Virus Nonstructural 5A Research Articles

Functional Role of Hepatitis C Virus NS5A in the Regulation of Autophagy.

Many types of RNA viruses, including the hepatitis C virus (HCV), activate autophagy in infected cells to promote viral growth and counteract the host defense response. Autophagy acts as a catabolic pathway in which unnecessary materials are removed via the lysosome, thus maintaining cellular homeostasis. The HCV non-structural 5A (NS5A) protein is a phosphoprotein required for viral RNA replication, virion assembly, and the determination of interferon (IFN) sensitivity. Recently, increasing evidence has shown that HCV NS5A can induce autophagy to promote mitochondrial turnover and the degradation of hepatocyte nuclear factor 1 alpha (HNF-1α) and diacylglycerol acyltransferase 1 (DGAT1). In this review, we summarize recent progress in understanding the detailed mechanism by which HCV NS5A triggers autophagy, and outline the physiological significance of the balance between host-virus interactions.

Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV.

Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.

Orally bioavailable HCV NS5A inhibitors of unsymmetrical structural class

A novel unsymmetrical structural class of orally bioavailable hepatitis C virus (HCV) nonstructural 5A protein (NS5A) inhibitors has been generated by improving both the solubility and membrane permeability of the lead compound found in our previous work. The representative compound 14, with a 5-hydroxymethylpyrazine group and a 3-t-butylpropargyl group on each side of the molecule, exhibited the best oral bioavailability in this study, inhibiting not only the HCV genotype 1a, 1b, 2a, and 3a replicons with EC50 values in the picomolar range, but also inhibited 1a Q30 mutants induced by launched symmetrical inhibitors with EC50 values in the low nanomolar range.

HCV NS5A hyperphosphorylation is involved in viral translation modulation

Hepatitis C virus (HCV) non-structural (NS) 5A protein is a multifunctional phosphoprotein. NS5A exists as hypo- and hyper-phosphorylated forms and the dynamic transitions between these two states are involved in the functions of NS5A. Hyperphosphorylation occurs primarily at six serine residues within the low complexity sequence I of NS5A. We previously showed that NS5A downregulates viral translation. In this study, we investigated the role of NS5A hyperphosphorylation in translation modulation. By analyzing the effects of phospho-ablative and phospho-mimetic mutants of the six serine residues on translation, we showed that NS5A hyperphospho-ablative mutation at all six serine residues can no longer downregulate viral translation. We then studied the effects of phospho-mutations at each of the six serine residues on translation. We found that phosphorylation of S222, S225, S235 is not involved in translation downregulation by NS5A. In contrast, NS5A with alanine mutations at S229 or S238 can no longer downregulate translation, whereas S229D or S238D mutations have no effect. Interestingly, S232D NS5A, but not S232A, abrogates translation downregulation by NS5A. Since dimerization of NS5A plays an important role in its functions, we also studied the effects of phospho-mutants of S229, S232, and S238 on dimerization in a protein-protein interaction assay. We showed that phopho-mimetic S229D or S238D mutations enhances NS5A dimerization, whereas the phospho-ablative mutations of these two residues have no effect. Neither phospho-ablative nor phopho-mimetic mutations of S232 affect dimerization. These results indicate that phosphorylation of NS5A at S229, S232, and S238 is involved in viral translation regulation and NS5A dimerization.

Viperin inhibits classical swine fever virus replication by interacting with viral nonstructural 5A protein.

