Comparison of structural architecture of HCV NS3 genotype 1 versus Pakistani genotype 3a.
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ABSTRACT:
SUBMITTER: Fatima K
PROVIDER: S-EPMC4221965 | biostudies-literature | 2014
REPOSITORIES: biostudies-literature
Publications
Comparison of structural architecture of HCV NS3 genotype 1 versus Pakistani genotype 3a.
Fatima Kaneez K Azhar Esam E Mathew Shilu S Damanhouri Ghazi G Qadri Ishtiaq I
BioMed research international 20141021
This study described the structural characterization of Pakistani HCV NS3 GT3a in parallel with genotypes 1a and 1b NS3. We investigated the role of amino acids and their interaction patterns in different HCV genotypes by crystallographic modeling. Different softwares were used to study the interaction pattern, for example, CLCBIO sequence viewer, MODELLER, NMRCLUST, ERRAT score, and MODELLER. Sixty models were produced and clustered into groups and the best model of PK-NCVI/Pk3a NS3 was selecte ...[more]
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Project description:BackgroundHepatitis C virus infection is the main cause of liver ailments across the globe. Several HCV genotypes have been identified in different parts of the world. Effective drugs for combating HCV infections are available but not affordable, particularly to infected individuals from resource-limited countries. Hence, cost-effective drugs need to be developed against important HCV drug targets. As Citrus fruits naturally contain bioactive compounds with antiviral activities, the current study was designed to identify antiviral inhibitors from Citrus fruit extracts against an important drug target, NS3 protease, of HCV genotype 3a which is found predominantly in South Asian countries.MethodsThe full-length NS3 protease alone and the NS3 protease domain in fusion with the cognate NS4A cofactor were expressed in Escherichia coli, and purified by chromatographic techniques. Using the purified protein as a drug target, Citrus extracts were evaluated in a FRET assay, and active ingredients, identified using ESI-MS/MS, were docked to observe the interaction with active site residues of NS3. The best interacting compound was further confirmed through the FRET assay as the inhibitor of NS3 protease.ResultsFusion of the NS3 protease domain to the NS4A cofactor significantly improved the purification yield, and NS3-NS4A was functionally more active than the full-length NS3 alone. The purified protein (NS3-NS4A) was successfully employed in a validated FRET assay to evaluate 14 Citrus fruit extracts, revealing that the mesocarp extract of Citrus paradisi, and whole fruit extracts of C. sinesis, C. aurantinum, and C. reticulata significantly inhibited the protease activity of HCV NS3 protease (IC50 values of 5.79 ± 1.44 µg/mL, 37.19 ± 5.92 µg/mL, 42.62 ± 6.89 µg/mL, and 57.65 ± 3.81 µg/mL, respectively). Subsequent ESI-MSn analysis identified a flavonoid, hesperidin, abundantly present in all the afore-mentioned Citrus extracts. Importantly, docking studies suggested that hesperidin interacts with active site residues, and acts as a potent inhibitor of NS3 protease, exhibiting an IC50 value of 11.34 ± 3.83 µg/mL.ConclusionsA FRET assay was developed using NS3-NS4A protease, which was successfully utilized for the evaluation of Citrus fruit extracts. Hesperidin, a compound present in the Citrus extracts, was identified as the main flavonoid, which can serve as a cost-effective potent inhibitor of NS3 protease, and could be developed as a drug for antiviral therapy against HCV genotype 3a.
| S-EPMC8976278 | biostudies-literature
Project description:BackgroundHepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma and infects about 3% world population. Response to interferon therapy depends upon the genotype of the virus and factors associated with the host. Despite a good response to interferon therapy, a considerable number of genotype 3a infected patients remains unalleviated.ResultsIn total forty-nine patients including twenty-five non-responders (non-SVR) and twenty-four responders (SVR) were recruited. Patients were tested for viral status at different intervals and the isolated RNA was sequenced for the NS5A region in both groups. The comparison of PKRBD of HCV between the SVR and non-SVR patients did not confirm any significant difference in the number of mutations. However, when the sequence downstream to the PKRBD of NS5A was compared, two important statistically significant mutations were observed; at positions 2309 (Ala to Ser) and 2326 (Gly to Ala). These mutations were then analysed for tertiary protein structure and important structural changes were observed. Statistically significant difference was also observed when age groups of patients were compared; younger patients showed better response than the older ones.ConclusionsThe region between PKRBD and IRRDR may be important for prediction of response to IFN therapy for genotype 3a. ISDR and PKRBD have not shown any involvement in treatment response. Further functional analyses of these findings can help in understanding the involvement of the NS5A region in interferon treatment of HCV-3a infected patients.
