Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza.

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic "swine" H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance.

Advanced Hepatitis C Virus Replication PDE Models within a Realistic Intracellular Geometric Environment.

The hepatitis C virus (HCV) RNA replication cycle is a dynamic intracellular process occurring in three-dimensional space (3D), which is difficult both to capture experimentally and to visualize conceptually. HCV-generated replication factories are housed within virus-induced intracellular structures termed membranous webs (MW), which are derived from the Endoplasmatic Reticulum (ER). Recently, we published 3D spatiotemporal resolved diffusion⁻reaction models of the HCV RNA replication cycle by means of surface partial differential equation (sPDE) descriptions. We distinguished between the basic components of the HCV RNA replication cycle, namely HCV RNA, non-structural viral proteins (NSPs), and a host factor. In particular, we evaluated the sPDE models upon realistic reconstructed intracellular compartments (ER/MW). In this paper, we propose a significant extension of the model based upon two additional parameters: different aggregate states of HCV RNA and NSPs, and population dynamics inspired diffusion and reaction coefficients instead of multilinear ones. The combination of both aspects enables realistic modeling of viral replication at all scales. Specifically, we describe a replication complex state consisting of HCV RNA together with a defined amount of NSPs. As a result of the combination of spatial resolution and different aggregate states, the new model mimics a cis requirement for HCV RNA replication. We used heuristic parameters for our simulations, which were run only on a subsection of the ER. Nevertheless, this was sufficient to allow the fitting of core aspects of virus reproduction, at least qualitatively. Our findings should help stimulate new model approaches and experimental directions for virology.

The thymidine dideoxynucleoside analog, alovudine, inhibits the mitochondrial DNA polymerase γ, impairs oxidative phosphorylation and promotes monocytic differentiation in acute myeloid leukemia.

Mitochondrial DNA encodes 13 proteins that comprise components of the respiratory chain that maintain oxidative phosphorylation. The replication of mitochondrial DNA is performed by the sole mitochondrial DNA polymerase γ. As acute myeloid leukemia (AML) cells and stem cells have an increased reliance on oxidative phosphorylation, we sought to evaluate polymerase γ inhibitors in AML. The thymidine dideoxynucleoside analog, alovudine, is an inhibitor of polymerase γ. In AML cells, alovudine depleted mitochondrial DNA, reduced mitochondrial encoded proteins, decreased basal oxygen consumption, and decreased cell proliferation and viability. To evaluate the effects of polymerase γ inhibition with alovudine in vivo, mice were xenografted with OCI-AML2 cells and then treated with alovudine. Systemic administration of alovudine reduced leukemic growth without evidence of toxicity and decreased levels of mitochondrial DNA in the leukemic cells. We also showed that alovudine increased the monocytic differentiation of AML cells. Genetic knockdown and other chemical inhibitors of polymerase γ also promoted AML differentiation, but the effects on AML differentiation were independent of reductions in oxidative phosphorylation or respiratory chain proteins. Thus, we have identified a novel mechanism by which mitochondria regulate AML fate and differentiation independent of oxidative phosphorylation. Moreover, we highlight polymerase γ inhibitors, such as alovudine, as novel therapeutic agents for AML.

Quantitative Analysis of Hepatitis C NS5A Viral Protein Dynamics on the ER Surface.

Exploring biophysical properties of virus-encoded components and their requirement for virus replication is an exciting new area of interdisciplinary virological research. To date, spatial resolution has only rarely been analyzed in computational/biophysical descriptions of virus replication dynamics. However, it is widely acknowledged that intracellular spatial dependence is a crucial component of virus life cycles. The hepatitis C virus-encoded NS5A protein is an endoplasmatic reticulum (ER)-anchored viral protein and an essential component of the virus replication machinery. Therefore, we simulate NS5A dynamics on realistic reconstructed, curved ER surfaces by means of surface partial differential equations (sPDE) upon unstructured grids. We match the in silico NS5A diffusion constant such that the NS5A sPDE simulation data reproduce experimental NS5A fluorescence recovery after photobleaching (FRAP) time series data. This parameter estimation yields the NS5A diffusion constant. Such parameters are needed for spatial models of HCV dynamics, which we are developing in parallel but remain qualitative at this stage. Thus, our present study likely provides the first quantitative biophysical description of the movement of a viral component. Our spatio-temporal resolved ansatz paves new ways for understanding intricate spatial-defined processes central to specfic aspects of virus life cycles.

