Journal articles on the topic 'NS5b'
To see the other types of publications on this topic, follow the link: NS5b.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'NS5b.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Wong, Mun-Teng, and Steve S. Chen. "Human Choline Kinase-α Promotes Hepatitis C Virus RNA Replication through Modulation of Membranous Viral Replication Complex Formation." Journal of Virology 90, no. 20 (August 3, 2016): 9075–95. http://dx.doi.org/10.1128/jvi.00960-16.
Full textAbstract:
ABSTRACTHepatitis C virus (HCV) infection reorganizes cellular membranes to create an active viral replication site named the membranous web (MW). The role that human choline kinase-α (hCKα) plays in HCV replication remains elusive. Here, we first showed that hCKα activity, not the CDP-choline pathway, promoted viral RNA replication. Confocal microscopy and subcellular fractionation of HCV-infected cells revealed that a small fraction of hCKα colocalized with the viral replication complex (RC) on the endoplasmic reticulum (ER) and that HCV infection increased hCKα localization to the ER. In the pTM-NS3-NS5B model, NS3-NS5B expression increased the localization of the wild-type, not the inactive D288A mutant, hCKα on the ER, and hCKα activity was required for effective trafficking of hCKα and NS5A to the ER. Coimmunoprecipitation showed that hCKα was recruited onto the viral RC presumably through its binding to NS5A domain 1 (D1). hCKα silencing or treatment with CK37, an hCKα activity inhibitor, abolished HCV-induced MW formation. In addition, hCKα depletion hindered NS5A localization on the ER, interfered with NS5A and NS5B colocalization, and mitigated NS5A-NS5B interactions but had no apparent effect on NS5A-NS4B and NS4B-NS5B interactions. Nevertheless, hCKα activity was not essential for the binding of NS5A to hCKα or NS5B. These findings demonstrate that hCKα forms a complex with NS5A and that hCKα activity enhances the targeting of the complex to the ER, where hCKα protein, not activity, mediates NS5A binding to NS5B, thereby promoting functional membranous viral RC assembly and viral RNA replication.IMPORTANCEHCV infection reorganizes the cellular membrane to create an active viral replication site named the membranous web (MW). Here, we report that human choline kinase-α (hCKα) acts as an essential host factor for HCV RNA replication. A fraction of hCKα colocalizes with the viral replication complex (RC) on the endoplasmic reticulum (ER) in HCV-infected cells. NS3-NS5B expression increases ER localization of wild-type, but not D288A mutant, hCKα, and hCKα activity facilitates the transport of itself and NS5A to the ER. Silencing or inactivation of hCKα abrogates MW formation. Moreover, hCKα is recruited by NS5A independent of hCKα activity, presumably through binding to NS5A D1. hCKα activity then mediates the ER targeting of the hCKα-NS5A complex. On the ER membrane, hCKα protein,per se, induces NS5A binding to NS5B, thereby promoting membranous RC formation and viral RNA replication. Our study may benefit the development of hCKα-targeted anti-HCV therapeutics.
2
Chatterji, Udayan, Michael Bobardt, Andrew Tai, Malcolm Wood, and Philippe A. Gallay. "Cyclophilin and NS5A Inhibitors, but Not Other Anti-Hepatitis C Virus (HCV) Agents, Preclude HCV-Mediated Formation of Double-Membrane-Vesicle Viral Factories." Antimicrobial Agents and Chemotherapy 59, no. 5 (February 9, 2015): 2496–507. http://dx.doi.org/10.1128/aac.04958-14.
Full textAbstract:
ABSTRACTAlthough the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.
3
Sheng, Chun, Jing Wang, Jing Xiao, Jun Xiao, Yan Chen, Lin Jia, Yimiao Zhi, Guangyuan Li, and Ming Xiao. "Classical swine fever virus NS5B protein suppresses the inhibitory effect of NS5A on viral translation by binding to NS5A." Journal of General Virology 93, no. 5 (May 1, 2012): 939–50. http://dx.doi.org/10.1099/vir.0.039495-0.
Full textAbstract:
In order to investigate molecular mechanisms of internal ribosome entry site (IRES)-mediated translation in classical swine fever virus (CSFV), an important pathogen of pigs, the expression level of NS3 was evaluated in the context of genomic RNAs and reporter RNA fragments. All data showed that the NS5A protein has an inhibitory effect on IRES-mediated translation and that NS5B proteins suppress the inhibitory effect of NS5A on viral translation, but CSFV NS5B GDD mutants do not. Furthermore, glutathione S-transferase pull-down assay and immunoprecipitation analysis, associated with deletion and alanine-scanning mutations, were performed. Results showed that NS5B interacts with NS5A and that the region aa 390–414, located in the C-terminal half of NS5A, is important for binding of NS5B to NS5A. Furthermore, amino acids K399, T401, E406 and L413 in the region were found to be essential for NS5A–NS5B interaction, virus rescue and infection. The above-mentioned region and four amino acids were observed to overlap with the site responsible for inhibition of IRES-mediated translation by the NS5A protein. We also found that aa 63–72, aa 637–653 and the GDD motif of NS5B were necessary for the interaction between NS5A and NS5B. These findings suggest that the repression activity of the NS5B protein toward the role of NS5A in translation might be achieved by NS5A–NS5B interaction, for which aa 390–414 of NS5A and aa 63–72, aa 637–653 and the GDD motif of NS5B are indispensable. This is important for understanding the role of NS5A–NS5B interaction in the virus life cycle.
4
Gao, Lu, Hideki Aizaki, Jian-Wen He, and Michael M. C. Lai. "Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft." Journal of Virology 78, no. 7 (April 1, 2004): 3480–88. http://dx.doi.org/10.1128/jvi.78.7.3480-3488.2004.
Full textAbstract:
ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.
5
Choi, Soo-Ho, Kyu-Jin Park, Byung-Yoon Ahn, Guhung Jung, Michael M. C. Lai, and Soon B. Hwang. "Hepatitis C Virus Nonstructural 5B Protein Regulates Tumor Necrosis Factor Alpha Signaling through Effects on Cellular IκB Kinase." Molecular and Cellular Biology 26, no. 8 (April 15, 2006): 3048–59. http://dx.doi.org/10.1128/mcb.26.8.3048-3059.2006.
Full textAbstract:
ABSTRACT Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNA-dependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-α)-induced NF-κB and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-α-induced NF-κB and JNK activation. In this study, we have demonstrated that TNF-α-induced NF-κB activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2- and IKK-induced NF-κB activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKKα. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKKα, and then TNF-α-mediated IKKα kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-α-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-α signaling pathways and may contribute to HCV pathogenesis.
