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Abstract Local cell densities and positioning within cellular monolayers and stratified epithelia have important implications for cell interactions and the functionality of various biological processes. To analyze the relationship between cell localization and tissue physiology, density-based clustering algorithms, such as DBSCAN, allow for a detailed characterization of the spatial distribution and positioning of individual cells. However, these methods rely on predefined parameters that influence the outcome of the analysis. With varying cell densities in cell cultures or tissues impacting cell sizes and, thus, cellular proximities, these parameters need to be carefully chosen. In addition, standard DBSCAN approaches generally come short in appropriately identifying individual cell positions. We therefore developed three extensions to the standard DBSCAN-algorithm that provide: (i) an automated parameter identification to reliably identify cell clusters, (ii) an improved identification of cluster edges; and (iii) an improved characterization of the relative positioning of cells within clusters. We apply our novel methods, which are provided as a user-friendly OpenSource-software package (DBSCAN-CellX), to cellular monolayers of different cell lines. Thereby, we show the importance of the developed extensions for the appropriate analysis of cell culture experiments to determine the relationship between cell localization and tissue physiology.

Double membrane vesicles (DMV) are used as replication organelles by several RNA viruses. Applying proteomics and lipidomics, Tabata and Prasad et al. find that two cellular acyltransferases (AGPAT1/2), responsible for synthesis of phosphatidic acid, play a role in the DMV-biogenesis of HCV and SARS-CoV-2, highlighting a common biogenesis mechanism for evolutionary distant positive-strand RNA viruses.

A genetic variant in the IFN-lambda 4 gene has been associated with poor hepatitis C virus prognosis but it is not clear how this functions. Here the authors show that IFN-lambda 4 promotes ER stress and inhibits presentation of HCV epitopes to CD8+ T cells.

Cellular immunotherapies based on T cell receptor (TCR) transfer are promising approaches for the treatment of cancer and chronic viral infections. The discovery of novel receptors is expanding considerably; however, the clinical development of TCR-T cell therapies still lags. Here we provide a pipeline for process development and clinical-scale manufacturing of TCR-T cells in academia. We utilized two TCRs specific for hepatitis C virus (HCV) as models because of their marked differences in avidity and functional profile in TCR-redirected cells. With our clinical-scale pipeline, we reproduced the functional profile associated with each TCR. Moreover, the two TCR-T cell products demonstrated similar yield, purity, transduction efficiency as well as phenotype. The TCR-T cell products had a highly reproducible yield of over 1.4 × 109 cells, with an average viability of 93%; 97.8–99% of cells were CD3+, of which 47.66 ± 2.02% were CD8+ T cells; the phenotype was markedly associated with central memory (CD62L+CD45RO+) for CD4+ (93.70 ± 5.23%) and CD8+ (94.26 ± 4.04%). The functional assessments in 2D and 3D cell culture assays showed that TCR-T cells mounted a polyfunctional response to the cognate HCV peptide target in tumor cell lines, including killing. Collectively, we report a solid strategy for the efficient large-scale manufacturing of TCR-T cells.

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines.

The liver-specific microRNA-122 is an essential proviral host factor of Hepatitis C virus replication. Here the authors show that microRNA-122 functions as an RNA chaperone that guides the formation of a functional internal ribosome entry site by preventing energetically more favorable secondary structures within the HCV RNA genome.

Most studies investigating the biology of Hepatitis C virus (HCV) have used the human hepatoma cell line Huh-7 or subclones thereof, as these are the most permissive cell lines for HCV infection and replication. Other cell lines also support replication of HCV, most notably the human hepatoblastoma cell line HuH6. HCV replication in cell culture is generally highly sensitive to interferons (IFNs) and differences in the IFN-mediated inhibition of virus replication may reflect alterations in the IFN-induced antiviral response inherent to different host cells. For example, HCV replication is highly sensitive to IFN-γ treatment in Huh-7, but not in HuH6 cells. In this study, we used microarray-based gene expression profiling to compare the response of Huh-7 and HuH6 cells to stimulation with IFN-α and IFN-γ. Furthermore, we determined whether the resistance of HCV replication in HuH6 cells can be linked to differences in the expression profile of IFN-regulated genes. Although both cells lines responded to IFNs with rapid changes in gene expression, thereby demonstrating functional type I and type II signaling pathways, differences were observed for a number of genes. Raw and normalized expression data have been deposited in GEO under accession number GSE68927. Keywords: Interferon, IFN-α, IFN-γ, Huh-7, HuH6, Gene expression profile