Classical swine fever virus (CSFV) is a single-stranded RNA flavivirus that can cause serious diseases in porcine species, including symptoms of infarction, systemic hemorrhage, high fever, or depression. Viperin is an important interferon-inducible antiviral gene that has been shown to inhibit CSFV, but the exact mechanisms by which it is able to do so remain poorly characterized. In the present study, we determined that CSFV infection led to viperin upregulation in PK-15 cells (porcine kidney cell). When viperin was overexpressed in these cells, this markedly attenuated CSFV replication, with clear reductions in viral copy number after 12 to 48 hours postinfection. Immunofluorescence microscopy revealed that the viral NS5A protein colocalized with viperin in infected cells, and this was confirmed via confocal laser scanning microscopy using labeled versions of these proteins, and by co-immunoprecipitation which confirmed that NS5A directly interacts with viperin. When NS5A was overexpressed, this inhibited the replication of CSFV, and we determined that the radical SAM domain and N-terminal domain of viperin was critical for its ability to bind to NS5A, with the latter being most important for this interaction. Together, our in vitro results highlight a potential mechanism whereby viperin is able to inhibit CSFV replication. These results have the potential to assist future efforts to prevent or treat systemic CSFV-induced disease, and may also offer more general insights into the antiviral role of viperin in innate immunity.

HepatitisC virus nonstructural5A protein inhibits the starvation‑induced apoptosis of hepatoblastoma cells by increasing Beclin1 expression.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) modulates cellular apoptosis, which is involved in the occurrence and development of liver cancer. The mechanisms of apoptosis inhibition by NS5A in liver cancer cells remains unclear. Beclin 1, which functions upstream of autophagosome formation, is upregulated by NS5A. Autophagy, an evolutionarily conserved catabolic process, has a crucial role in tumor initiation and progression. Autophagy was blocked by inhibitors 3‑methyladenine and chloroquine, or via knockdown of Beclin 1. Flow cytometric analysis and western blotting were used to detect apoptosis. It was found that inhibition of autophagy attenuated the NS5A‑mediated apoptosis inhibition of HepG2 cells. Furthermore, it was confirmed that Beclin 1 expression by NS5A was involved in the negative regulation of starvation‑induced liver cancer apoptosis, which was accompanied by reduced p53 and apoptosis regulator Bax expression, as well as decreased caspase‑3/-7 activation. Therefore, inhibition of autophagy may be promising therapeutic strategy in the treatment of HCV‑associated liver cancer.

Identification of two distinct peptide-binding pockets in the SH3 domain of human mixed-lineage kinase 3

Mixed-lineage kinase 3 (MLK3; also known as MAP3K11) is a Ser/Thr protein kinase widely expressed in normal and cancerous tissues, including brain, lung, liver, heart, and skeletal muscle tissues. Its Src homology 3 (SH3) domain has been implicated in MLK3 autoinhibition and interactions with other proteins, including those from viruses. The MLK3 SH3 domain contains a six-amino-acid insert corresponding to the n-Src insert, suggesting that MLK3 may bind additional peptides. Here, affinity selection of a phage-displayed combinatorial peptide library for MLK3's SH3 domain yielded a 13-mer peptide, designated “MLK3 SH3–interacting peptide” (MIP). Unlike most SH3 domain peptide ligands, MIP contained a single proline. The 1.2-Å crystal structure of the MIP-bound SH3 domain revealed that the peptide adopts a β-hairpin shape, and comparison with a 1.5-Å apo SH3 domain structure disclosed that the n-Src loop in SH3 undergoes an MIP-induced conformational change. A 1.5-Å structure of the MLK3 SH3 domain bound to a canonical proline-rich peptide from hepatitis C virus nonstructural 5A (NS5A) protein revealed that it and MIP bind the SH3 domain at two distinct sites, but biophysical analyses suggested that the two peptides compete with each other for SH3 binding. Moreover, SH3 domains of MLK1 and MLK4, but not MLK2, also bound MIP, suggesting that the MLK1–4 family may be differentially regulated through their SH3 domains. In summary, we have identified two distinct peptide-binding sites in the SH3 domain of MLK3, providing critical insights into mechanisms of ligand binding by the MLK family of kinases.

Sulfur(vi) fluoride exchange as a key reaction for synthesizing biaryl sulfate core derivatives as potent hepatitis C virus NS5A inhibitors and their structure-activity relationship studies.