6
Grassmann, Claus W., Olaf Isken, Norbert Tautz, and Sven-Erik Behrens. "Genetic Analysis of the Pestivirus Nonstructural Coding Region: Defects in the NS5A Unit Can Be Complemented intrans." Journal of Virology 75, no. 17 (September 1, 2001): 7791–802. http://dx.doi.org/10.1128/jvi.75.17.7791-7802.2001.
Full textAbstract:
ABSTRACT The functional analysis of molecular determinants which control the replication of pestiviruses was considerably facilitated by the finding that subgenomic forms of the positive-strand RNA genome of BVDV (bovine viral diarrhea virus) are capable of autonomous replication in transfected host cells. The prototype replicon, BVDV DI9c, consists of the genomic 5′ and 3′ untranslated regions and a truncated open reading frame (ORF) encoding mainly the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To gain insight into which of these proteins are essential for viral replication and whether they act in cisor in trans, we introduced a large spectrum of in-frame mutations into the DI9c ORF. Tests of the mutant RNAs in terms of their replication capacity and their ability to support translation and cleavage of the nonstructural polyprotein, and whether defects could be rescued in trans, yielded the following results. (i) RNA replication was found to be dependent on the expression of each of the DI9c-encoded mature proteins NS3 to NS5B (and the known associated enzymatic activities). In the same context, a finely balanced molar ratio of the diverse proteolytic processing products was indicated to be crucial for the formation of an active catalytic replication complex. (ii) Synthesis of negative-strand intermediate and progeny positive-strand RNA was observed to be strictly coupled with all functional DI9c ORF derivatives. NS3 to NS5B were hence suggested to play a pivotal role even during early steps of the viral replication pathway. (iii) Mutations in the NS3 and NS4B units which generated nonfunctional or less functional RNAs were determined to becis dominant. Likewise, lethal alterations in the NS4A and NS5B regions were invariably noncomplementable. (iv) In surprising contrast, replication of functional and nonfunctional NS5A mutants could be clearly enhanced and restored, respectively. In summary, our data provide initial insights into the organization of the pestivirus replication machinery.
7
Shimakami, Tetsuro, Makoto Hijikata, Hong Luo, Yuan Yuan Ma, Shuichi Kaneko, Kunitada Shimotohno, and Seishi Murakami. "Effect of Interaction between Hepatitis C Virus NS5A and NS5B on Hepatitis C Virus RNA Replication with the Hepatitis C Virus Replicon." Journal of Virology 78, no. 6 (March 15, 2004): 2738–48. http://dx.doi.org/10.1128/jvi.78.6.2738-2748.2004.
Full textAbstract:
ABSTRACT Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.
8
Svitkin, Yuri V., Arnim Pause, Marcelo Lopez-Lastra, Sandra Perreault, and Nahum Sonenberg. "Complete Translation of the Hepatitis C Virus Genome In Vitro: Membranes Play a Critical Role in the Maturation of All Virus Proteins except for NS3." Journal of Virology 79, no. 11 (June 1, 2005): 6868–81. http://dx.doi.org/10.1128/jvi.79.11.6868-6881.2005.
Full textAbstract:
ABSTRACT We developed an in vitro translation extract from Krebs-2 cells that translates the entire open reading frame of the hepatitis C virus (HCV) strain H77 and properly processes the viral protein precursors when supplemented with canine microsomal membranes (CMMs). Translation of the C-terminal portion of the viral polyprotein in this system is documented by the synthesis of NS5B. Evidence for posttranslational modification of the viral proteins, the N-terminal glycosylation of E1 and the E2 precursor (E2-p7), and phosphorylation of NS5A is presented. With the exception of NS3, efficient generation of all virus-specific proteins is CMM dependent. A time course of the appearance of HCV products indicates that the viral polyprotein is cleaved cotranslationally. A competitive inhibitor of the NS3 protease inhibited accumulation of NS3, NS4B, NS5A, and NS5B, but not that of NS2 or structural proteins. CMMs also stabilized HCV mRNA during translation. Finally, the formyl-[35S]methionyl moiety of the initiator tRNAMet was incorporated exclusively into the core protein portion of the polyprotein, demonstrating that translation initiation in this system occurs with high fidelity.
9
Nutt, Jamie, James Grantham, Marilyn Smith, Emily Smith, Ashley Wedin, Aaron Tyler, Mauricio Miralles, Steve Kleiboeker, and Mark Wissel. "Performance of NS3, NS5a AND NS5b Hepatitis C Virus (HCV) Antiviral Resistance Sequencing Assays." Open Forum Infectious Diseases 4, suppl_1 (2017): S40. http://dx.doi.org/10.1093/ofid/ofx162.097.
Full textAbstract:
Abstract Background HCV genotype 1a (HCV g1a) followed by HCV g1b, –g2, and –g3 are the most common etiologic agents in the ~3 million current HCV-infections in the US. To achieve effective therapy, antiviral drug resistance testing is often essential but not fully available. Knowledge of both the genotype and the presence of HCV mutations in the genes of the major drug targets (NS3, NS5A and NS5B) can assist in optimal treatment selection. Methods The HCV genotype of 1000 HCV positive clinical plasmas and sera was determined (HCVg Direct, GenMark). Ten independent Sanger sequencing assays detecting antiviral drug resistance mutations in the genes encoding NS3, NS5a, and NS5b were developed. Six of these assays address mutations in all three genes in the two most common genotypes (HCV g1a and g1b). In addition, four more assays address mutations in NS5a and NS5b of HCV g2 and g3. These mutations are resistance determinants against 11 anti-HCV drugs as shown in Figure 1. A streamlined workflow employs conventional reverse transcriptase PCR, gel electrophoresis, spectrophotometry, bi-directional Sanger sequencing and reporting. The assays were designed to cover hot spot regions and capturing all known resistance mutations in NS3, NS5a and NS5b. Results Consistent with previous US HCV incidence reports, g1a, g1b, g2, and g3 comprised 99% of 1000 sequentially tested HCV patient specimens (62%, 12%, 11%, and 14%, respectively). Testing of more than 20 clinical samples each for g1a, g1b, g2, and g3 resulted in successful detection of NS3, NS5a, and NS5b mutations that confer drug resistance. The design successfully permitted detection of relevant mutations known to date for all 11 drugs. The number of reportable mutations range from 20 ‒ 36, 9 ‒ 49, and 10 ‒ 29 for the NS3, NS5a, and NS5b inhibitors, respectively (Figure 1). Conclusion These assays provide the most comprehensive commercially available antiviral drug resistance information to date for mutations in HCV NS3, NS5A, and NS5B. This testing will assist physicians in deciding on the most appropriate treatment options for their patients. Disclosures J. Nutt, Viracor Eurofins Laboratories: Employee, Salary; 
J. Grantham, Viracor Eurofins Laboratories: Employee, Salary; M. Smith, Viracor Eurofins Laboratories: Employee, Salary; E. Smith, Viracor Eurofins Laboratories: Employee, Salary; A. Wedin, Viracor Eurofins Laboratories: Employee, Salary; 
A. Tyler, Viracor Eurofins Laboratories: Employee, Salary; M. Miralles, Viracor Eurofins Laboratories: Employee, Salary; S. Kleiboeker, Viracor Eurofins Laboratories: Employee, Salary; M. Wissel, Viracor Eurofins Laboratories: Employee, Salary
10
Aldunate, Fabián, Natalia Echeverría, Daniela Chiodi, Pablo López, Adriana Sánchez-Cicerón, Alvaro Fajardo, Martín Soñora, Juan Cristina, Nelia Hernández, and Pilar Moreno. "Pretreatment Hepatitis C Virus NS5A/NS5B Resistance-Associated Substitutions in Genotype 1 Uruguayan Infected Patients." Disease Markers 2018 (August 14, 2018): 1–9. http://dx.doi.org/10.1155/2018/2514901.