| S-EPMC4029318 | biostudies-literature
Project description:The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) helicase is believed to be essential for viral replication and has become an attractive target for the development of antiviral drugs. The study of helicase is useful for elucidating its involvement in positive sense single-stranded RNA virus replication and to serve as templates for the design of novel antiviral drugs. In recent years, several models have been proposed on the conformational change leading to protein movement and RNA unwinding. Some compounds have been recently reported to inhibit the helicase and these include small molecules, RNA aptamers and antibodies. The current study is designed to help gain insights for the consideration of potential inhibitors for Pakistani HCV NS3 helicase protein. We have cloned, expressed and purified HCV NS3 helicase from Pakistani HCV serum samples and determined its 3D structure and employed it further in computational docking analysis to identify inhibitors against HCV genotype 3a (GT3a),including six antiviral key molecules such as quercetin, beta-carotene, resveratrol, catechins, lycopene and lutein. The conformation obtained after docking showed good hydrogen bond (HBond) interactions with best docking energy for quercetin and catechins followed by resveratrol and lutein. These anti-helicase key molecules will offer an alternative attraction to target the viral helicase, due to the current limitation with the interferon resistance treatment and presences of high rate of resistance in anti-protease inhibitor classes.
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Project description:BACKGROUND:Hepatitis C virus (HCV) is the cause of high morbidity and mortality worldwide, inflicting around one million people in Pakistan alone. The HCV genomic RNA harbors conserved structural elements that are indispensable for its replication. The 3' untranslated region (UTR) contains several of these elements essentially involved in regulating the major steps of the viral life cycle. OBJECTIVES:Differences in regulatory elements of HCV may contribute towards differential infectivity of local isolates. The present study explicates sequence analysis and secondary structure prediction of HCV 3'UTR region of subtype 3a from Pakistan to characterize this particular region. PATIENTS AND METHODS:HCV 3'UTR region was amplified, cloned and sequenced from five different patients. Sequence and structural analysis was performed and phylogenetic analysis was carried out using the 3'UTR sequence reported in NCBI nucleotide data base (http://www.ncbi.nlm.nih.gov/nuccore) by other studies. RESULTS:Sequence analysis of the amplified fragment from five patients indicated that the 3'UTR is composed of 214-235 nts. Its sequence contains a type-specific variable region followed by a poly U/UC region and a highly conserved X-tail of 98 nts. The variable region reported here has 26 nts and one stem loop at the secondary structure that differentiate it from HCV genotype 1a ( GT1a) 3'UTR which contains additional 14 nts and two stem loops. The poly U/UC region varied in length (100-79 nts) and nucleotide sequence within the Pakistani isolates, and among different genotypes. Some substitutions found in the X-tail do not affect secondary structure of this element suggesting that this region might play an important role in replication, stabilization and packaging of HCV genome. Additionally, U residues are not present at the end of the X-tail in Pakistani 3a isolates as otherwise reported for the variants of genotype 1b. CONCLUSIONS:Sequence and structural diversity of the 3'UTR variable region and Poly U/UC region found in the local isolates indicate specificity in the regulating elements of 3'UTR that might be associated with differential replication efficacy of the HCV Pakistani isolates. The study necessitates functional characterization of these regulating elements to elucidate variable viral efficiency and pathogenicity associated with inter-geographical isolates.
