Nonstructural Protein 5A Expression Research Articles

Mitophagy induced by classical swine fever virus nonstructural protein 5A promotes viral replication

The classical swine fever virus (CSFV) is one of the most harmful pathogens of swine and causes considerable economic loss. Mitophagy is a selective form of autophagy that degrades damaged mitochondria by combining with lysosomes. Previous studies have been reported that CSFV infection can induce mitophagy, but which effector protein is responsible for this process remains unclear. Herein, we revealed here that the CSFV nonstructural protein 5A (NS5A) plays a critical role in inducing cellular mitophagy. Specifically, the expression of CSFV NS5A in the PK-15 cells induces membrane potential loss and mitochondrial fission, and the quantities of mitophagosomes, the expression of Parkin and PINK1 were significantly increased compared with mock cells. Intriguingly, we found that Parkin-overexpression promotes CSFV propagation. Furthermore, the expression level of reactive oxygen species (ROS) was increased by CSFV NS5A protein, while NS5A-induced mitophagy correlated with the quantity of ROS production. In summary, our results reveal a new function of NS5A in inducing cellular mitophagy and broaden our understanding of the mechanism of CSFV-induced mitophagy, which may provide a new way to develop an antiviral strategy.

Autophagy Induced by the N-Terminus of the Classic Swine Fever Virus Nonstructural Protein 5A Protein Promotes Viral Replication.

Although classic swine fever virus (CSFV) infection has been reported to induce autophagy, the specific induced mechanism remains unrevealed. Nonstructural protein 5A (NS5A) of CSFV is a multiphosphorylated protein with multiple functions to regulate viral replication and the host cell immune responses. Herein, we demonstrated that CSFV NS5A could induce cellular autophagy and promote viral replication. In the current study, we showed that NS5A expression significantly increased the levels of autophagy-related genes (ATGs), including light chain 3 (LC3), ATG5, and Beclin 1; conversely, degradation of P62/sequestosome 1 (SQSTM1) was observed by Western blotting. The number of autophagy-like vesicles was also obviously increased in NS5A-expressing cells, as analyzed by transmission electron microscopy (TEM). Furthermore, we observed the co-localization of the NS5A and LC3 proteins by confocal immunofluorescence analysis. Direct binding of NS5A to the autophagy-related LC3 protein was confirmed by coimmunoprecipitation in vivo and by a GST pulldown assay in vitro. Through segmentation and point mutation research on the NS5A protein, we found that the N-terminal region and the phosphorylation of amino acids 81 and 92 of the NS5A protein were essential for inducing autophagy. Finally, we demonstrated that the LC3 protein had a positive effect on CSFV replication. These findings emphasize a previously unascertained interaction relationship between NS5A and LC3 in the autophagy process. Furthermore, our research revealed a new role of CSFV NS5A, particularly its N-terminal amino acids serine 81 and serine 92, as a critical regulator of CSFV-induced autophagy and have significance for extending our understanding of the CSFV-autophagy interplay.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective: To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase (MAPK) signaling pathway modulated by hepatitis C virus (HCV) nonstructural protein 5A (NS5A). Methods: A total of ten plant extracts were initially screened for their toxicities against HepG2 cells. The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both mRNA and protein levels using real-time PCR and Western blotting assays, respectively. The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR. Subsequently, the identification of secondary metabolites was carried out by phytochemical and HPLC analysis. Results: The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids, phenols, saponins, tannins, flavonoids, carbohydrates, terpenoids, steroids, and glycosides. Similarly, quercetin, myricetin, gallic acid, caffeic acid, and ferulic acid were identified through HPLC analysis. The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44 µg/mL. RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dose- dependent manner. Berberis lyceum extract also attenuated NS5A- induced dysregulation of the MAPK signaling pathway. Conclusions: Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5A- induced perturbation of MAPK signaling.

Hepatitis C Virus Non-Structural Protein 5A (NS5A) Disrupts Mitochondrial Dynamics and Induces Mitophagy.