Full textAbstract:
Hepatitis C Virus (HCV) infection treatment has dramatically changed with the advent of direct-acting antiviral agents (DAAs). However, the efficacy of DAAs can be attenuated by the presence of resistance-associated substitutions (RASs) before and after treatment. Indeed, RASs detected in DAA treatment-naïve HCV-infected patients could be useful for clinical management and outcome prediction. Although the frequency of naturally occurring HCV NS5A and NS5B RASs has been addressed in many countries, there are only a few reports on their prevalence in the South American region. The aim of this study was to investigate the presence of RASs to NS5A and NS5B inhibitors in a DAA treatment naïve cohort of Uruguayan patients infected with chronic hepatitis C and compare them with reports from other South American countries. Here, we found that naturally occurring substitutions conferring resistance to NS5A and NS5B inhibitors were present in 8% and 19.2%, respectively, of treatment-naïve HCV genotype 1 infected patients. Importantly, the baseline substitutions in NS5A and NS5B herein identified differ from the studies previously reported in Brazil. Furthermore, Uruguayan strains subtype 1a clustered within all major world clades, showing that HCV variants currently circulating in this country are characterized by a remarkable genetic diversity.
11
Hamamoto, Itsuki, Yorihiro Nishimura, Toru Okamoto, Hideki Aizaki, Minyi Liu, Yoshio Mori, Takayuki Abe, et al. "Human VAP-B Is Involved in Hepatitis C Virus Replication through Interaction with NS5A and NS5B." Journal of Virology 79, no. 21 (November 1, 2005): 13473–82. http://dx.doi.org/10.1128/jvi.79.21.13473-13482.2005.
Full textAbstract:
ABSTRACT The hepatitis C virus (HCV) nonstructural protein (NS) 5A is a phosphoprotein that associates with various cellular proteins and participates in the replication of the HCV genome. Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) is known to be a host factor essential for HCV replication by binding to both NS5A and NS5B. To obtain more information on the NS5A protein in HCV replication, we screened human brain and liver libraries by a yeast two-hybrid system using NS5A as bait and identified VAP-B as an NS5A-binding protein. Immunoprecipitation and mutation analyses revealed that VAP-B binds to both NS5A and NS5B in mammalian cells and forms homo- and heterodimers with VAP-A. VAP-A interacts with VAP-B through the transmembrane domain. NS5A interacts with the coiled-coil domain of VAP-B via 70 residues in the N-terminal and 341 to 344 amino acids in the C-terminal polyproline cluster region. NS5A was colocalized with VAP-B in the endoplasmic reticulum and Golgi apparatus. The specific antibody to VAP-B suppressed HCV RNA replication in a cell-free assay. Overexpression of VAP-B, but not of a mutant lacking its transmembrane domain, enhanced the expression of NS5A and NS5B and the replication of HCV RNA in Huh-7 cells harboring a subgenomic replicon. In the HCV replicon cells, the knockdown of endogenous VAP-B by small interfering RNA decreased expression of NS5B, but not of NS5A. These results suggest that VAP-B, in addition to VAP-A, plays an important role in the replication of the HCV genome.
12
Foster, Toshana L., Tamara Belyaeva, Nicola J. Stonehouse, Arwen R. Pearson, and Mark Harris. "All Three Domains of the Hepatitis C Virus Nonstructural NS5A Protein Contribute to RNA Binding." Journal of Virology 84, no. 18 (June 30, 2010): 9267–77. http://dx.doi.org/10.1128/jvi.00616-10.
Full textAbstract:
ABSTRACT The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3′ untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3′ UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3′ UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3′ UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3′ UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.
13
Binder, Marco, Doris Quinkert, Olga Bochkarova, Rahel Klein, Nikolina Kezmic, Ralf Bartenschlager, and Volker Lohmann. "Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras." Journal of Virology 81, no. 10 (March 7, 2007): 5270–83. http://dx.doi.org/10.1128/jvi.00032-07.
Full textAbstract:
ABSTRACT The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant nonstructural protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.
14
Cheng, Ju-Chien, Ming-Fu Chang, and Shin C. Chang. "Specific Interaction between the Hepatitis C Virus NS5B RNA Polymerase and the 3′ End of the Viral RNA." Journal of Virology 73, no. 8 (August 1, 1999): 7044–49. http://dx.doi.org/10.1128/jvi.73.8.7044-7049.1999.
Full textAbstract:
ABSTRACT Hepatitis C virus (HCV) NS5B protein is the viral RNA-dependent RNA polymerase capable of directing RNA synthesis. In this study, an electrophoretic mobility shift assay demonstrated the interaction between a partially purified recombinant NS5B protein and a 3′ viral genomic RNA with or without the conserved 98-nucleotide tail. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA but not by the viral 5′ noncoding region and very poorly by the 3′ conserved 98-nucleotide tail. A 3′ coding region with conserved stem-loop structures rather than the 3′ noncoding region of the HCV genome is critical for the specific binding of NS5B. Nevertheless, no direct interaction between the 3′ coding region and the HCV NS5A protein was detected. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1, from amino acid residues 83 to 194, and RBD2, from residues 196 to 298. Interestingly, the conserved motifs of RNA-dependent RNA polymerase for putative RNA binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. Nevertheless, the RNA-binding activity of RBD2 was abolished when it was linked to the carboxy-terminal half of the NS5B. These results provide some clues to understanding the initiation of HCV replication.
15
Marascio, Nadia, Angela Costantino, Stefania Taffon, Alessandra Lo Presti, Michele Equestre, Roberto Bruni, Giulio Pisani, et al. "Phylogenetic and Molecular Analyses of More Prevalent HCV1b Subtype in the Calabria Region, Southern Italy." Journal of Clinical Medicine 10, no. 8 (April 13, 2021): 1655. http://dx.doi.org/10.3390/jcm10081655.