| S-EPMC3732663 | biostudies-literature
Project description:Chronic Hepatitis C Virus (HCV) infection is still a major health issue especially in endemic areas where fewer direct-acting virals (DAAs) are treatment options. Some HCV variants are associated with resistance and it reduces DAAs success where pre-existing variants prevail. In this study, we investigated resistance-associated polymorphisms (RAPs) in the HCV NS3 region from DAAs naïve Pakistani patients. 277 chronic HCV treatment naïve patients infected with genotype 1a, 3a and 3b were selected from various clinical centers in the capital city of Khyber Pakhtunkhwa province Pakistan. All the patients were included in this study after taking informed consent. HCV NS3 region was amplified and Sanger sequencing was performed to analyze RAPs to NS3 protease inhibitors. Of the total 29.24% (81/277) patients had detected with known RAPs viz V36A/G/L, T54S, V55A/D/I, Q80K/R, S122G/T/R, R155K/T/I, V158I, D168T/Q, and I170V. Among HCV-1a subjects overall RAPs found were 26.09% (12/46) and most prevalent substitutions were V36A/G (10.87%, 5/46) and R155K/T/I (8.70%, 4/46). Of the total HCV-3a infected patients, 30.95% were observed with RAPS. Ammon these, the most frequent substitutions were Q80R (13.69%, 23/168) followed by V36L (18.33%, 14/168) and V55I (5.95%, 10/168). Among HCV-3b patients, 26.98% were found with RAPs and S122R and Q80R were the dominant variants detected in 17.46 (11/63) and 12.70% (8/63) patients respectively. All these substitutions were associated with Boceprevir, Simeprevir, Telaprevir, and Paritaprevir. Single substitution in one sequence was found in 18.77% (52/277) and multiple in 10.46% (29/277). More than one RAP was frequent in HCV-3a sequences. Natural RAPs are common in chronic HCV patients infected with genotype 1a, 3a and 3b, the most prevalent subtypes in Pakistan. High prevalence of HCV NS3 RAPs suggested a large scale study of the NS3 gene before the introduction of NS3 protease inhibitors in Pakistan.
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Project description:BackgroundHepatitis C virus (HCV) genotype 3a is known to show comparatively better response to combination therapy than genotype 1 and 4. Mutations within NS5A gene of HCV have earlier been implicated with response to interferon (IFN) therapies in chronic HCV patients among various populations. As response to therapy are available in different populations because of the ethnic and viral factors and there was no study available on the phenomenon of resistivity to IFN.ResultsChronic HCV 3a infected Pakistani patients were kept on IFN-α and ribavirin therapy for six months. NS5A gene of HCV was amplified and sequenced in the case of all the patients prior to therapy and the sequences were analysed for mutations. Out of the total 27 patients, 20 (74.07%) were observed with sustained virological response (SVR), 4 (14.81%) patients were non responder (NR) while 3 (11.11%) patients exhibited in end of treatment response (ETR). Three (3/20) (15%) SVR patients and two (2/3) ETR patients had mutations (ranging from I-V amino acids) within the NS5A ISDR regions. While the rest of the SVR patients (85%) and the NR had no mutations at ISDR region when compared with HCV K3a ISDR.ConclusionsMutations within the NS5A gene of HCV 3a genotype may not influence the outcome of combination therapy in Pakistani populations.
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Project description:Asunaprevir (ASV), an isoquinoline-based competitive inhibitor targeting the hepatitis C virus (HCV) NS3/4A protease, is very potent in vivo. However, the potency is significantly compromised by the drug resistance mutations R155K and D168A. In this study three crystal structures of ASV and an analogue were determined to analyze the structural basis of drug resistance susceptibility. These structures revealed that ASV makes extensive contacts with Arg155 outside the substrate envelope. Arg155 in turn is stabilized by Asp168, and thus when either residue is mutated, the enzyme's interaction with ASV's P2* isoquinoline is disrupted. Adding a P1-P3 macrocycle to ASV enhances the inhibitor's resistance barrier, likely due to poising the inhibitor to its bound conformation. Macrocyclic inhibitors with P2* extension moieties avoiding interaction with the protease S2 residues including Arg155 must be chosen for future design of more robust protease inhibitors.
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Project description:Hepatitis C virus (HCV) is a major health problem in developing countries including Pakistan. Chronic HCV infection results in progressive liver disease including fibrosis, cirrhosis, insulin resistance and eventually hepatocellular carcinoma (HCC). Ionotrophic purinergic (P2X) receptors are identified to involve in a spectrum of physiological and pathophysiological processes. However, the role of P2X receptors in HCV liver associated diseases still remains to be investigated. The current study was designed to identify the presence of P2X receptors in human liver cells. Furthermore, it investigates the response of P2X receptors towards HCV structural proteins (E1E2). To determine that how many isoforms of P2X receptors are expressed in human liver cells, human hepatoma cell line (Huh-7) was used. Transcripts (mRNA) of five different isoforms of P2X receptors were identified in Huh-7 cells. To examine the gene expression of identified isoforms of P2X receptors in presence of HCV structural proteins E1E2, Huh-7/E1E2 cell line (stably expressing HCV structural proteins E1E2) was used. The results showed significant increase (6.2 fold) in gene expression of P2X4 receptors in Huh-7/E1E2 cells as compared to control Huh-7 cells. The findings of present study confirmed the presence of transcripts of five different isoforms of P2X receptors in human liver cells and suggest that P2X4 receptors could be represented an important component of the purinergic signaling complex in HCV induced liver pathogenesis.