Extremely potent, new hepatitis C virus (HCV) nonstructural 5A (NS5A) featuring substituted biaryl sulfate core structures was designed and synthesized. Based on the previously reported novel HCV NS5A inhibitors featuring biaryl sulfate core structures which exhibit two-digit picomolar half-maximal effective concentration (EC50) values against HCV genotype 1b and 2a, the new inhibitors equipped with the sulfate core structures containing diversely substituted aryl groups were explored. In this study, highly efficient, chemoselective coupling reactions between an arylsulfonyl fluoride and an aryl silyl ether, known as the sulfur(vi) fluoride exchange (SuFEx) reaction, were utilized. Among the inhibitors prepared based on the SuFEx chemistry, compounds 14, 15 and 29 exhibited two-digit picomolar EC50 values against GT-1b and single digit or sub nanomolar activities against the HCV GT-2a strain. Nonsymmetrical inhibitors containing an imidazole and amide moieties on each side of the sulfate core structures were also synthesized. In addition, a biotinylated probe targeting NS5A protein was prepared for labeling using the same synthetic methodology.

Regulation of hepatitis C virus replication via threonine phosphorylation of the NS5A protein.

The hepatitis C virus non-structural 5A (NS5A) protein is highly phosphorylated and plays roles in both virus genome replication and assembly of infectious virus particles. NS5A comprises three domains separated by low complexity sequences (LCS). Mass spectrometry analysis of NS5A revealed the existence of a singly phosphorylated tryptic peptide corresponding to the end of LCS I and the beginning of domain II that contained a number of potential phosphorylatable residues (serines and threonines). Here we use a mutagenic approach to investigate the potential role of three of these threonine residues. Phosphomimetic mutations of two of these (T242E and T244E) resulted in significant reductions in virus genome replication and the production of infectious virus, suggesting that the phosphorylation of these residues negatively regulated virus RNA synthesis. Mutation of T245 had no effect, however when T245E was combined with the other two phosphomimetic mutations (TripleE) the inhibitory effect on replication was less pronounced. Effects of the mutations on the ratio of basally/hyperphosphorylated NS5A, together with the apparent molecular weight of the basally phosphorylated species were also observed. Lastly, two of the mutations (T245A and TripleE) resulted in a perinuclear restricted localization of NS5A. These data add further complexity to NS5A phosphorylation and suggest that this analysis be extended outwith the serine-rich cluster within LCS I.

Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions.

ABSTRACTHepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes.IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.

H-Prune as a novel binding protein of NS5A that regulates ERK1/2 activation

Hepatitis C virus (HCV) non-structural 5A (NS5A) protein is associated with a wide variety of host signaling pathways by binding to C-terminal polyproline (PxxP) motifs of various proteins. In this study, we used yeast two-hybrid analysis and a GST pull-down assay to screen a novel NS5A interacting protein and elucidate the binding site and cellular signaling by focusing on recombinant human epidermal growth factor (rhEGF)-mediated ERK1/2 activation. Screening a liver cDNA library revealed that h-prune, a member of the DHH (Asp-His-His) protein superfamily, directly interacted with HCV NS5A C-terminus. In particular, a mutation of five proline amino acids to alanine in this region revealed that these two proteins produced strong interaction through this domain. It is known that h-prune possesses a highly conserved DHH motif, which has exopolyphosphatase activity that accelerates hydrolysis of inorganic polyphosphate. A time-chasing analysis after rhEGF treatment demonstrated that h-prune overexpression almost restored NS5A-mediated attenuation of ERK1/2 phosphorylation, but h-prune itself did not alter this signaling. Although the detailed mechanisms need to be clarified, this study demonstrates that h-prune interacts directly with the PxxP motif of the HCV NS5A C-terminus and that this binding alters the rhEGF-mediated ERK1/2 signaling cascade in liver cells.

FKBP8 interact with classical swine fever virus NS5A protein and promote virus RNA replication.