Full textAbstract:
Hepatitis C virus subtype 1b (HCV1b) is still the most prevalent subtype worldwide, with massive expansion due to poor health care standards, such as blood transfusion and iatrogenic procedures. Despite safe and effective new direct antiviral agents (DAA), treatment success can depend on resistance-associated substitutions (RASs) carried in target genomic regions. Herein we investigated transmission clusters and RASs among isolates from HCV1b positive subjects in the Calabria Region. Forty-one NS5B and twenty-two NS5A sequences were obtained by Sanger sequencing. Phylogenetic analysis was performed using the maximum likelihood method and resistance substitutions were analyzed with the Geno2pheno tool. Phylogenetic analysis showed sixteen statistically supported clusters, with twelve containing Italian sequences mixed with foreign HCV1b isolates and four monophyletic clusters including only sequences from Calabria. Interestingly, HCV1b spread has been maintained by sporadic infections in geographically limited areas and by dental treatment or surgical intervention in the metropolitan area. The L159F NS5B RAS was found in 15 isolates and in particular 8/15 also showed the C316N substitution. The Y93H and L31M NS5A RASs were detected in three and one isolates, respectively. The A92T NS5A RAS was found in one isolate. Overall, frequencies of detected NS5B and NS5A RASs were 36.6% and 22.7%, respectively. For the eradication of infection, improved screening policies should be considered and the prevalence of natural RASs carried on viral strains.
16
Vajdy, Michael, Mark Selby, Angelica Medina-Selby, Doris Coit, John Hall, Laura Tandeske, David Chien, et al. "Hepatitis C virus polyprotein vaccine formulations capable of inducing broad antibody and cellular immune responses." Journal of General Virology 87, no. 8 (August 1, 2006): 2253–62. http://dx.doi.org/10.1099/vir.0.81849-0.
Full textAbstract:
Although approximately 3 % of the world's population is infected with Hepatitis C virus (HCV), there is no prophylactic vaccine available. This study reports the design, cloning and purification of a single polyprotein comprising the HCV core protein and non-structural proteins NS3, NS4a, NS4b, NS5a and NS5b. The immunogenicity of this polyprotein, which was formulated in alum, oil-in-water emulsion MF59 or poly(dl-lactide co-glycolide) in the presence or absence of CpG adjuvant, was then determined in a murine model for induction of B- and T-cell responses. The addition of adjuvants or a delivery system to the HCV polyprotein enhanced serum antibody and T-cell proliferative responses, as well as IFN-γ responses, by CD4+ T cells. The antibody responses were mainly against the NS3 and NS5 components of the polyprotein and relatively poor responses were elicited against NS4 and the core components. IFN-γ responses, however, were induced against all of the individual components of the polyprotein. These data suggest that the HCV polyprotein delivered with adjuvants induces broad B- and T-cell responses and could be a vaccine candidate against HCV.
17
Martin, Morgan M., Stephanie A. Condotta, Jeremy Fenn, Andrea D. Olmstead, and François Jean. "In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile." Biological Chemistry 392, no. 10 (October 1, 2011): 927–35. http://dx.doi.org/10.1515/bc.2011.076.
Full textAbstract:
AbstractThe need to identify anti-Flaviviridaeagents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.
18
Ikeda, Masanori, MinKyung Yi, Kui Li, and Stanley M. Lemon. "Selectable Subgenomic and Genome-Length Dicistronic RNAs Derived from an Infectious Molecular Clone of the HCV-N Strain of Hepatitis C Virus Replicate Efficiently in Cultured Huh7 Cells." Journal of Virology 76, no. 6 (March 15, 2002): 2997–3006. http://dx.doi.org/10.1128/jvi.76.6.2997-3006.2002.
Full textAbstract:
ABSTRACT Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005→I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.
19
Ku, Karin S., Ramakrishna K. Chodavarapu, Ross Martin, Michael D. Miller, Hongmei Mo, and Evguenia S. Svarovskaia. "Sequencing Analysis of NS3/4A, NS5A, and NS5B Genes from Patients Infected with Hepatitis C Virus Genotypes 5 and 6." Journal of Clinical Microbiology 54, no. 7 (May 4, 2016): 1835–41. http://dx.doi.org/10.1128/jcm.00238-16.
Full textAbstract:
Direct-acting antivirals (DAAs) with activity against multiple genotypes of the hepatitis C virus (HCV) were recently developed and approved for standard-of-care treatment. However, sequencing assays to support HCV genotype 5 and 6 analysis are not widely available. Here, we describe the development of a sequencing assay for the NS3/4A, NS5A, and NS5B genes from HCV genotype 5 and 6 patient isolates. Genotype- and subtype-specific primers were designed to target NS3/4A, NS5A, and NS5B for cDNA synthesis and nested PCR amplification. Amplification was successfully performed for a panel of 32 plasma samples from HCV-infected genotype 5 and 6 patients with sequencing data obtained for all attempted samples. LiPA 2.0 (Versant HCV genotype 2.0) is a reverse hybridization line probe assay that is commonly used for genotyping HCV-infected patients enrolled in clinical studies. Using NS3/4A, NS5A, and NS5B consensus sequences, HCV subtypes were determined that were not available for the initial LiPA 2.0 result for genotype 6 samples. Samples amplified here included the following HCV subtypes: 5a, 6a, 6e, 6f, 6j, 6i, 6l, 6n, 6o, and 6p. The sequencing data generated allowed for the determination of the presence of variants at amino acid positions previously characterized as associated with resistance to DAAs. The simple and robust sequencing assay for genotypes 5 and 6 presented here may lead to a better understanding of HCV genetic diversity and prevalence of resistance-associated variants.
20
Liang, Yisha, Guigen Zhang, Qiheng Li, Lin Han, Xiaoyou Hu, Yu Guo, Wanyin Tao, et al. "TRIM26 is a critical host factor for HCV replication and contributes to host tropism." Science Advances 7, no. 2 (January 2021): eabd9732. http://dx.doi.org/10.1126/sciadv.abd9732.
Full textAbstract:
Hepatitis C virus (HCV) remains a major human pathogen that requires better understanding of virus-host interactions. In this study, we performed a genome-wide CRISPR-Cas9 screening and identified TRIM26, an E3 ligase, as a critical HCV host factor. Deficiency of TRIM26 specifically impairs HCV genome replication. Mechanistic studies showed that TRIM26 interacts with HCV-encoded NS5B protein and mediates its K27-linked ubiquitination at residue K51, and thus promotes the NS5B-NS5A interaction. Moreover, mouse TRIM26 does not support HCV replication because of its unique six–amino acid insert that prevents its interaction with NS5B. Ectopic expression of human TRIM26 in a mouse hepatoma cell line that has been reconstituted with other essential HCV host factors promotes HCV infection. In conclusion, we identified TRIM26 as a host factor for HCV replication and a new determinant of host tropism. These results shed light on HCV-host interactions and may facilitate the development of an HCV animal model.