Human noroviruses (huNoV) are the most frequent cause of non-bacterial acute gastroenteritis worldwide, particularly genogroup II genotype 4 (GII.4) variants. The viral nonstructural (NS) proteins encoded by the ORF1 polyprotein induce vesical clusters harboring the viral replication sites. Little is known so far about the ultrastructure of these replication organelles or the contribution of individual NS proteins to their biogenesis. We compared the ultrastructural changes induced by expression of norovirus ORF1 polyproteins with those induced upon infection with murine norovirus (MNV). Characteristic membrane alterations induced by ORF1 expression resembled those found in MNV infected cells, consisting of vesicle accumulations likely built from the endoplasmic reticulum (ER) which included single membrane vesicles (SMVs), double membrane vesicles (DMVs) and multi membrane vesicles (MMVs). In-depth analysis using electron tomography suggested that MMVs originate through the enwrapping of SMVs with tubular structures similar to mechanisms reported for picornaviruses. Expression of GII.4 NS1-2, NS3 and NS4 fused to GFP revealed distinct membrane alterations when analyzed by correlative light and electron microscopy. Expression of NS1-2 induced proliferation of smooth ER membranes forming long tubular structures that were affected by mutations in the active center of the putative NS1-2 hydrolase domain. NS3 was associated with ER membranes around lipid droplets (LDs) and induced the formation of convoluted membranes, which were even more pronounced in case of NS4. Interestingly, NS4 was the only GII.4 protein capable of inducing SMV and DMV formation when expressed individually. Our work provides the first ultrastructural analysis of norovirus GII.4 induced vesicle clusters and suggests that their morphology and biogenesis is most similar to picornaviruses. We further identified NS4 as a key factor in the formation of membrane alterations of huNoV and provide models of the putative membrane topologies of NS1-2, NS3 and NS4 to guide future studies.

Innate immune cells can sense hepatitis C virus (HCV)-infected cells and respond with anti-viral actions including secretion of interferons (IFNs). In previous studies, the response of individual innate immune cells against HCV was analyzed in detail. We hypothesized that interaction of multiple innate immune cells increases the magnitude of the immune response and eventually leads to clearance of HCV-infected cells. To investigate this, we co-cultured Huh-7 HCV subgenomic replicon (SGR) cells with peripheral blood mononuclear cells (PBMCs). We confirm secretion of IFNα by plasmacytoid dendritic cells (pDCs) and IFNγ by natural killer (NK) cells in the co-culture setup. Moreover, we observed that also monocytes contribute to the anti-viral response. Flow cytometry and ImageStream analysis demonstrated that monocytes take up material from HCV SGR cells in co-culture with PBMCs. Preceding the uptake, PBMCs caused apoptosis of HCV SGR cells by tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression on NK cells. We observed that only the interplay of monocytes, pDCs, and NK cells resulted in efficient clearance of HCV SGR cells, while these cell populations alone did not kill HCV SGR cells. Despite similar TRAIL receptor expression on Huh-7 control cells and HCV SGR cells, HCV activated PBMCs specifically killed HCV SGR cells and did not target Huh-7 control cells. Finally, we showed that HCV replicating cells per se are sensitive toward TRAIL-induced apoptosis. Our results highlight the importance of the interplay of different innate immune cells to initiate an efficient, rapid, and specific response against HCV-infected cells.

