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Translation initiation of the Hepatitis C virus (HCV) genome is driven by an internal ribosome entry site (IRES), located within the 5' non-coding region. Several studies have suggested that different cellular non canonical proteins or viral proteins can regulate the HCV IRES activity. However, the role of the viral proteins on HCV translation remains controversial. In this report, we confirmed previous studies showing that NS5A down-regulates IRES activity in HepG2 but not in Huh7 cells suggesting that the NS5A effect on HCV IRES is cell-type dependent. Additionally, we provide strong evidence that activated PKR up-regulates the IRES activity while silencing of endogenous PKR had the opposite effect. Furthermore, we present data indicating that the NS5A-mediated inhibitory effect on IRES-dependent translation could be linked with the PKR inactivation. Finally, we show that NS5A from GBV-C but not from GBV-B down-regulates HCV IRES activity in the absence or the presence of PKR over expression. Notably, HCV and GBV-C but not GBV-B NS5A contains a previously identified PKR interacting protein domain., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: According to previous data, the antibodies produced during natural hepatitis C virus (HCV) infection frequently recognize amino acids 10-43 in the core protein and 1689-1740 or 1921-1940 in the non-structural 4B (NS4B) protein. The reactivity of these peptides with the corresponding antibodies has mainly been evaluated using serum samples from Western countries where HCV genotype 1 (HCV-1) is predominant, and no information is available concerning samples from sub-Saharan countries where high HCV variability has been reported. OBJECTIVE OF THIS STUDY: To evaluate the performance of HCV core and NS4B peptide-based immunoassays in the serodiagnosis of HCV infection in Cameroon subjects., Study Design: Three core and four NS4B-based synthetic peptides derived from HCV genotypes 1b and 2a were designed and tested against a panel of 151 serum samples from Cameroon (40 positive for HCV-1, 32 for HCV-2, 39 HCV-4, and 40 HCV-negative)., Results: The three core peptides all demonstrated strong immunoreactivity, regardless of the HCV genotype from which they were derived, with greater than 90% and 92% sensitivity and specificity. In contrast, the NS4B-derived peptides exhibited lower sensitivities (24.3-65.8% depending on the HCV genotype) but higher specificities (100% for all four peptides tested)., Conclusions: Our findings indicate that an HCV core peptide could be used for the diagnosis of chronic HCV infection. Among the NS4B peptides tested, a chimeric NS4B peptide encompassing both N- and C-terminal portions of the NS4B protein gave a much better performance than the two component N- and C-terminal peptides used individually., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

The Hepatitis C virus (HCV) NS4B protein, a multispanning endoplasmic reticulum (ER) membrane protein, generates intracellular rearrangements of ER-derived membranes, essential for HCV replication. In this study, we characterized NS4B elements involved in the process of targeting, association and retention in the ER membrane. We investigated the localization and membrane association of a number of C- or N-terminal NS4B deletions expressed as GFP chimeras by biochemical and fluorescence microscopy techniques. A second set of GFP-NS4B chimeras containing the plasma membrane ecto-ATPase CD39 at the C-terminus of each NS4B deletion mutant was used to further examine the role of N-terminal NS4B sequences in ER retention. Several structural elements, besides the first two transmembrane domains (TMs), within the NS4B N-terminal half (residues 1-130) were found to mediate association of the NS4B-GFP chimeras with ER membranes. Both TM1 and TM2 are required for ER anchoring and retention but are not sufficient for ER retention. Sequences upstream of TM1 are also required. These include two putative amphipathic alpha-helices and a Leucine Rich Repeat-like motif, a sequence highly conserved in all HCV genotypes. The N-terminal 55peptidic sequence, containing the 1st amphipathic helix, mediates association of the 55N-GFP chimera with cellular membranes including the ER, but is dispensable for ER targeting of the entire NS4B molecule. Importantly, the C-terminal 70peptidic sequence can associate with membranes positive for ER markers in the absence of any predicted TMs. In conclusion, HCV NS4B targeting and retention in the ER results from the concerted action of several NS4B structural elements.

It is well established that HCV NS5A protein when expressed in mammalian cells perturbs the extracellular signal regulated kinase (ERK) pathway. The protein serine/threonine phosphatase 2A controls the phosphorylation of numerous proteins involved in cell signaling and one characterized function is the regulation of Ras-Raf mitogen activated protein (MAP) kinase signaling pathways. Our results showed that expression of HCV NS5A protein stimulates phosphatase 2A (PP2A) activity in cells, indicating the relevance of NS5A as a regulator of PP2A in vivo. We found that transient expression of the full length NS5A protein in different cell lines leads to a significant increase of the PP2A activity and this activity is specifically inhibited by the addition of okadaic acid, a PP2A inhibitor, in living cells. Further investigation showed that NS5A protein interacts in vivo and in vitro with the scaffolding A and the catalytic C subunits of PP2A. We propose that HCV NS5A represents a viral PP2A regulatory protein. This is a novel function for the NS5A protein which may have a key role in the ability of the virus to deregulate cell growth and survival.