3D Spatially Resolved Models of the Intracellular Dynamics of the Hepatitis C Genome Replication Cycle.

Mathematical models of virus dynamics have not previously acknowledged spatial resolution at the intracellular level despite substantial arguments that favor the consideration of intracellular spatial dependence. The replication of the hepatitis C virus (HCV) viral RNA (vRNA) occurs within special replication complexes formed from membranes derived from endoplasmatic reticulum (ER). These regions, termed membranous webs, are generated primarily through specific interactions between nonstructural virus-encoded proteins (NSPs) and host cellular factors. The NSPs are responsible for the replication of the vRNA and their movement is restricted to the ER surface. Therefore, in this study we developed fully spatio-temporal resolved models of the vRNA replication cycle of HCV. Our simulations are performed upon realistic reconstructed cell structures-namely the ER surface and the membranous webs-based on data derived from immunostained cells replicating HCV vRNA. We visualized 3D simulations that reproduced dynamics resulting from interplay of the different components of our models (vRNA, NSPs, and a host factor), and we present an evaluation of the concentrations for the components within different regions of the cell. Thus far, our model is restricted to an internal portion of a hepatocyte and is qualitative more than quantitative. For a quantitative adaption to complete cells, various additional parameters will have to be determined through further in vitro cell biology experiments, which can be stimulated by the results deccribed in the present study.

Discovery of ß-d-2'-deoxy-2'-dichlorouridine nucleotide prodrugs as potent inhibitors of hepatitis C virus replication.

Discovery of sofosbuvir has radically changed hepatitis C treatment and nucleoside/tide NS5B inhibitors are now viewed as one of the key components in combination therapies with other direct-acting antiviral agents. As part of our program to identify new nucleoside inhibitors of HCV replication, we now wish to report on the discovery of ß-d-2'-deoxy-2'-dichlorouridine nucleotide prodrugs as potent inhibitors of HCV replication. Although, cytidine analogues have long been recognized to be metabolized to both cytidine and uridine triphosphates through the action of cytidine deaminase, uridine analogues are generally believed to produce exclusively uridine triphosphate. Detailed investigation of the intracellular metabolism of our newly discovered uridine prodrugs, as well as of sofosbuvir, has now revealed the formation of both uridine and cytidine triphosphates. This occurs, not only in vitro in cell lines, but also in vivo upon oral dosing to dogs.

Transient replication of a hepatitis C virus genotype 1b replicon chimera encoding NS5A-5B from genotype 3a.

Although hepatitis C virus (HCV) is a pathogen of global significance, experimental therapies in current clinical development include highly efficacious all-oral combinations of HCV direct-acting antivirals (DAAs). If approved for use, these new treatment regimens will impact dramatically upon our capacity to eradicate HCV in the majority of virus-infected patients. However, recent data from late-stage clinical evaluations demonstrated that individuals infected with HCV genotype (GT) 3 responded less well to all-oral DAA combinations than patients infected with other HCV GTs. In light of these observations, the present study sought to expand the number of molecular tools available to investigate small molecule-mediated inhibition of HCV GT3 NS5A and NS5B proteins in preclinical tissue-culture systems. Accordingly, a novel subgenomic HCV replicon chimera was created by utilizing a GT1b backbone modified to produce NS5A and NS5B proteins from a consensus sequence generated from HCV GT3a genomic sequences deposited online at the European Hepatitis C Virus database. This approach avoided the need to isolate and amplify HCV genomes from sera derived from HCV-infected patients. The replicon chimera, together with a version engineered to express NS5A encoding a Y93H mutation, demonstrated levels of replication in transient assays robust enough to assess accurate antiviral activities of inhibitors representing different HCV DAA classes. Thus, the replicon chimera represents a new simple molecular tool suitable for drug discovery programmes aimed at investigating, understanding, and improving GT3a activities of HCV DAAs targeting NS5A or NS5B.