ABSTRACT As obligate parasites, viruses strictly depend on host cell translation for the production of new progeny, yet infected cells also synthesize antiviral proteins to limit virus infection. Modulation of host cell translation therefore represents a frequent strategy by which viruses optimize their replication and spread. Here we sought to define how host cell translation is regulated during infection of human cells with dengue virus (DENV) and Zika virus (ZIKV), two positive-strand RNA flaviviruses. Polysome profiling and analysis of de novo protein synthesis revealed that flavivirus infection causes potent repression of host cell translation, while synthesis of viral proteins remains efficient. Selective repression of host cell translation was mediated by the DENV polyprotein at the level of translation initiation. In addition, DENV and ZIKV infection suppressed host cell stress responses such as the formation of stress granules and phosphorylation of the translation initiation factor eIF2α (α subunit of eukaryotic initiation factor 2). Mechanistic analyses revealed that translation repression was uncoupled from the disruption of stress granule formation and eIF2α signaling. Rather, DENV infection induced p38-Mnk1 signaling that resulted in the phosphorylation of the eukaryotic translation initiation factor eIF4E and was essential for the efficient production of virus particles. Together, these results identify the uncoupling of translation suppression from the cellular stress responses as a conserved strategy by which flaviviruses ensure efficient replication in human cells. IMPORTANCE For efficient production of new progeny, viruses need to balance their dependency on the host cell translation machinery with potentially adverse effects of antiviral proteins produced by the infected cell. To achieve this, many viruses evolved mechanisms to manipulate host cell translation. Here we find that infection of human cells with two major human pathogens, dengue virus (DENV) and Zika virus (ZIKV), leads to the potent repression of host cell translation initiation, while the synthesis of viral protein remains unaffected. Unlike other RNA viruses, these flaviviruses concomitantly suppress host cell stress responses, thereby uncoupling translation suppression from stress granule formation. We identified that the p38-Mnk1 cascade regulating phosphorylation of eIF4E is a target of DENV infection and plays an important role in virus production. Our results define several molecular interfaces by which flaviviruses hijack host cell translation and interfere with stress responses to optimize the production of new virus particles.

ABSTRACT Positive-strand RNA [(+)RNA] viruses are true masters of reprogramming host lipid trafficking and synthesis to support virus genome replication. Via their membrane-associated 3A protein, picornaviruses of the genus Enterovirus (e.g., poliovirus, coxsackievirus, and rhinovirus) subvert Golgi complex-localized phosphatidylinositol 4-kinase IIIβ (PI4KB) to generate “replication organelles” (ROs) enriched in phosphatidylinositol 4-phosphate (PI4P). PI4P lipids serve to accumulate oxysterol-binding protein (OSBP), which subsequently transfers cholesterol to the ROs in a PI4P-dependent manner. Single-point mutations in 3A render enteroviruses resistant to both PI4KB and OSBP inhibition, indicating coupled dependency on these host factors. Recently, we showed that encephalomyocarditis virus (EMCV), a picornavirus that belongs to the Cardiovirus genus, also builds PI4P/cholesterol-enriched ROs. Like the hepatitis C virus (HCV) of the Flaviviridae family, it does so by hijacking the endoplasmic reticulum (ER)-localized phosphatidylinositol 4-kinase IIIα (PI4KA). Here we provide genetic evidence for the critical involvement of EMCV protein 3A in this process. Using a genetic screening approach, we selected EMCV mutants with single amino acid substitutions in 3A, which rescued RNA virus replication upon small interfering RNA (siRNA) knockdown or pharmacological inhibition of PI4KA. In the presence of PI4KA inhibitors, the mutants no longer induced PI4P, OSBP, or cholesterol accumulation at ROs, which aggregated into large cytoplasmic clusters. In contrast to the enterovirus escape mutants, we observed little if any cross-resistance of EMCV mutants to OSBP inhibitors, indicating an uncoupled level of dependency of their RNA replication on PI4KA and OSBP activities. This report may contribute to a better understanding of the roles of PI4KA and OSBP in membrane modifications induced by (+)RNA viruses. IMPORTANCE Positive-strand RNA viruses modulate lipid homeostasis to generate unique, membranous “replication organelles” (ROs) where viral genome replication takes place. Hepatitis C virus, encephalomyocarditis virus (EMCV), and enteroviruses have convergently evolved to hijack host phosphatidylinositol 4-kinases (PI4Ks), which produce PI4P lipids, to recruit oxysterol-binding protein (OSBP), a PI4P-binding protein that shuttles cholesterol to ROs. Consistent with the proposed coupling between PI4K and OSBP, enterovirus mutants resistant to PI4KB inhibitors are also resistant to OSBP inhibitors. Here, we show that EMCV can replicate without accumulating PI4P/cholesterol at ROs, by acquiring point mutations in nonstructural protein 3A. Remarkably, the mutations conferred resistance to PI4K but not OSBP inhibitors, thereby uncoupling the levels of dependency of EMCV RNA replication on PI4K and OSBP. This work may contribute to a deeper understanding of the roles of PI4K/PI4P and OSBP/cholesterol in membrane modifications induced by positive-strand RNA viruses.