21
Lang Kuhs, Krystle A., Roberta Toporovski, Arielle A. Ginsberg, Devon J. Shedlock, and David B. Weiner. "Induction of Intrahepatic HCV NS4B, NS5A and NS5B-Specific Cellular Immune Responses following Peripheral Immunization." PLoS ONE 7, no. 12 (December 21, 2012): e52165. http://dx.doi.org/10.1371/journal.pone.0052165.
Full text
22
Krishnan, Preethi, Rakesh Tripathi, Gretja Schnell, Thomas Reisch, Jill Beyer, Michelle Irvin, Wangang Xie, et al. "Resistance Analysis of Baseline and Treatment-Emergent Variants in Hepatitis C Virus Genotype 1 in the AVIATOR Study with Paritaprevir-Ritonavir, Ombitasvir, and Dasabuvir." Antimicrobial Agents and Chemotherapy 59, no. 9 (June 22, 2015): 5445–54. http://dx.doi.org/10.1128/aac.00998-15.
Full textAbstract:
ABSTRACTAVIATOR, a phase 2 clinical trial, evaluated ritonavir-boosted paritaprevir (a protease inhibitor), ombitasvir (an NS5A inhibitor), and dasabuvir (a nonnucleoside polymerase inhibitor) (the three-drug [3D] regimen) with or without ribavirin (RBV) for 8, 12, or 24 weeks in 406 HCV genotype 1 (GT1)-infected patients. The rate of sustained virologic response 24 weeks after treatment ranged from 88% to 100% across the arms of the 3D regimen with or without RBV; 20 GT1a-infected patients and 1 GT1b-infected patient experienced virologic failure (5.2%). Baseline resistance-conferring variants in NS3 were rare. M28V in GT1a and Y93H in GT1b were the most prevalent preexisting variants in NS5A, and C316N in GT1b and S556G in both GT1a and GT1b were the most prevalent variants in NS5B. Interestingly, all the GT1a sequences encoding M28V in NS5A were from the United States, while GT1b sequences encoding C316N and S556G in NS5B were predominant in the European Union. Variants preexisting at baseline had no significant impact on treatment outcome. The most prevalent treatment-emergent resistance-associated variants (RAVs) in GT1a were R155K and D168V in NS3, M28T and Q30R in NS5A, and S556G in NS5B. The single GT1b-infected patient experiencing virologic failure had no RAVs in any target. A paritaprevir-ritonavir dose of 150/100 mg was more efficacious in suppressing R155K in NS3 than a 100/100-mg dose. In patients who failed after receiving 12 or more weeks of treatment, RAVs were selected in all 3 targets, while most patients who relapsed after 8 weeks of treatment did so without any detectable RAVs. Results from this study guided the selection of the optimal treatment regimen, treatment duration, and paritaprevir dose for further development of the 3D regimen. (This study has been registered at ClinicalTrials.gov under registration number NCT01464827.)
23
Bukhtiyarova, Marina, Christopher J. Rizzo, Charles A. Kettner, Bruce D. Korant, Helen T. Scarnati, and Robert W. King. "Inhibition of the Bovine Viral Diarrhoea Virus NS3 Serine Protease by a Boron-Modified Peptidyl Mimetic of its Natural Substrate." Antiviral Chemistry and Chemotherapy 12, no. 6 (December 2001): 367–73. http://dx.doi.org/10.1177/095632020101200607.
Full textAbstract:
Bovine viral diarrhoea virus (BVDV) is closely related to hepatitis C virus (HCV), and has been used as a surrogate virus in drug development for HCV infection. Similar to HCV, BVDV-encoded NS3 serine proteinase is responsible for multiple cleavages in the viral polyprotein, generating mature NS4A, NS4B, NS5A and NS5B proteins. NS3-dependent cleavage sites of BVDV contain a strictly conserved leucine at P1, and either serine or alanine at P1′. The full length BVDV NS3/4A serine protease has been cloned and expressed in bacterial cells. The enzyme has been purified from the soluble portion of Escherichia coli via a two-step purification procedure employing chromatography on heparin resin and gel filtration. The protease activity was characterized using in vitro translated BVDV NS4A/B and NS5A/B polyprotein substrates. A boronic acid analogue of the BVDV NS4A/NS4B cleavage site was synthesized and shown to be an efficient inhibitor of the NS3 serine protease in vitro. The compound, designated DPC-AB9144–00, inhibited approximately 75% of the NS3/4 activity at 10 μM with the NS4A/B substrate. However, no antiviral activity was detected with DPC-AB9144–00 in BVDV-infected Madin—Darby bovine kidney cells at concentrations as great as 90 μM, suggesting permeability or that other cellular-derived limitations were present.
24
Graziani, Rita, and Giacomo Paonessa. "Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon." Journal of General Virology 85, no. 7 (July 1, 2004): 1867–75. http://dx.doi.org/10.1099/vir.0.80006-0.
Full textAbstract:
An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture. It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B. However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins. The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons. This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture. With the use of a β-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.
25
Friborg, Jacques, Steven Levine, Chaoqun Chen, Amy K. Sheaffer, Susan Chaniewski, Stacey Voss, Julie A. Lemm, and Fiona McPhee. "Combinations of Lambda Interferon with Direct-Acting Antiviral Agents Are Highly Efficient in Suppressing Hepatitis C Virus Replication." Antimicrobial Agents and Chemotherapy 57, no. 3 (December 28, 2012): 1312–22. http://dx.doi.org/10.1128/aac.02239-12.
Full textAbstract:
ABSTRACTThe clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-λ1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alfa interferon (referred to herein as alfa). In the study described in this report, an assessment of thein vitroantiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-λ1 (rIFN-λ1) exerted the most robust effect, while rIFN-λ3 exhibited greater activity than rIFN-λ2. More importantly, cross-resistance to rIFN-λ1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-α, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-λ1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-λ1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.
26
Moriyama, Masaru, Naoya Kato, Motoyuki Otsuka, Run-Xuan Shao, Hiroyoshi Taniguchi, Takao Kawabe, and Masao Omata. "Interferon-beta is activated by hepatitis C virus NS5B and inhibited by NS4A, NS4B, and NS5A." Hepatology International 1, no. 2 (May 31, 2007): 302–10. http://dx.doi.org/10.1007/s12072-007-9003-8.