The non-structural 5A (NS5A) protein of classical swine fever virus (CSFV) is proven to be involved in viral replication and can also modulate cellular signaling and host cellular responses via to its ability to interact with various cellular proteins. FKBP8 is also reported to promote virus replication. Here, we show that NS5A specifically interacts with FKBP8 through coimmunoprecipitation and GST-pulldown studies. Additionally, confocal microscopy study showed that NS5A and FKBP8 colocalized in the cytoplasm. Overexpression of FKBP8 via the eukaryotic expression plasmid pDsRED N1 significantly promoted viral RNA synthesis. The cells knockdown of FKBP8 by lentivirus-mediated shRNA markedly decreased the virus replication when infected with CSFV. These data suggest that FKBP8 plays a critical role in the viral life cycle, particularly during the virus RNA replication period. The investigation of FKBP8 protein functions may be beneficial for developing new strategies to treat CSFV infection.

Synergistic Activity of Combined NS5A Inhibitors.

Daclatasvir (DCV) is a first-in-class hepatitis C virus (HCV) nonstructural 5A replication complex inhibitor (NS5A RCI) that is clinically effective in interferon-free combinations with direct-acting antivirals (DAAs) targeting alternate HCV proteins. Recently, we reported NS5A RCI combinations that enhance HCV inhibitory potential in vitro, defining a new class of HCV inhibitors termed NS5A synergists (J. Sun, D. R. O'Boyle II, R. A. Fridell, D. R. Langley, C. Wang, S. Roberts, P. Nower, B. M. Johnson F. Moulin, M. J. Nophsker, Y. Wang, M. Liu, K. Rigat, Y. Tu, P. Hewawasam, J. Kadow, N. A. Meanwell, M. Cockett, J. A. Lemm, M. Kramer, M. Belema, and M. Gao, Nature 527:245-248, 2015, doi:10.1038/nature15711). To extend the characterization of NS5A synergists, we tested new combinations of DCV and NS5A synergists against genotype (gt) 1 to 6 replicons and gt 1a, 2a, and 3a viruses. The kinetics of inhibition in HCV-infected cells treated with DCV, an NS5A synergist (NS5A-Syn), or a combination of DCV and NS5A-Syn were distinctive. Similar to activity observed clinically, DCV caused a multilog drop in HCV, followed by rebound due to the emergence of resistance. DCV-NS5A-Syn combinations were highly efficient at clearing cells of viruses, in line with the trend seen in replicon studies. The retreatment of resistant viruses that emerged using DCV monotherapy with DCV-NS5A-Syn resulted in a multilog drop and rebound in HCV similar to the initial decline and rebound observed with DCV alone on wild-type (WT) virus. A triple combination of DCV, NS5A-Syn, and a DAA targeting the NS3 or NS5B protein cleared the cells of viruses that are highly resistant to DCV. Our data support the observation that the cooperative interaction of DCV and NS5A-Syn potentiates both the genotype coverage and resistance barrier of DCV, offering an additional DAA option for combination therapy and tools for explorations of NS5A function.

Serine Phosphorylation of the Hepatitis C Virus NS5A Protein Controls the Establishment of Replication Complexes

ABSTRACTThe hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold reduction in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex.IMPORTANCE The mechanisms by which hepatitis C virus replicates its RNA genome remain poorly characterized. We show here that phosphorylation of the viral nonstructural protein NS5A at serine residues is important for the efficient assembly of a complex that is able to replicate the viral genome. This research implicates cellular protein kinases in the control of virus replication and highlights the need to further understand the interplay between the virus and the host cell in order to develop potential avenues for future antiviral therapy.

In vitro activity and resistance profile of samatasvir, a novel NS5A replication inhibitor of hepatitis C virus.

