenin-responsive transcription.

The hepatitis C virus (HCV) nonstructural NS5A protein has been shown to bind to and activate phosphoinositide 3-kinase (PI3K), resulting in activation of the downstream effector serine/threonine kinase Akt/protein kinase B. Here we present data pertaining to the effects of NS5A-mediated Akt activation on its downstream targets. Using a recombinant baculovirus to deliver the complete HCV polyprotein to human hepatoma cells in a tetracycline-regulable fashion, we confirm that expression of the complete HCV polyprotein also activates PI3K and Akt. We further show that this results in the inhibition of the Akt substrate Forkhead transcription factor and the stimulation of phosphorylation of a second key Akt substrate, glycogen synthase kinase-3beta (GSK-3beta). Phosphorylation of GSK-3beta results in its inactivation; consistent with this, we show that expression of the HCV polyprotein results in the accumulation of beta-catenin. Finally, we show that levels of beta-catenin-dependent transcription are also elevated in the presence of the HCV polyprotein. Given the prevalence of beta-catenin mutations in many human tumors, especially colon and hepatocellular carcinomas, these data implicate NS5A-mediated PI3K activation as a contributory factor in the increasingly common association between HCV infection and the development of hepatocellular carcinoma.

Further studies on hepatitis C virus NS5A-SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling.

The NS5A protein of hepatitis C virus has been shown to interact with a subset of Src homology 3 (SH3) domain-containing proteins. The molecular mechanisms underlying these observations have not been fully characterized, therefore a previous analysis of NS5A-SH3 domain interactions was extended. By using a semi-quantitative ELISA assay, a hierarchy of binding between various SH3 domains for NS5A was demonstrated. Molecular modelling of a polyproline motif within NS5A (termed PP2.2) bound to the FynSH3 domain predicted that the specificity-determining RT-loop region within the SH3 domain did not interact directly with the PP2.2 motif. However, it was demonstrated that the RT loop did contribute to the specificity of binding, implicating the involvement of other intermolecular contacts between NS5A and SH3 domains. The modelling analysis also predicted a critical role for a conserved arginine located at the C terminus of the PP2.2 motif; this was confirmed experimentally. Finally, it was demonstrated that, in comparison with wild-type replicon cells, inhibition of the transcription factor AP-1, a function previously assigned to NS5A, was not observed in cells harbouring a subgenomic replicon containing a mutation within the PP2.2 motif. However, the ability of the mutated replicon to establish itself within Huh-7 cells was unaffected. The highly conserved nature of the PP2.2 motif within NS5A suggests that functions involving this motif are of importance, but are unlikely to play a role in replication of the viral RNA genome. It is more likely that they play a role in altering the cellular environment to favour viral persistence.