Full text
27
McCormick, Christopher J., David Brown, Stephen Griffin, Lisa Challinor, David J. Rowlands, and Mark Harris. "A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A." Journal of General Virology 87, no. 1 (January 1, 2006): 93–102. http://dx.doi.org/10.1099/vir.0.81180-0.
Full textAbstract:
Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.
28
Malov, V. A., E. A. Ubeeva, I. P. Ubeeva, S. M. Nikolaev, and K. T. Umbetova. "Treatment of chronic viral hepatitis C with direct acting antiviral agent: review." Terapevticheskii arkhiv 91, no. 11 (November 15, 2019): 86–89. http://dx.doi.org/10.26442/00403660.2019.11.000307.
Full textAbstract:
HCV infection treatment regimens are viewed from positions of HCV life cycle and replication, effects of NS3/4A protease inhibitors and NS5A/NS5B inhibitors on HCV strain replication. Evolution of HCV treatment regimens from its discovery to modern DAA agents had led to substantial progress although drug resistance poses a new issue to be addressed.
29
Hillung, Julia, Elena Ruiz-López, Itxaso Bellón-Echeverría, Pilar Clemente-Casares, and Antonio Mas. "Characterization of the interaction between hepatitis C virus NS5B and the human oestrogen receptor alpha." Journal of General Virology 93, no. 4 (April 1, 2012): 780–85. http://dx.doi.org/10.1099/vir.0.039396-0.
Full textAbstract:
The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is part of the viral replicative complex and plays a crucial role in HCV replication. It has been described that NS5B interacts with cellular proteins, and that interactions between NS5B and host proteins are crucial for viral replication. Some of the host factors involved in the HCV replication cycle include the oestrogen receptor alpha (ESR1), protein kinases (c-Src) and chaperones (Hsp70). In this report, we determine the requirements for the interplay between NS5B and the domain C of ESR1 (ESR1C) by using Förster Resonance Energy Transfer. NS5B–ESR1C and ESR1C–ESR1C interactions are dependent on ionic strength, indicating that contacts are mainly electrostatic. Additionally, NS5B residues involved in NS5B oligomerization were also essential for NS5B–ESR1C interaction. The study of the interactions among viral and host factors will provide data to establish innovative therapeutic strategies and the development of new antiviral drugs.
30
Esposito, Isabella, Sebastián Marciano, Leila Haddad, Omar Galdame, Alejandra Franco, Adrián Gadano, Diego Flichman, and Julieta Trinks. "Prevalence and Factors Related to Natural Resistance-Associated Substitutions to Direct-Acting Antivirals in Patients with Genotype 1 Hepatitis C Virus Infection." Viruses 11, no. 1 (December 21, 2018): 3. http://dx.doi.org/10.3390/v11010003.
Full textAbstract:
This study aimed to assess the prevalence of natural resistance-associated substitutions (RASs) to NS3, NS5A and NS5B inhibitors in 86 genotype 1 Hepatitis C Virus (HCV)-infected patients from Buenos Aires, Argentina, and to determine their effect on therapy outcome. Additionally, virological, clinical and host genetic factors were explored as predictors of the presence of baseline RASs. NS3 RASs (39.2%) were more prevalent than NS5A RASs (25%) and NS5B RASs (8.9%). In the three regions, the frequencies of RASs were significantly higher in HCV-1b than in HCV-1a. The prevalence of Y93H, L159F and Q80K were 1.3%, 6.3% and 2.5%, respectively. IFNL3 CC genotype was identified as an independent predictor of the presence of baseline RASs in NS5A and NS3 genes (p = 0.0005 and p = 0.01, respectively). Sustained virologic response was achieved by 93.3% of the patients after receiving direct-acting antivirals (DAAs), although 48.7% of them showed baseline RASs related to the DAA-regimen. Notably, the prevalence of clinically relevant RASs in the three genes was lower than that observed around the world. The baseline presence of RASs in both subtypes did not appear to affect therapy outcome. These results support the need to evaluate resistance patterns in each particular country since RASs´ prevalence significantly vary worldwide.
31
Wang, Q. May, Michelle A. Hockman, Kirk Staschke, Robert B. Johnson, Katharine A. Case, Jirong Lu, Steve Parsons, et al. "Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase." Journal of Virology 76, no. 8 (April 15, 2002): 3865–72. http://dx.doi.org/10.1128/jvi.76.8.3865-3872.2002.
Full textAbstract:
ABSTRACT The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, Kd , of ≈22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.
32
Appel, Nicole, Ulrike Herian, and Ralf Bartenschlager. "Efficient Rescue of Hepatitis C Virus RNA Replication by trans-Complementation with Nonstructural Protein 5A." Journal of Virology 79, no. 2 (January 15, 2005): 896–909. http://dx.doi.org/10.1128/jvi.79.2.896-909.2005.
Full textAbstract:
ABSTRACT Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.
33
Kukihara, Hiroshi, Kohji Moriishi, Shuhei Taguwa, Hideki Tani, Takayuki Abe, Yoshio Mori, Tetsuro Suzuki, et al. "Human VAP-C Negatively Regulates Hepatitis C Virus Propagation." Journal of Virology 83, no. 16 (June 10, 2009): 7959–69. http://dx.doi.org/10.1128/jvi.00889-09.
Full textAbstract:
ABSTRACT Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) and subtype B (VAP-B) are involved in the regulation of membrane trafficking, lipid transport and metabolism, and the unfolded protein response. VAP-A and VAP-B consist of the major sperm protein (MSP) domain, the coiled-coil motif, and the C-terminal transmembrane anchor and form homo- and heterodimers through the transmembrane domain. VAP-A and VAP-B interact with NS5B and NS5A of hepatitis C virus (HCV) through the MSP domain and the coiled-coil motif, respectively, and participate in the replication of HCV. VAP-C is a splicing variant of VAP-B consisting of the N-terminal half of the MSP domain of VAP-B followed by the subtype-specific frameshift sequences, and its biological function has not been well characterized. In this study, we have examined the biological functions of VAP-C in the propagation of HCV. VAP-C interacted with NS5B but not with VAP-A, VAP-B, or NS5A in immunoprecipitation analyses, and the expression of VAP-C inhibited the interaction of NS5B with VAP-A or VAP-B. Overexpression of VAP-C impaired the RNA replication of the HCV replicon and the propagation of the HCV JFH1 strain, whereas overexpression of VAP-A and VAP-B enhanced the replication. Furthermore, the expression of VAP-C was observed in various tissues, whereas it was barely detected in the liver. These results suggest that VAP-C acts as a negative regulator of HCV propagation and that the expression of VAP-C may participate in the determination of tissue tropism of HCV propagation.