Mitophagy is a selective form of autophagy, targeting damaged mitochondria for lysosomal degradation. Although HCV infection has been shown to induce mitophagy, the precise underlying mechanism and the effector protein responsible remain unclear. Herein, we demonstrated that the HCV non-structural protein 5A (NS5A) plays a key role in regulating cellular mitophagy. Specifically, the expression of HCV NS5A in the hepatoma cells triggered hallmarks of mitophagy including mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin translocation to the mitochondria. Furthermore, mitophagy induction through the expression of NS5A led to an increase in autophagic flux as demonstrated by an accumulation of LC3II in the presence of bafilomycin and a time-dependent decrease in p62 protein level. Intriguingly, the expression of NS5A concomitantly enhanced reactive oxygen species (ROS) production, and treatment with an antioxidant attenuated the NS5A-induced mitophagy event. These phenomena are similarly recapitulated in the NS5A-expressing HCV subgenomic replicon cells. Finally, we demonstrated that expression of HCV core, which has been documented to inhibit mitophagy, blocked the mitophagy induction both in cells harboring HCV replicating subgenomes or expressing NS5A alone. Our results, therefore, identified a new role for NS5A as an important regulator of HCV-induced mitophagy and have implications to broadening our understanding of the HCV-mitophagy interplay.

Molecular Neuroprotection Induced by Zinc-Dependent Expression of Hepatitis C-Derived Protein NS5A Targeting Kv2.1 Potassium Channels.

We present the design of an innovative molecular neuroprotective strategy and provide proof-of-concept for its implementation, relying on the injury-mediated activation of an ectopic gene construct. As oxidative injury leads to the intracellular liberation of zinc, we hypothesize that tapping onto the zinc-activated metal regulatory element (MRE) transcription factor 1 system to drive expression of the Kv2.1-targeted hepatitis C protein NS5A (hepatitis C nonstructural protein 5A) will provide neuroprotection by preventing cell death-enabling cellular potassium loss in rat cortical neurons in vitro. Indeed, using biochemical and morphologic assays, we demonstrate rapid expression of MRE-driven products in neurons. Further, we report that MRE-driven NS5A expression, induced by a slowly evolving excitotoxic stimulus, functionally blocks injurious, enhanced Kv2.1 potassium whole-cell currents and improves neuronal viability. We suggest this form of "on-demand" neuroprotection could provide the basis for a tenable therapeutic strategy to prevent neuronal cell death in neurodegeneration.

Effect of hepatitis C virus nonstructural protein 5A and its domains II on hepatocyte gluconeogenesis in mice

Objective: To investigate the role of hepatitis C virus nonstructural protein 5A (NS5A) and its domains I, II, and III in regulating gluconeogenesis in mice and the underlying mechanism. Methods: A total of 60 male C57BL/6J mice were randomly divided into six groups. Recombinant lentiviral particles with specific expression of full-length NS5A, NS5A domain I, NS5A domain II, or NS5A domain III were injected via the caudal vein to establish a mouse model, and the group without injection and the group with the injection of the lentiviral particles containing enhanced green fluorescent protein (EGFP) were established as negative control. The effect of full-length NS5A protein and its domains on fasting blood glucose (FBG) and fasting serum insulin (FINS) were measured. Liver tissue was collected to prepare a paraffin section. Immunohistochemistry was used to measure the expression of phosphoenolpyruvate carboxykinase (PEPCK) in hepatocytes, quantitative real-time PCR and/or Western blot were used to measure the expression of NS5A, phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK), sterol regulatory element-binding protein-1 (SREBP-1), and PEPCK. Results: Compared with the group without injection and the group with the injection of the lentiviral particles containing EGFP, the groups with the injection of the lentiviral particles containing full-length NS5A and NS5A domain II had significant increases in FBG and homeostasis model assessment of insulin resistance index (P < 0.01). Immunohistochemistry and quantitative real-time PCR showed a significant increase in the expression of PEPCK, a key enzyme involved in gluconeogenesis. Western blot showed that full-length NS5A protein and NS5A domain II inhibited the level of p-AMPK and increased the levels of SREBP-1 and PEPCK. Conclusion: NS5A protein and NS5A domain II may affect glucose metabolism in hepatocytes in mice by regulating AMPK/SREBP-1/PEPCK, and NS5A domain II may play an important role in insulin resistance in hepatocytes caused by HCV infection.

Hepatitis C Virus Subverts Human Choline Kinase-α To Bridge Phosphatidylinositol-4-Kinase IIIα (PI4KIIIα) and NS5A and Upregulates PI4KIIIα Activation, Thereby Promoting the Translocation of the Ternary Complex to the Endoplasmic Reticulum for Viral Replication.