Small molecule inhibitors of the hepatitis C virus-encoded NS5A protein.

Hepatitis C virus (HCV) is a modern-day pandemic; 2-3% of the world's population are thought to be infected with the virus and are subsequently at risk of developing end-stage liver diseases. The traditional standard of care (SOC) for HCV-infected patients has been limited to a regimen of pegylated-interferon alpha (pegIFN) and ribavirin; displaying low cure rates in a majority of patients and severe side effects. However, in 2011 the first direct-acting antivirals (DAA) were licensed to treat HCV-infected patients in combination with SOC, which served to elevate treatment response rates. The HCV drug development pipeline is currently populated with many additional and improved DAAs; primarily molecules that target the virus-encoded protease or polymerase enzymes. These molecules are being evaluated both in combination with the traditional SOC and together with other DAAs as all-oral pegIFN-free regimens with the ultimate goal of developing multiple DAA-containing HCV therapies that do not rely on an pegIFN backbone. A recent addition to the arsenal of HCV inhibitors in development is represented by an entirely new DAA class; molecules that target the HCV-encoded non-enzymatic NS5A protein. NS5A is essential for HCV propagation and, although its actual functions are largely unknown, it is likely a key regulator of viral genome replication and virion assembly. The protein is exquisitely sensitive to small molecule-mediated inhibition; NS5A-targeting molecules are probably the most potent antiviral molecules ever discovered and exhibit a number of other attractive drug-like properties, including activity against many HCV genotypes/subtypes and once-daily dosing potential. Although their mechanism of action is unclear, NS5A-targeting molecules are already proving their utility in clinical evaluation; particularly as components of pegIFN-sparring DAA combination regimens. This review will aim to amalgamate our current understanding and knowledge of NS5A-targeting molecules; their discovery, properties, applications, and insight into their future impact as components of all-oral pegIFN-free DAA combination therapies to combat HCV infection.

Correlation between NS5A dimerization and hepatitis C virus replication.

Hepatitis C virus (HCV) is the main agent of acute and chronic liver diseases leading to cirrhosis and hepatocellular carcinoma. The current standard therapy has limited efficacy and serious side effects. Thus, the development of alternate therapies is of tremendous importance. HCV NS5A (nonstructural 5A protein) is a pleiotropic protein with key roles in HCV replication and cellular signaling pathways. Here we demonstrate that NS5A dimerization occurs through Domain I (amino acids 1-240). This interaction is not mediated by nucleic acids because benzonase, RNase, and DNase treatments do not prevent NS5A-NS5A interactions. Importantly, DTT abrogates NS5A-NS5A interactions but does not affect NS5A-cyclophilin A interactions. Other reducing agents such as tris(2-carboxyethyl)phosphine and 2-mercaptoethanol also abrogate NS5A-NS5A interactions, implying that disulfide bridges may play a role in this interaction. Cyclophilin inhibitors, cyclosporine A, and alisporivir and NS5A inhibitor BMS-790052 do not block NS5A dimerization, suggesting that their antiviral effects do not involve the disruption of NS5A-NS5A interactions. Four cysteines, Cys-39, Cys-57, Cys-59, and Cys-80, are critical for dimerization. Interestingly, the four cysteines have been proposed to form a zinc-binding motif. Supporting this notion, NS5A dimerization is greatly facilitated by Zn(2+) but not by Mg(2+) or Mn(2+). Importantly, the four cysteines are vital not only for viral replication but also critical for NS5A binding to RNA, revealing a correlation between NS5A dimerization, RNA binding, and HCV replication. Altogether our data suggest that NS5A-NS5A dimerization and/or multimerization could represent a novel target for the development of HCV therapies.