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is a clinically validated target for drugs designed to treat chronic HCV infection. This study evaluated the in vitro activity, selectivity, and resistance profile of a novel anti-HCV compound, samatasvir (IDX719), alone and in combination with other antiviral agents. Samatasvir was effective and selective against infectious HCV and replicons, with 50% effective concentrations (EC50s) falling within a tight range of 2 to 24 pM in genotype 1 through 5 replicons and with a 10-fold EC50 shift in the presence of 40% human serum in the genotype 1b replicon. The EC90/EC50 ratio was low (2.6). A 50% cytotoxic concentration (CC50) of >100 μM provided a selectivity index of >5 × 10(7). Resistance selection experiments (with genotype 1a replicons) and testing against replicons bearing site-directed mutations (with genotype 1a and 1b replicons) identified NS5A amino acids 28, 30, 31, 32, and 93 as potential resistance loci, suggesting that samatasvir affects NS5A function. Samatasvir demonstrated an overall additive effect when combined with interferon alfa (IFN-α), ribavirin, representative HCV protease, and nonnucleoside polymerase inhibitors or the nucleotide prodrug IDX184. Samatasvir retained full activity in the presence of HIV and hepatitis B virus (HBV) antivirals and was not cross-resistant with HCV protease, nucleotide, and nonnucleoside polymerase inhibitor classes. Thus, samatasvir is a selective low-picomolar inhibitor of HCV replication in vitro and is a promising candidate for future combination therapies with other direct-acting antiviral drugs in HCV-infected patients.

Discovery and Development of Hepatitis C Virus NS5A Replication Complex Inhibitors

Lead inhibitors that target the function of the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein have been identified by phenotypic screening campaigns using HCV subgenomic replicons. The demonstration of antiviral activity in HCV-infected subjects by the HCV NS5A replication complex inhibitor (RCI) daclatasvir (1) spawned considerable interest in this mechanistic approach. In this Perspective, we summarize the medicinal chemistry studies that led to the discovery of 1 and other chemotypes for which resistance maps to the NS5A protein and provide synopses of the profiles of many of the compounds currently in clinical trials. We also summarize what is currently known about the NS5A protein and the studies using NS5A RCIs and labeled analogues that are helping to illuminate aspects of both protein function and inhibitor interaction. We conclude with a synopsis of the results of notable clinical trials with HCV NS5A RCIs.

Sandwich Immunoassay for Hepatitis C Virus Non-Structural 5A Protein Using a Glassy Carbon Electrode Modified with an Au-MoO3/Chitosan Nanocomposite

A novel electrochemical immunosensor was prepared for the detection of the hepatitis C virus non-structural 5A protein. A glassy carbon electrode was modified with an Au-MoO3/Chitosan nanocomposite that warranted good conductivity and biocompatibility. Mesoporous silica with a large specific surface served as a nanocarrier for horseradish peroxidase and the polyclonal antibody as the reporter probe. The immunosensor was characterized by scanning electron microscopy, electrochemical impedance spectroscopy, and cyclic voltammetry. Following the sandwich-type immunoreaction, horseradish peroxidase was efficiently captured on the surface of the electrode to catalyze the decomposition of hydrogen peroxide. The analytical signal was obtained as an amperometric i-t curve (chronoamperometry). The assay reported here had a wide detection range (1 ng mL−1 −50 µg mL−1) and detection limit as low as 1 ng mL−1 of hepatitis C virus non-structural 5A protein. The electrochemical biosensor experiments showed excellent reproducibility, high selectivity, and outstanding stability for the determination of hepatitis C virus non-structural 5A protein, and it was successfully applied to the detection of the analyte in real serum samples.

A Synthetic Codon-Optimized Hepatitis C Virus Nonstructural 5A DNA Vaccine Primes Polyfunctional CD8+ T Cell Responses in Wild-Type and NS5A-Transgenic Mice