34
Carrasco, Teresa, David Barquín, Adolphe Ndarabu, Mirian Fernández-Alonso, Marina Rubio-Garrido, Silvia Carlos, Benit Makonda, África Holguín, and Gabriel Reina. "HCV Diagnosis and Sequencing Using Dried Blood Spots from Patients in Kinshasa (DRC): A Tool to Achieve WHO 2030 Targets." Diagnostics 11, no. 3 (March 15, 2021): 522. http://dx.doi.org/10.3390/diagnostics11030522.
Full textAbstract:
The World Health Organization has established an elimination plan for hepatitis C virus (HCV) by 2030. In Sub-Saharan Africa (SSA) access to diagnostic tools is limited, and a number of genotype 4 subtypes have been shown to be resistant to some direct-acting antivirals (DAAs). This study aims to analyze diagnostic assays for HCV based on dried blood spots (DBS) specimens collected in Kinshasa and to characterize genetic diversity of the virus within a group of mainly HIV positive patients. HCV antibody detection was performed on 107 DBS samples with Vidas® anti-HCV and Elecsys anti-HCV II, and on 31 samples with INNO-LIA HCV. Twenty-six samples were subjected to molecular detection. NS3, NS5A, and NS5B regions from 11 HCV viremic patients were sequenced. HCV seroprevalence was 12.2% (72% with detectable HCV RNA). Both Elecsys Anti-HCV and INNO-LIA HCV were highly sensitive and specific, whereas Vidas® anti-HCV lacked full sensitivity and specificity when DBS sample was used. NS5B/NS5A/NS3 sequencing revealed exclusively GT4 isolates (50% subtype 4r, 30% 4c and 20% 4k). All 4r strains harbored NS5A resistance-associated substitutions (RAS) at positions 28, 30, and 31, but no NS3 RAS was detected. Elecsys Anti-HCV and INNO-LIA HCV are reliable methods to detect HCV antibodies using DBS. HCV subtype 4r was the most prevalent among our patients. RASs found in subtype 4r in NS5A region confer unknown susceptibility to DAA.
35
Lemm, Julie A., Mengping Liu, Robert G. Gentles, Min Ding, Stacey Voss, Lenore A. Pelosi, Ying-Kai Wang, et al. "Preclinical Characterization of BMS-791325, an Allosteric Inhibitor of Hepatitis C Virus NS5B Polymerase." Antimicrobial Agents and Chemotherapy 58, no. 6 (April 14, 2014): 3485–95. http://dx.doi.org/10.1128/aac.02495-13.
Full textAbstract:
ABSTRACTBMS-791325 is an allosteric inhibitor that binds to thumb site 1 of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase. BMS-791325 inhibits recombinant NS5B proteins from HCV genotypes 1, 3, 4, and 5 at 50% inhibitory concentrations (IC50) below 28 nM. In cell culture, BMS-791325 inhibited replication of HCV subgenomic replicons representing genotypes 1a and 1b at 50% effective concentrations (EC50s) of 3 nM and 6 nM, respectively, with similar (3 to 18 nM) values for genotypes 3a, 4a, and 5a. Potency against genotype 6a showed more variability (9 to 125 nM), and activity was weaker against genotype 2 (EC50, 87 to 925 nM). Specificity was demonstrated by the absence of activity (EC50s of >4 μM) against a panel of mammalian viruses, and cytotoxic concentrations (50%) were >3,000-fold above the HCV EC50. Resistance substitutions selected by BMS-791325 in genotype 1 replicons mostly mapped to a single site, NS5B amino acid 495 (P495A/S/L/T). Additive or synergistic activity was observed in combination studies using BMS-791325 with alfa interferon plus ribavirin, inhibitors of NS3 protease or NS5A, and other classes of NS5B inhibitor (palm site 2-binding or nucleoside analogs). Plasma and liver exposuresin vivoin several animal species indicated that BMS-791325 has a hepatotropic disposition (liver-to-plasma ratios ranging from 1.6- to 60-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥10-fold above the inhibitor EC50s observed with HCV genotype 1 replicons. These findings support the evaluation of BMS-791325 in combination regimens for the treatment of HCV. Phase 3 studies are ongoing.
36
Liu, Mengping, Maria Tuttle, Min Gao, and Julie A. Lemm. "Potency and Resistance Analysis of Hepatitis C Virus NS5B Polymerase Inhibitor BMS-791325 on All Major Genotypes." Antimicrobial Agents and Chemotherapy 58, no. 12 (September 29, 2014): 7416–23. http://dx.doi.org/10.1128/aac.03851-14.
Full textAbstract:
ABSTRACTBMS-791325 is a hepatitis C virus (HCV) inhibitor binding to the thumb domain of the NS5B RNA-dependent RNA polymerase. BMS-791325 is well characterized in genotype 1 (GT1) and exhibits good inhibitory activity (50% effective concentration [EC50], <10 nM) against hybrid replicons containing patient NS5B sequences from GT3a, -4a, and -5a while potency against GT2 is significantly reduced (J. A. Lemm et al., Antimicrob. Agents Chemother. 58:3485–3495, 2014, doi:http://dx.doi.org/10.1128/AAC.02495-13). BMS-791325 potency against GT6a hybrid replicons is more variable, with two of three hybrid clones having EC50s similar to that for GT1 while a third patient clone was ∼10 times less susceptible to BMS-791325. To characterize the resistance profile of BMS-791325 beyond GT1, curing studies were performed across GT1a and -3a to -6a and demonstrated that GT1a has the highest resistance barrier versus BMS-791325 while GT6a has the lowest. Selection of GT3 to -6 NS5B chimeric replicon cells at different concentrations of BMS-791325 revealed substitutions in the thumb domain of NS5B at residues 494 and 495 that conferred different levels of resistance to BMS-791325 but remained susceptible to NS5A or NS3 protease inhibitors. In addition, we demonstrate that the reduced potency of BMS-791325 against one GT6a patient is due to an A494 polymorphism present in ∼21% of sequences in the European HCV database. The results from this report suggest that BMS-791325 is a candidate for combination treatment of HCV GT3 to -6 chronic infections, and the resistance profiles identified will provide useful information for future clinical development.
37
Kati, Warren, Gennadiy Koev, Michelle Irvin, Jill Beyer, Yaya Liu, Preethi Krishnan, Thomas Reisch, et al. "In VitroActivity and Resistance Profile of Dasabuvir, a Nonnucleoside Hepatitis C Virus Polymerase Inhibitor." Antimicrobial Agents and Chemotherapy 59, no. 3 (December 22, 2014): 1505–11. http://dx.doi.org/10.1128/aac.04619-14.