In-cell click labelling of small molecules to determine subcellular localisation.

UNLABELLED: Small molecule fluorometric boron dipyrromethene probes were developed to bind hepatitis C virus-encoded NS5A protein and aid subcellular distribution studies. These molecules did not co-locate with NS5A, therefore alternative 'silent' azide reporters were used to obtain a more relevant picture of their distribution. Following pre-incubation with replicon cells, click chemistry was used to append a fluorophore to the azide that confirmed the co-localisation of the small molecule with the NS5A protein, thus providing greater insight into the antiviral mode of action of this chemotype. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12154-010-0047-1) contains supplementary material, which is available to authorized users.

An integrated transcriptomic and meta-analysis of hepatoma cells reveals factors that influence susceptibility to HCV infection.

Hepatitis C virus (HCV) is a global problem. To better understand HCV infection researchers employ in vitro HCV cell-culture (HCVcc) systems that use Huh-7 derived hepatoma cells that are particularly permissive to HCV infection. A variety of hyper-permissive cells have been subcloned for this purpose. In addition, subclones of Huh-7 which have evolved resistance to HCV are available. However, the mechanisms of susceptibility or resistance to infection among these cells have not been fully determined. In order to elucidate mechanisms by which hepatoma cells are susceptible or resistant to HCV infection we performed genome-wide expression analyses of six Huh-7 derived cell cultures that have different levels of permissiveness to infection. A great number of genes, representing a wide spectrum of functions are differentially expressed between cells. To focus our investigation, we identify host proteins from HCV replicase complexes, perform gene expression analysis of three HCV infected cells and conduct a detailed analysis of differentially expressed host factors by integrating a variety of data sources. Our results demonstrate that changes relating to susceptibility to HCV infection in hepatoma cells are linked to the innate immune response, secreted signal peptides and host factors that have a role in virus entry and replication. This work identifies both known and novel host factors that may influence HCV infection. Our findings build upon current knowledge of the complex interplay between HCV and the host cell, which could aid development of new antiviral strategies.

Small molecules targeting hepatitis C virus-encoded NS5A cause subcellular redistribution of their target: insights into compound modes of action.

The current standard of care for hepatitis C virus (HCV)-infected patients consists of lengthy treatment with interferon and ribavirin. To increase the effectiveness of HCV therapy, future regimens will incorporate multiple direct-acting antiviral (DAA) drugs. Recently, the HCV-encoded NS5A protein has emerged as a promising DAA target. Compounds targeting NS5A exhibit remarkable potency in vitro and demonstrate early clinical promise, suggesting that NS5A inhibitors could feature in future DAA combination therapies. Since the mechanisms through which these molecules operate are unknown, we have used NS5A inhibitors as tools to investigate their modes of action. Analysis of replicon-containing cells revealed dramatic phenotypic alterations in NS5A localization following treatment with NS5A inhibitors; NS5A was redistributed from the endoplasmic reticulum to lipid droplets. The NS5A relocalization did not occur in cells treated with other classes of HCV inhibitors, and NS5A-targeting molecules did not cause similar alterations in the localization of other HCV-encoded proteins. Time course analysis of the redistribution of NS5A revealed that the transfer of protein to lipid droplets was concomitant with the onset of inhibition, as judged by the kinetic profiles for these compounds. Furthermore, analysis of the kinetic profile of inhibition for a panel of test molecules permitted the separation of compounds into different kinetic classes based on their modes of action. Results from this approach suggested that NS5A inhibitors perturbed the function of new replication complexes, rather than acting on preformed complexes. Taken together, our data reveal novel biological consequences of NS5A inhibition, which may help enable the development of future assay platforms for the identification of new and/or different NS5A inhibitors.