ABSTRACTIn this study, we elucidated the mechanism by which human choline kinase-α (hCKα) interacts with nonstructural protein 5A (NS5A) and phosphatidylinositol-4-kinase IIIα (PI4KIIIα), the lipid kinase crucial for maintaining the integrity of virus-induced membranous webs, and modulates hepatitis C virus (HCV) replication. hCKα activity positively modulated phosphatidylinositol-4-phosphate (PI4P) levels in HCV-expressing cells, and hCKα-mediated PI4P accumulation was abolished by AL-9, a PI4KIIIα-specific inhibitor. hCKα colocalized with NS5A and PI4KIIIα or PI4P; NS5A expression increased hCKα and PI4KIIIα colocalization; and hCKα formed a ternary complex with PI4KIIIα and NS5A, supporting the functional interplay of hCKα with PI4KIIIα and NS5A. PI4KIIIα inactivation by AL-9 or hCKα inactivation by CK37, a specific hCKα inhibitor, impaired the endoplasmic reticulum (ER) localization and colocalization of these three molecules. Interestingly, hCKα knockdown or inactivation inhibited PI4KIIIα-NS5A binding. In an in vitro PI4KIIIα activity assay, hCKα activity slightly increased PI4KIIIα basal activity but greatly augmented NS5A-induced PI4KIIIα activity, supporting the essential role of ternary complex formation in robust PI4KIIIα activation. Concurring with the upregulation of PI4P production and viral replication, overexpression of active hCKα-R (but not the D288A mutant) restored PI4KIIIα and NS5A translocation to the ER in hCKα stable knockdown cells. Furthermore, active PI4KIIIα overexpression restored PI4P production, PI4KIIIα and NS5A translocation to the ER, and viral replication in CK37-treated cells. Based on our results, hCKα functions as an indispensable regulator that bridges PI4KIIIα and NS5A and potentiates NS5A-stimulated PI4KIIIα activity, which then facilitates the targeting of the ternary complex to the ER for viral replication.IMPORTANCE The mechanisms by which hCKα activity modulates the transport of the hCKα-NS5A complex to the ER are not understood. In the present study, we investigated how hCKα interacts with PI4KIIIα (a key element that maintains the integrity of the “membranous web” structure) and NS5A to regulate viral replication. We demonstrated that HCV hijacks hCKα to bridge PI4KIIIα and NS5A, forming a ternary complex, which then stimulates PI4KIIIα activity to produce PI4P. Pronounced PI4P synthesis then redirects the translocation of the ternary complex to the ER-derived, PI4P-enriched membrane for assembly of the viral replication complex and viral replication. Our study provides novel insights into the indispensable modulatory role of hCKα in the recruitment of PI4KIIIα to NS5A and in NS5A-stimulated PI4P production and reveals a new perspective for understanding the impact of profound PI4KIIIα activation on the targeting of PI4KIIIα and NS5A to the PI4P-enriched membrane for viral replication complex formation.

Hepatitis C Virus Nonstructural Protein 5A Inhibits MG132-Induced Apoptosis of Hepatocytes in Line with NF-κB-Nuclear Translocation.

BackgroundHepatitis C virus (HCV) infection is one of the major causes of cirrhosis and hepatocellular carcinoma. HCV nonstructural protein 5A (NS5A) is an attractive antiviral target and plays an important role in HCV replication as well as hepatocarcinogenesis. The aim of this study was to assess the effect of HCV NS5A protein in the abrogation of apoptotic cell death induced by the proteasome inhibitor MG132.MethodsApoptotic responses to MG132 and the expression of molecules involved in NF-κB signaling pathways in human hepatocytes were investigated with or without the expression of HCV NS5A.ResultsHCV NS5A protected HepG2 cells against MG132-induced apoptosis, in line with NF-κB-nuclear translocation. A similar NF-κB-nuclear translocation was observed in Huh7 cells infected with HCV JFH1. In agreement with this, after treatment with MG132, HCV NS5A could elevate the transcription of several NF-κB target genes such as BCL2 and BCLXL to inhibit MG132-induced apoptosis in hepatocytes. HCV HCV NS5A also enhanced phosphorylation of IκBα. Consistent with a conferred prosurvival advantage, HCV NS5A reduced MG132-induced poly(adenosine diphosphate-ribose) polymerase cleavage.ConclusionsHCV NS5A expression enhances phosphorylation of IκBα, liberates NF-κB for nuclear translocation and downregulates MG132-induced apoptotic pathways in human hepatocytes. It is possible that the disruption of proteasome-associated apoptosis plays a role in the pathogenesis of HCV infection.