The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional protein that is implicated in viral replication and pathogenesis. We report here that NS5A of HCV-1a is cleaved at multiple sites by caspase proteases in transfected cells. Two cleavage sites at positions Asp154 and 248DXXD251 were mapped. Cleavage at Asp154 has been previously recognized as one of the caspase cleavage sites for the NS5A protein of HCV genotype 1b (1, 2) and results in the production of a 17-kDa fragment. The sequence 248DXXD251 is a novel caspase recognition motif for NS5A and is responsible for the production of a 31-kDa fragment. Furthermore, we show that Arg217 is implicated in the production of the previously described 24-kDa product, whose accumulation is affected by both calpain and caspase inhibitors. We also showed that caspase-mediated cleavage occurs in the absence of exogenous proapoptotic stimuli and is not related to the accumulation of the protein in the endoplasmic reticulum. Interestingly, our data indicate that NS5A is targeted by at least two different caspases and suggest that caspase 6 is implicated in the production of the 17-kDa fragment. Most importantly, we report that, all the detectable NS5A fragments following caspase-mediated cleavage are C-terminal-truncated forms of NS5A and are mainly localized in the cytosol. Thus, in sharp contrast to the current view we found no evidence supporting a role for caspase-mediated cleavage in the transport of the NS5A protein to the nucleus, which could lead to transcriptional activation.

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located mainly within the 5' non-translated region of the viral genome. In this study, the effect of the HCV non-structural 5A (NS5A) protein on the HCV IRES-dependent translation was investigated by using a transient transfection system. Three different cell lines (HepG2, WRL-68 and BHK-21) were co-transfected with a plasmid vector containing a bicistronic transcript carrying the chloramphenicol acetyltransferase (CAT) and the firefly luciferase genes separated by the HCV IRES sequences, and an expression vector producing the NS5A protein. Here, it was shown that the HCV NS5A protein inhibited HCV IRES-dependent translation in a dose-dependent manner. In contrast, NS5A had no detectable effect on cap-dependent translation of the upstream gene (CAT) nor on translation from another viral IRES. Further analysis using deleted forms of the NS5A protein revealed that a region of about 120 aa located just upstream of the nuclear localization signal of the protein is critical for this suppression. Overall, these results suggest that HCV NS5A protein negatively modulates the HCV IRES activity in a specific manner.

The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional phosphoprotein that is implicated in viral replication and HCV-mediated pathogenesis. We report here that the NS5A protein from the HCV genotype 1a is processed into shorter distinct forms when expressed in mammalian cells (Vero, HepG2, HuH-7, and WRL68) infected with an NS5A-expressing HSV-1-based amplicon vector or when transiently transfected with NS5A-expressing plasmids in the absence of exogenous apoptotic stimuli. Inhibitor studies combined with cell-free cleavage assays suggest that calcium-dependent calpain proteases, in addition to caspase-like proteases, are involved in NS5A processing. Interestingly, His-tagging experiments indicated that all the detectable NS5A-cleaved products are N-terminal forms of the protein. Additionally, immunofluorescence studies showed that, despite proteolytic cleavage, the NS5A protein exhibits a cytoplasm-perinuclear localization similar to that of the full-length protein. Thus, our results are consistent with recent data that demonstrated that NS5A is capable of perturbing intracellular calcium homeostasis and suggest that NS5A is both an inducer and a substrate of the calcium-dependent calpain protease(s). This may imply that cleavage of NS5A by calpain(s) could play a role in the modulation of NS5A function.

Two herpes simplex type 1 (HSV-1) recombinant viruses carrying the hepatitis C virus (HCV) NS5A open reading frame under the control of the cytomegalovirus immediate early (IE) or a herpes simplex chimeric promoter (alpha 4 gamma 1UL19) were constructed and characterized. Expression studies showed that both HSV-NS5A recombinant viruses were able to express high levels of the NS5A protein in infected cells. Most importantly, using this system, we demonstrated that the NS5A protein interacts with the growth receptor-bound protein 2 (Grb2) and inhibits the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2) in HeLa, NIH3T3 or liver infected cells. Thus, our studies confirm the ability of the NS5A protein to perturb the extracellular signal-regulated kinase (ERK) pathway in HeLa cells by the use of an alternative system for NS5A expression and extend this observation to additional cell lines. We conclude that HSV-based viral vectors may provide a useful system for studying the expression and selected functional properties of the HCV NS5A protein.

In this study different forms of the hepatitis C virus (HCV) NS5A protein, including a nearly full-length, an amino-terminal and a carboxy-terminal truncated form were produced in E. coli as fusion proteins with the MBP or the GST protein. The chimeric proteins were tested for their reactivity with sera from HCV infected patients by immunoblot and ELISA assays. A panel of 110 sera specimens, including 39 HCV-positive sera, 27 sera from patients with non-HCV-associated liver disease and 44 healthy individuals were analyzed for the presence of antibodies to NS5A. Twenty four (61 %) out of the 39 HCV positive sera, showed reactivity against the nearly full length NS5A, 21 (54 %) against the amino-terminal part of NS5A and 20 (51 %) against the carboxy-terminal part of the NS5A protein in immunoblot assays, suggesting that immunoreactive epitopes are present both at the carboxy- and the amino- terminal part of the protein. None of the 71 HCV-negative serum samples showed any reactivity against the NS5A antigens. With the exception of one patient, similar data were obtained with an ELISA assay based on the use of the nearly full-length NS5A antigen. The data indicate that new forms of NS5A may be potentially valuable antigens for the development of serological assays for HCV.