ABSTRACT Induction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identify cis- and trans-acting factors of DMV formation, we established a trans cleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses. IMPORTANCE Like all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends on cis cleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.

Cardioviruses, including encephalomyocarditis virus (EMCV) and the human Saffold virus, are small non-enveloped viruses belonging to the Picornaviridae, a large family of positive-sense RNA [(+)RNA] viruses. All (+)RNA viruses remodel intracellular membranes into unique structures for viral genome replication. Accumulating evidence suggests that picornaviruses from different genera use different strategies to generate viral replication organelles (ROs). For instance, enteroviruses (e.g. poliovirus, coxsackievirus, rhinovirus) rely on the Golgi-localized phosphatidylinositol 4-kinase III beta (PI4KB), while cardioviruses replicate independently of the kinase. By which mechanisms cardioviruses develop their ROs is currently unknown. Here we show that cardioviruses manipulate another PI4K, namely the ER-localized phosphatidylinositol 4-kinase III alpha (PI4KA), to generate PI4P-enriched ROs. By siRNA-mediated knockdown and pharmacological inhibition, we demonstrate that PI4KA is an essential host factor for EMCV genome replication. We reveal that the EMCV nonstructural protein 3A interacts with and is responsible for PI4KA recruitment to viral ROs. The ensuing phosphatidylinositol 4-phosphate (PI4P) proved important for the recruitment of oxysterol-binding protein (OSBP), which delivers cholesterol to EMCV ROs in a PI4P-dependent manner. PI4P lipids and cholesterol are shown to be required for the global organization of the ROs and for viral genome replication. Consistently, inhibition of OSBP expression or function efficiently blocked EMCV RNA replication. In conclusion, we describe for the first time a cellular pathway involved in the biogenesis of cardiovirus ROs. Remarkably, the same pathway was reported to promote formation of the replication sites of hepatitis C virus, a member of the Flaviviridae family, but not other picornaviruses or flaviviruses. Thus, our results highlight the convergent recruitment by distantly related (+)RNA viruses of a host lipid-modifying pathway underlying formation of viral replication sites.