Hepatitis C virus NS5A promotes insulin resistance through IRS-1 serine phosphorylation and increased gluconeogenesis.

To investigate the mechanisms of insulin resistance in human hepatoma cells expressing hepatitis C virus (HCV) nonstructural protein 5A (NS5A). The human hepatoma cell lines, Huh7 and Huh7.5, were infected with HCV or transiently-transfected with a vector expressing HCV NS5A. The effect of HCV NS5A on the status of the critical players involved in insulin signaling was analyzed using real-time quantitative polymerase chain reaction and Western blot assays. Data were analyzed using Graph Pad Prism version 5.0. To investigate the effect of insulin treatment on the players involved in insulin signaling pathway, we analyzed the status of insulin receptor substrate-1 (IRS-1) phosphorylation in HCV infected cells or Huh7.5 cells transfected with an HCV NS5A expression vector. Our results indicated that there was an increased phosphorylation of IRS-1 (Ser(307)) in HCV infected or NS5A transfected Huh7.5 cells compared to their respective controls. Furthermore, an increased phosphorylation of Akt (Ser(473)) was observed in HCV infected and NS5A transfected cells compared to their mock infected cells. In contrast, we observed decreased phosphorylation of Akt Thr308 phosphorylation in HCV NS5A transfected cells. These results suggest that Huh7.5 cells either infected with HCV or ectopically expressing HCV NS5A alone have the potential to induce insulin resistance by the phosphorylation of IRS-1 at serine residue (Ser(307)) followed by decreased phosphorylation of Akt Thr(308), Fox01 Ser(256) and GSK3β Ser(9), the downstream players of the insulin signaling pathway. Furthermore, increased expression of PECK and glucose-6-phosphatase, the molecules involved in gluconeogenesis, in HCV NS5A transfected cells was observed. Taken together, our results suggest the role of HCV NS5A in the induction of insulin resistance by modulating various cellular targets involved in the insulin signaling pathway.

Hepatitis C virus nonstructural protein 5A favors upregulation of gluconeogenic and lipogenic gene expression leading towards insulin resistance: a metabolic syndrome

Chronic hepatitis C is a lethal blood-borne infection often associated with a number of pathologies such as insulin resistance and other metabolic abnormalities. Insulin is a key hormone that regulates the expression of metabolic pathways and favors homeostasis. In this study, we demonstrated the molecular mechanism of hepatitis C virus (HCV) nonstructural protein 5A (NS5A)-induced metabolic dysregulation. We showed that transient expression of HCV NS5A in human hepatoma cells increased lipid droplet formation through enhanced lipogenesis. We also showed increased transcriptional expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and diacylglycerol acyltransferase-1 (DGAT-1) in NS5A-expressing cells. On the other hand, there was significantly reduced transcriptional expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor γ (PPARγ) in cells expressing HCV NS5A. Furthermore, increased gluconeogenic gene expression was observed in HCV-NS5A-expressing cells. In addition, it was also shown that HCV-NS5A-expressing hepatoma cells show serine phosphorylation of IRS-1, thereby hampering metabolic activity and contributing to insulin resistance. Therefore, this study reveals that HCV NS5A is involved in enhanced gluconeogenic and lipogenic gene expression, which triggers metabolic abnormality and impairs insulin signaling pathway.