Colony-forming assays reveal enhanced suppression of hepatitis C virus replication using combinations of direct-acting antivirals.

The current standard of care for patients infected with hepatitis C virus (HCV) is not effective universally and is associated with severe side effects. Direct-acting antiviral molecules have potential to transform treatment of HCV-infected individuals but emergence of drug-resistant virus will be problematic. It is anticipated that, to limit the emergence of drug-resistant virus, future HCV therapies must consist of multiple direct-acting antivirals. In the present study, cell culture-based colony-forming assays were used to demonstrate enhanced suppression of HCV RNA replication following simultaneous treatment of HCV replicon-containing cells with two direct-acting antivirals. Specifically, combinations of NS5Ai and Filibuvir (small molecule inhibitors of HCV-encoded NS5A and NS5B proteins respectively) were able to suppress colony formation fully at concentrations that individually they could not. HCV replicon RNA isolated from colonies that emerged following treatment with suboptimal concentrations of NS5Ai were found to encode resistance substitutions in the NS5A gene, which rendered them insensitive to subsequent high doses of NS5Ai. Furthermore, both NS5Ai and Filibuvir were effective at suppressing colony formation in combination with BILN 2061, an inhibitor of HCV-encoded NS3. Collectively, these data underscore the increased inhibitory capacity of direct-acting antivirals to suppress HCV RNA replication when present in combination.

Comparison of U2OS and Huh-7 cells for identifying host factors that affect hepatitis C virus RNA replication.

Host cell factors are critical to all stages of the hepatitis C virus (HCV) life cycle. While many cellular proteins that regulate HCV genome synthesis have been identified, the mechanisms engaged in this process are incompletely understood. To identify novel cellular proteins involved in HCV RNA replication, we screened a library of small interfering RNAs (siRNAs) targeting 299 cellular factors, which principally function in RNA interactions. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tricistronic subgenomic replicons (SGRs). We found that the U2OS cell line gave lower levels of intracellular HCV RNA replication compared with Huh-7 cells and was more readily transfected by siRNAs. Consequently, increased gene silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells provided a suitable and more sensitive alternative to Huh-7 cells for siRNA studies on HCV RNA replication. From the screen, several cellular proteins that enhanced and suppressed HCV RNA replication were identified. One of the genes found to downregulate viral RNA synthesis, ISG15, is expressed in response to alpha interferon and may therefore partly contribute to the clearance of virus from infected individuals. A second gene that inhibited HCV RNA levels was the 5'-3' exoRNase XRN1, which suggested a role for cellular RNA degradation pathways in modulating the abundance of viral genomes. Therefore, this study provides an important framework for future detailed analyses of these and other cellular proteins.

HCV resistance to cyclosporin A does not correlate with a resistance of the NS5A-cyclophilin A interaction to cyclophilin inhibitors.

BACKGROUND & AIMS: The cyclophilin (Cyp) inhibitors - cyclosporine A (CsA), NIM811, Debio 025, and SCY 635 - block HCV replication both in vitro and in vivo, and represent a novel class of potent anti-HCV agents. We and others showed that HCV relies on cyclophilin A (CypA) to replicate. We demonstrated that the hydrophobic pocket of CypA, where Cyp inhibitors bind, and which controls the isomerase activity of CypA, is critical for HCV replication. Recent studies showed that under Cyp inhibitor selection, mutations arose in the HCV nonstructural 5A (NS5A) protein. This led us to postulate that CypA assists HCV by acting on NS5A. METHODS: We tested this hypothesis by developing several interaction assays including GST pull-down assays, ELISA, and mammalian two-hybrid binding assays. RESULTS: We demonstrated that full-length NS5A and CypA form a stable complex. Remarkably, CsA prevents the CypA-NS5A interaction in a dose-dependent manner. Importantly, the CypA-NS5A interaction is conserved among genotypes and is interrupted by CsA. Surprisingly, the NS5A mutant protein, which arose in CsA-resistant HCV variants, behaves similarly to wild-type NS5A in terms of both CypA binding and CsA-mediated release from CypA. This latter finding suggests that HCV resistance to CsA does not correlate with a resistance of the CypA-NS5A interaction to Cyp inhibitors. Moreover, we found that CypA, devoid of its isomerase activity, fails to bind NS5A. CONCLUSIONS: Altogether these data suggest that CypA, via its isomerase pocket, binds directly to NS5A, and most importantly, that disrupting this interaction stops HCV replication.