The hepatitis C virus (HCV) nonstructural (NS) 5A protein has been shown to promote viral persistence by interfering with both innate and adaptive immunity. At the same time, the HCV NS5A protein has been suggested as a target for antiviral therapy. In this study, we performed a detailed characterization of HCV NS5A immunogenicity in wild-type (wt) and immune tolerant HCV NS5A-transgenic (Tg) C57BL/6J mice. We evaluated how efficiently HCV NS5A-based genetic vaccines could activate strong T cell responses. Truncated and full-length wt and synthetic codon-optimized NS5A genotype 1b genes were cloned into eukaryotic expression plasmids, and the immunogenicity was determined after i.m. immunization in combination with in vivo electroporation. The NS5A-based genetic vaccines primed high Ab levels, with IgG titers of >10(4) postimmunization. With respect to CD8(+) T cell responses, the coNS5A gene primed more potent IFN-γ-producing and lytic cytotoxic T cells in wt mice compared with NS5A-Tg mice. In addition, high frequencies of NS5A-specific CD8(+) T cells were found in wt mice after a single immunization. To test the functionality of the CTL responses, the ability to inhibit growth of NS5A-expressing tumor cells in vivo was analyzed after immunization. A single dose of coNS5A primed tumor-inhibiting responses in both wt and NS5A-Tg mice. Finally, immunization with the coNS5A gene primed polyfunctional NS5A-specific CD8(+) T cell responses. Thus, the coNS5A gene is a promising therapeutic vaccine candidate for chronic HCV infections.

Dissimilar expression of multidrug resistance mdr1 and bcrp by the replication of hepatitis C virus: role of the nonstructural 5A protein

Multidrug resistance associated with the overexpression of ATP-dependent binding cassette (ABC) proteins is widely accepted as an important cause of treatment failure in patients with neoplastic or infectious diseases. Some of them play also a pivotal role in detoxification processes. Herein, we investigated the effect of hepatitis C virus (HCV) replication and nonstructural 5A (NS5A) protein on the expression and functional activity of two ABC transport proteins: MDR1 and BCRP. RT-quantitative real-time polymerase chain reaction (qPCR) was carried out for mdr1 and bcrp mRNAs in both Huh7 cells expressing NS5A and Huh7.5 cells containing either full-length- or subgenomic-HCV replicon systems. The functional activity of these pumps was studied by performing a dye efflux assay with DiOC2 and Rhodamine 123. A dose-dependent down-regulation of mdr1 expression was documented in Huh7 cells expressing the NS5A protein, as well as in both replicon systems. In contrast, a significant increase of bcrp expression in both systems was recorded, which were in full agreement with the dye efflux assay results. These results warrant further in vivo studies in HCV patients with cholestasis and/or patients that are refractive to the pharmacotherapy due to the activity of these pumps.

P53 controls hepatitis C virus non-structural protein 5A-mediated downregulation of GADD45α expression via the NF-κB and PI3K–Akt pathways

Growth arrest and DNA-damage-inducible gene 45-α (GADD45α) protein has been shown to be a tumour suppressor and is implicated in cell-cycle arrest and suppression of cell growth. The hepatitis C virus (HCV) non-structural 5A (NS5A) protein plays an important role in cell survival and is linked to the development of hepatocellular carcinoma (HCC). However, the role of HCV NS5A in the development of HCC remains to be clarified. This study sought to determine whether GADD45α mediates HCV NS5A-induced cellular survival and to elucidate the molecular mechanism of GADD45α expression regulated by HCV NS5A. It was found that HCV NS5A downregulated GADD45α expression at the transcriptional level by decreasing promoter activity, mRNA transcription and protein levels. Knockdown of p53 resulted in a similar decrease in GADD45α expression to that caused by HCV NS5A, whilst overexpression of p53 reversed the HCV NS5A-mediated downregulation of GADD45α. HCV NS5A repressed p53 expression, which was followed by a subsequent decrease in GADD45α expression. Further evidence was provided showing that HCV NS5A led to increases of phosphorylated nuclear factor-κB and Akt levels. Inhibition of these pathways using pharmacological inhibitors or specific small interfering RNAs rescued HCV NS5A-mediated downregulation of p53 and GADD45α. It was also found that HCV NS5A protein and depletion of GADD45α increased cell growth, whereas ectopic expression of GADD45α eliminated HCV NS5A-induced cell proliferation. These results indicated that HCV NS5A downregulates GADD45α expression and subsequently triggers cellular proliferation. These findings provide new insights suggesting that HCV NS5A could contribute to the occurrence of HCV-related HCC.