NS5ATP9 Contributes to Inhibition of Cell Proliferation by Hepatitis C Virus (HCV) Nonstructural Protein 5A (NS5A) via MEK/Extracellular Signal Regulated Kinase (ERK) Pathway

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a remarkable protein as it clearly plays multiple roles in mediating viral replication, host-cell interactions and viral pathogenesis. However, on the impact of cell growth, there have been different study results. NS5ATP9, also known as KIAA0101, p15PAF, L5, and OEACT-1, was first identified as a proliferating cell nuclear antigen-binding protein. Earlier studies have shown that NS5ATP9 might play an important role in HCV infection. The aim of this study is to investigate the function of NS5ATP9 on hepatocellular carcinoma (HCC) cell lines proliferation under HCV NS5A expression. The results showed that overexpression of NS5ATP9 inhibited the proliferation of Bel7402 cells, whereas knockdown of NS5ATP9 by interfering RNA promoted the growth of HepG2 cells. Under HCV NS5A expression, RNA interference (RNAi) targeting of NS5ATP9 could reverse the inhibition of HepG2 cell proliferation, suggesting that NS5ATP9 might be an anti-proliferation gene that plays an important role in the suppression of cell growth mediated by HCV NS5A via MEK/ERK signaling pathway. These findings might provide new insights into HCV NS5A and NS5ATP9.

Hepatitis C Virus NS5A Binds to the mRNA Cap-binding Eukaryotic Translation Initiation 4F (eIF4F) Complex and Up-regulates Host Translation Initiation Machinery through eIF4E-binding Protein 1 Inactivation

Initiation, a major rate-limiting step of host protein translation, is a critical target in many viral infections. Chronic hepatitis C virus (HCV) infection results in hepatocellular carcinoma. Translation initiation, up-regulated in many cancers, plays a critical role in tumorigenesis. mTOR is a major regulator of host protein translation. Even though activation of PI3K-AKT-mTOR by HCV non-structural protein 5A (NS5A) is known, not much is understood about the regulation of host translation initiation by this virus. Here for the first time we show that HCV up-regulates host cap-dependent translation machinery in Huh7.5 cells through simultaneous activation of mTORC1 and eukaryotic translation initiation factor 4E (eIF4E) by NS5A. NS5A, interestingly, overexpressed and subsequently hyperphosphorylated 4EBP1. NS5A phosphorylated eIF4E through the p38 MAPK-MNK pathway. Both HCV infection and NS5A expression augmented eIF4F complex assembly, an indicator of cap-dependent translation efficiency. Global translation, however, was not altered by HCV NS5A. 4EBP1 phosphorylation, but not that of S6K1, was uniquely resistant to rapamycin in NS5A-Huh7.5 cells, indicative of an alternate phosphorylation mechanism of 4EBP1. Resistance of Ser-473, but not Thr-308, phosphorylation of AKT to PI3K inhibitors suggested an activation of mTORC2 by NS5A. NS5A associated with eIF4F complex and polysomes, suggesting its active involvement in host translation. This is the first report that implicates an HCV protein in the up-regulation of host translation initiation apparatus through concomitant regulation of multiple pathways. Because both mTORC1 activation and eIF4E phosphorylation are involved in tumorigenesis, we propose that their simultaneous activation by NS5A might contribute significantly to the development of hepatocellular carcinoma.

HCV NS5A protein down-regulates hepcidin gene expression and increases hepatic intracellular iron storage

To investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene. HCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level. HCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells. HCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.

The oncogenic role of NS5A of hepatitis C virus is mediated by up-regulation of survivin gene expression in the hepatocellular cell through p53 and NF-κB pathways

Approx. 4% of patients experiencing chronic infection of human HCV (hepatitis C virus) ultimately develop HCC (hepatocellular carcinoma). The NS5A (non-structural protein 5A) encoded by HCV has been reported to have an oncogenic role during HCV infection, but the precise mechanism remains largely unclear. The aim of this study is to investigate the signal transduction pathways that mediate the role of NS5A in hepatocarcinogenesis. HepG2 cells were transfected with a plasmid expressing HCV NS5A protein. Subsequently, cell proliferation was analysed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay and cell counting, apoptosis was analysed by Hoechst 33342 staining, and the gene expression profile was identified by microarray and subsequently validated by RT-PCR (reverse transcription-PCR). The protein levels of survivin, p53, NOS2A (nitric oxide synthase 2A), cyclin D1 and NF-κB (nuclear factor κB) were monitored by Western blotting. Our results showed that transfection of HCV NS5A expression plasmid significantly down-regulated the expression of nine genes and up-regulated the expression of ten genes among the 104 genes detectable by the microarray associated with signalling transduction. The increased expression of survivin mRNA and protein, down-regulated p53 protein levels and increased NOS2A, cyclin D1 and NF-κB protein levels were further identified. Our results suggested that HCV NS5A protein can enhance survivin transcription by increasing p53 degradation and stimulating NOS2A expression as well as NF-κB relocation to the nucleus. The functions of survivin in anti-apoptosis and regulation of cell division might mediate the role of NS5A in HCV-induced HCC.