Hepatitis C virus (HCV) is a major cause of chronic liver disease affecting around 130 million people worldwide. While great progress has been made to define the principle steps of the viral life cycle, detailed knowledge how HCV interacts with its host cells is still limited. To overcome this limitation we conducted a comprehensive whole-virus RNA interference-based screen and identified 40 host dependency and 16 host restriction factors involved in HCV entry/replication or assembly/release. Of these factors, heterogeneous nuclear ribonucleoprotein K (HNRNPK) was found to suppress HCV particle production without affecting viral RNA replication. This suppression of virus production was specific to HCV, independent from assembly competence and genotype, and not found with the related Dengue virus. By using a knock-down rescue approach we identified the domains within HNRNPK required for suppression of HCV particle production. Importantly, HNRNPK was found to interact specifically with HCV RNA and this interaction was impaired by mutations that also reduced the ability to suppress HCV particle production. Finally, we found that in HCV-infected cells, subcellular distribution of HNRNPK was altered; the protein was recruited to sites in close proximity of lipid droplets and colocalized with core protein as well as HCV plus-strand RNA, which was not the case with HNRNPK variants unable to suppress HCV virion formation. These results suggest that HNRNPK might determine efficiency of HCV particle production by limiting the availability of viral RNA for incorporation into virions. This study adds a new function to HNRNPK that acts as central hub in the replication cycle of multiple other viruses.

HMG-CoA-reductase-inhibitors (statins) have been shown to interfere with HCV replication in vitro. We investigated the mechanism, requirements and contribution of heme oxygenase-1(HO-1)-induction by statins to interference with HCV replication.HO-1-induction by fluva-, simva-, rosuva-, atorva- or pravastatin was correlated to HCV replication, using non-infectious replicon systems as well as the infectious cell culture system. The mechanism of HO-1-induction by statins as well as its relevance for interference with HCV replication was investigated using transient or permanent knockdown cell lines. Polyacrylamide(PAA) gels of different density degrees or the Rho-kinase-inhibitor Hydroxyfasudil were used in order to mimic matrix conditions corresponding to normal versus fibrotic liver tissue.All statins used, except pravastatin, decreased HCV replication and induced HO-1 expression, as well as interferon response in vitro. HO-1-induction was mediated by reduction of Bach1 expression and induction of the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) cofactor Krueppel-like factor 2 (KLF2). Knockdown of KLF2 or HO-1 abrogated effects of statins on HCV replication. HO-1-induction and anti-viral effects of statins were more pronounced under cell culture conditions mimicking advanced stages of liver disease.Statin-mediated effects on HCV replication seem to require HO-1-induction, which is more pronounced in a microenvironment resembling fibrotic liver tissue. This implicates that certain statins might be especially useful to support HCV therapy of patients at advanced stages of liver disease.

The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.

Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency.

HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope.

BACKGROUND: Hepatitis C virus (HCV) patients with high serum levels of bile acids (BAs) respond poorly to IFN therapy. BAs have been shown to increase RNA-replication of genotype 1 but not genotype 2a replicons. Since BAs modulate lipid metabolism including lipoprotein secretion and as HCV depends on lipids and lipoproteins during RNA-replication, virus production and cell entry, BAs may affect multiple steps of the HCV life cycle. Therefore, we analyzed the influence of BAs on individual steps of virus replication. METHODS: We measured replication of subgenomic genotype (GT) 1b and 2a RNAs as well as full-length GT2a genomes in the presence of BAs using quantitative RT-PCR and luciferase assays. Cell entry was determined using HCV pseudoparticles (HCVpp). Virus assembly and release were quantified using a core-specific ELISA. Replicon chimeras were employed to characterize genotype-specific modulation of HCV by BAs. Lunet CD81/GFP-NLS-MAVS cells were used to determine infection of Con1 particles. RESULTS: BAs increased RNA-replication of GT1b replicons up to 10-fold but had no effect on subgenomic GT2a replicons both in Huh-7 and HuH6 cells. They did not increase viral RNA translation, virus assembly and release or cell entry. Lowering replication efficiency of GT2a replicons rendered them susceptible to stimulation by BAs. Moreover, replication of full length GT1b with or without replication enhancing mutations and GT2a genomes were also stimulated by BAs. CONCLUSIONS: Bile acids specifically enhance RNA-replication. This is not limited to GT1, but also holds true for GT2a full length genomes and subgenomic replicons with low replication capacity. The increase of HCV replication by BAs may influence the efficacy of antiviral treatment in vivo and may improve replication of primary HCV genomes in cell culture.

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.

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.