The use of AlphaLISA technology to detect interaction between hepatitis C virus-encoded NS5A and cyclophilin A.

Cyclosporine A (CsA) is an immunosuppressive molecule that also impedes replication of hepatitis C virus (HCV). CsA inhibits isomerase activity of cellular-encoded cyclophilin proteins, of which cyclophilin A (CypA) in particular is required for HCV replication. Evidence suggests that the HCV-encoded NS5A and NS5B proteins may govern dependence of the virus on CypA-mediated isomerase activity, although the molecular mechanisms involved are unclear. However, association of NS5A and NS5B, with CypA has been reported, raising the possibility that direct interaction between these proteins facilitates HCV replication. In the present study, mammalian two-hybrid and AlphaLISA technologies were utilized to detect interactions between NS5A and NS5B, with CypA. AlphaLISA analysis revealed associations between NS5A and CypA using purified proteins, and in cell lysates prepared from co-transfected cells. Importantly, the NS5A-CypA interactions were sensitive to CsA in a dose-responsive manner and an isomerase mutant of CypA interacted with NS5A less efficiently than wild-type CypA. These findings correlate the anti-HCV properties of CsA with an ability of the compound to disrupt NS5A-CypA interactions in vitro and in vivo, whilst providing the basis for development of assay platforms suitable to screen compound libraries for novel inhibitors of the NS5A-CypA interaction.

Lipid metabolism and HCV infection.

Chronic infection by hepatitis C virus (HCV) can lead to severe liver disease and is a global healthcare problem. The liver is highly metabolically active and one of its key functions is to control the balance of lipid throughout the body. A number of pathologies have been linked to the impact of HCV infection on liver metabolism. However, there is also growing evidence that hepatic metabolic processes contribute to the HCV life cycle. This review summarizes the relationship between lipid metabolism and key stages in the production of infectious HCV.

Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.

The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core-DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.

The hepatitis C virus NS4B protein can trans-complement viral RNA replication and modulates production of infectious virus.

Studies of the hepatitis C virus (HCV) life cycle have been aided by development of in vitro systems that enable replication of viral RNA and production of infectious virus. However, the functions of the individual proteins, especially those engaged in RNA replication, remain poorly understood. It is considered that NS4B, one of the replicase components, creates sites for genome synthesis, which appear as punctate foci at the endoplasmic reticulum (ER) membrane. In this study, a panel of mutations in NS4B was generated to gain deeper insight into its functions. Our analysis identified five mutants that were incapable of supporting RNA replication, three of which had defects in production of foci at the ER membrane. These mutants also influenced posttranslational modification and intracellular mobility of another replicase protein, NS5A, suggesting that such characteristics are linked to focus formation by NS4B. From previous studies, NS4B could not be trans-complemented in replication assays. Using the mutants that blocked RNA synthesis, defective NS4B expressed from two mutants could be rescued in trans-complementation replication assays by wild-type protein produced by a functional HCV replicon. Moreover, active replication could be reconstituted by combining replicons that were defective in NS4B and NS5A. The ability to restore replication from inactive replicons has implications for our understanding of the mechanisms that direct viral RNA synthesis. Finally, one of the NS4B mutations increased the yield of infectious virus by five- to sixfold. Hence, NS4B not only functions in RNA replication but also contributes to the processes engaged in virus assembly and release.