Enhanced Ca2+ leak from ER Ca2+ stores induced by hepatitis C NS5A protein

The hepatitis C non-structural protein 5A (NS5A) is a Zn2+-binding phosphoprotein essential for viral replication. Expression of NS5A perturbs intracellular Ca2+ levels by an undefined mechanism, activating transcription factors implicated in the chronic pathogenesis of hepatitis infections. Here, we demonstrate that regulated expression of NS5A enhanced the passive leak of Ca2+ from a subset of the endoplasmic reticulum (ER) Ca2+ stores. This action was not replicated by expression of the amphipathic NH2-membrane anchoring domain of NS5A alone, despite targeting to intracellular membranes. Depletion of the NS5A-targeted ER Ca2+ store was prevented under conditions of ample ATP supply suggesting compensatory Ca2+ ATPase activity, but observed under conditions of ATP insufficiency and in intact cells expressing NS5A.

Hepatitis C virus NS5A protein binds TBP and p53, inhibiting their DNA binding and p53 interactions with TBP and ERCC3

Among the hepatotropic viruses, hepatitis C virus (HCV) is considered to be the leading cause of liver disease in humans, affecting ∼2% of the world population. HCV-encoded nonstructural protein 5A (NS5A) is a 56–58-kDa phosphoprotein, which is produced from the processing of viral polyprotein. The potential mechanism(s) by which NS5A is able to influence key cellular processes are largely unknown. In this study, we investigated the functional properties of NS5A. In vivo co-immunoprecipitation and pull-down assays demonstrated that NS5A forms a heteromeric complex with TATA box binding protein (TBP) and tumor suppressor protein p53. Mutants of TBP and p53 showed reduced binding to NS5A. To determine the functional relevance of these associations, we found that NS5A inhibits the binding of both p53 and TBP to their DNA consensus binding sequences in vitro. NS5A also inhibited the p53–TBP and p53–excision repair cross complementing factor 3 (ERCC3) protein–protein complex formation. Furthermore, NS5A repressed the p53 regulated p21 (WAF1) promoter and a synthetic promoter containing multiple p53 responsive DNA elements binding sites in HCT116 p53 +/+ cell line. p53-mediated transcriptional activation from both promoters was reduced ∼3–5-fold following expression of NS5A. Taken together, these results suggest that NS5A may exert its influence on key cellular processes by functional associations with p53 and TBP. This could explain one of the possible mechanism(s) by which NS5A is able to exert its effect on cellular gene expression and cell growth regulation.

Endoplasmic reticulum (ER) stress: hepatitis C virus induces an ER-nucleus signal transduction pathway and activates NF-κB and STAT-3

Human hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. The HCV RNA genome codes for at least ten proteins. The HCV non-structural protein 5A (NS5A) has generated considerable interest due to its effect on interferon sensitivity via binding and inactivating the cellular protein kinase, PKR. It has been shown that NS5A engages in the endoplasmic reticulum (ER)-nucleus signal transduction pathway. The expression of NS5A in the ER induces an ER stress ultimately leading to the activation of STAT-3 and NF-κB. This pathway is sensitive to inhibitors of Ca 2+ uptake in the mitochondria (ruthenium red), Ca 2+ chelators (TMB-8, EGTA-AM), and antioxidants (PDTC, NAC, Mn-SOD). The inhibitory effect of protein tyrosine kinase (PTK) inhibitors indicates the involvement of PTK in NF-κB activation by NS5A. This implicates an alternate pathway of NF-κB activation by NS5A. The actions of NS5A have also been studied in the context of an HCV subgenomic replicon inducing a similar intracellular event. Thus, activation of NF-κB leads to the induction of cellular genes, which are largely antiapoptotic in function. These studies suggest a potential function of NS5A in inducing chronic liver disease and hepatocellular carcinoma associated with HCV infection.

Nonstructural Protein 5A of Hepatitis C Virus Inhibits the Function of Karyopherin β3

It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin beta3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin beta3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin beta3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin beta3 gene. Human karyopherin beta3 complemented the loss of the PSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin beta3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin beta3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.