| S-EPMC3177911 | biostudies-literature
Project description:BACKGROUND: Hepatitis C virus (HCV) is a common and leading cause for liver cirrhosis and hepatocellular carcinoma. Current therapies to treat HCV infection are shown to be partially effective and poorly tolerated. Therefore, ample efforts are underway to rationally design therapies targeting the HCV non-structural proteins. Most of the work carried out in this direction has been focusing mainly on HCV genotype 1. Two direct-acting antiviral agents (DAAs) Telaprevir and Boceprevir are being used against genotype 1a infection in combination therapy with interferon and ribavirin. Unfortunately these DAAs are not effective against genotype 3a. Considering the wide spread infection by HCV genotype 3a in developing countries especially South Asia, we have focused on the recombinant production of antiviral drug targets NS3 and NS5A from HCV genotype 3a. These protein targets are to be used for screening of inhibitors. RESULTS: High-level expression of NS3 and NS5A was achieved at 25°C, using ~1 and 0.5 mM Isopropyl ?-D-1-thiogalactopyranoside (IPTG), respectively. Yields of the purified NS3 and NS5A were 4 and 1 mg per liter culture volume, respectively. Although similar amounts of purified NS3 were obtained at 25 and 14°C, specificity constant (Kcat/Km) was somewhat higher at expression temperature of 25°C. Circular dichroism (CD) and Fourier-transform infrared (FT-IR) spectroscopy revealed that both NS3 and NS5A contain a mixture of alpha-helix and beta-sheet secondary structures. For NS3 protein, percentages of secondary structures were similar to the values predicted from homology modeling. CONCLUSIONS: NS3 and NS5A were over-expressed and using Nickel-affinity method both proteins were purified to?~?95% purity. Yield of the purified NS3 obtained is four fold higher than previous reports. CD spectroscopy revealed that difference in activity of NS3 expressed at various temperatures is not related to changes in global structural features of the protein. Moreover, CD and FT-IR analysis showed that NS3 and NS5A contain both alpha-helical and beta-sheet structures and for NS5A, the proportion is almost equal. The production of NS3 and NS5A in milligram quantities will allow their characterization by biophysical and biochemical means that will help in designing new strategies to fight against HCV infection.
| S-EPMC3842787 | biostudies-literature
Project description:BACKGROUND:Hepatitis C virus (HCV) genotype-3a infection is now the dominant strain in South Asia and the UK. Characteristic features include a favourable response to therapy; the reasons for this are unknown but may include distinct genotype-3a-specific T cell immunity. In contrast to genotype-1 infection, T cell immunity to this subtype is poorly defined. OBJECTIVES:The aims of the study were to (1) define the frequency, specificity and cross-reactivity of T cell immunity across the whole viral genome in genotype-3a infection and (2) assess the impact of interferon (IFN)-?/ribavirin on T cell immunity. DESIGN:T cell responses in chronic and resolved HCV genotype-3a were analysed in comparison with genotype-1 infection (total n=85) using specific peptide panels in IFN-? ELISpot assays. T cell responses were followed longitudinally in a subset of genotype-3a infected patients receiving therapy. Responses were further defined by CD4 and CD8 subset analysis, sequencing of autologous virus and cross-reactivity of genotype-3a with genotype-1a/-1b antigens. RESULTS:CD8 T cell responses commonly targeted the non-structural (NS) proteins in chronic genotype-3a infection whereas in genotype-1 infection CD4 responses targeting HCV core predominated (p=0.0183). Resolved infection was associated with CD4 T cells targeting NS proteins. Paradoxically, a sustained response to therapy was associated with a brisk decline in virus-specific and total lymphocyte counts that recovered after treatment. CONCLUSION:HCV genotype-3a exhibits a distinct T cell specificity with implications for vaccine design. However, our data do not support the theory that genotype-3a viral clearance with therapy is associated with an enhanced antiviral T cell response. Paradoxically, a reduction in these responses may serve as a biomarker of IFN responsiveness.
| S-EPMC3733295 | biostudies-literature