Full textAbstract:
ABSTRACTDasabuvir (ABT-333) is a nonnucleoside inhibitor of the RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) NS5B gene. Dasabuvir inhibited recombinant NS5B polymerases derived from HCV genotype 1a and 1b clinical isolates, with 50% inhibitory concentration (IC50) values between 2.2 and 10.7 nM, and was at least 7,000-fold selective for the inhibition of HCV genotype 1 polymerases over human/mammalian polymerases. In the HCV subgenomic replicon system, dasabuvir inhibited genotype 1a (strain H77) and 1b (strain Con1) replicons with 50% effective concentration (EC50) values of 7.7 and 1.8 nM, respectively, with a 13-fold decrease in inhibitory activity in the presence of 40% human plasma. This level of activity was retained against a panel of chimeric subgenomic replicons that contained HCV NS5B genes from 22 genotype 1 clinical isolates from treatment-naive patients, with EC50s ranging between 0.15 and 8.57 nM. Maintenance of replicon-containing cells in medium containing dasabuvir at concentrations 10-fold or 100-fold greater than the EC50resulted in selection of resistant replicon clones. Sequencing of the NS5B coding regions from these clones revealed the presence of variants, including C316Y, M414T, Y448C, Y448H, and S556G, that are consistent with binding to the palm I site of HCV polymerase. Consequently, dasabuvir retained full activity against replicons known to confer resistance to other polymerase inhibitors, including the S282T variant in the nucleoside binding site and the M423T, P495A, P495S, and V499A single variants in the thumb domain. The use of dasabuvir in combination with inhibitors targeting HCV NS3/NS4A protease (ABT-450 with ritonavir) and NS5A (ombitasvir) is in development for the treatment of HCV genotype 1 infections.
38
Gao, Lu, Hong Tu, Stephanie T. Shi, Ki-Jeong Lee, Miyuki Asanaka, Soon B. Hwang, and Michael M. C. Lai. "Interaction with a Ubiquitin-Like Protein Enhances the Ubiquitination and Degradation of Hepatitis C Virus RNA-Dependent RNA Polymerase." Journal of Virology 77, no. 7 (April 1, 2003): 4149–59. http://dx.doi.org/10.1128/jvi.77.7.4149-4159.2003.
Full textAbstract:
ABSTRACT To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.
39
Elfiky, Abdo A. "Novel Guanosine Derivatives as Anti-HCV NS5b Polymerase: A QSAR and Molecular Docking Study." Medicinal Chemistry 15, no. 2 (February 12, 2019): 130–37. http://dx.doi.org/10.2174/1573406414666181015152511.
Full textAbstract:
Background: IDX-184 is a guanosine derivative having a potent inhibitory performance against HCV NS5b polymerase. Objective: To test three different groups of 2'C - modified analogues of guanosine nucleotide against HCV polymerase. Method: Using combined Quantitative Structure-Activity Relationships (QSAR) and molecular docking, the suggested compounds are studied. Results: Examining the docked structures of the compounds with experimentally solved NS5b structure (PDB ID: 2XI3) revealed that most of the compounds have the same mode of interaction as that of guanosine nucleotide and hence, NS5b inhibition is possible. Conclusion: It is revealed that sixteen modifications have a better binding affinity to NS5b compared to guanosine. In addition, seven more compounds are better in NS5b binding compared to the approved drug, sofosbuvir, and the compound under clinical trials, IDX-184. Hence, these compounds could be potent HCV NS5b inhibitors. Summary Points: Novel guanosine modifications were introduced in silico and optimized using QM. QSAR and docking calculations are performed to test the binding affinity of the compounds to HCV NS5b active site. Comparison between the binding affinities and the mode of interactions of the compounds and both GTP and IDX-184 is performed. Structural mining to quantify the mode of binding of the compounds to NS5b active site pocket.
40
Ferrari, Eric, Jacquelyn Wright-Minogue, Jane W. S. Fang, Bahige M. Baroudy, Johnson Y. N. Lau, and Zhi Hong. "Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli." Journal of Virology 73, no. 2 (February 1, 1999): 1649–54. http://dx.doi.org/10.1128/jvi.73.2.1649-1654.1999.
Full textAbstract:
ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.
41
Chen, Ye, Alain Bopda-Waffo, Amartya Basu, Ramalingam Krishnan, Erica Silberstein, Deborah R. Taylor, Tanaji T. Talele, Payal Arora, and Neerja Kaushik-Basu. "Characterization of Aurintricarboxylic Acid as a Potent Hepatitis C Virus Replicase Inhibitor." Antiviral Chemistry and Chemotherapy 20, no. 1 (August 2009): 19–36. http://dx.doi.org/10.3851/imp1286.
Full textAbstract:
Background: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells. Methods: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations. Results: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC50] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with ≥fivefold decreased potency compared with continuous polymerization conditions. The IC50 value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects. Conclusions: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.
42
Ludmerer, Steven W., Donald J. Graham, Evelyn Boots, Edward M. Murray, Amy Simcoe, Eric J. Markel, Jay A. Grobler, et al. "Replication Fitness and NS5B Drug Sensitivity of Diverse Hepatitis C Virus Isolates Characterized by Using a Transient Replication Assay." Antimicrobial Agents and Chemotherapy 49, no. 5 (May 2005): 2059–69. http://dx.doi.org/10.1128/aac.49.5.2059-2069.2005.
Full textAbstract:
ABSTRACT The innate genetic variability characteristic of chronic hepatitis C virus (HCV) infection makes drug resistance a concern in the clinical development of HCV inhibitors. To address this, a transient replication assay was developed to evaluate the replication fitness and the drug sensitivity of NS5B sequences isolated from the sera of patients with chronic HCV infection. This novel assay directly compares replication between NS5B isolates, thus bypassing the potential sequence and metabolic differences which may arise with independent replicon cell lines. Patient-derived NS5B sequences were similar to those of the established HCV genotypes, but isolates from each patient shared genetic variability specific to that patient, with additional genetic variability observed across the individual isolates. Every sample provided functional NS5B isolates which supported subgenomic replication, frequently to levels comparable to that of laboratory-optimized replicons. All isolates were equivalently sensitive to an active-site nucleoside inhibitor, but the sensitivities to a panel of nonnucleoside inhibitors which targeted three distinct sites on NS5B varied among the isolates. In con1, the original laboratory-optimized replicon, the NS5B S282T substitution confers resistance to the nucleoside inhibitor but impairs replication. This substitution was engineered into both genotype 1a and genotype 1b isolates. Replication was severely debilitated, demonstrating that no compensatory residues were encoded within these genetically diverse sequences to increase the replication fitness of the mutated replicons. This work describes a transient replicon-based assay that can support the clinical development of compounds which target NS5B and demonstrates its utility by examining several patient-derived NS5B isolates for replication fitness and differential sensitivity to NS5B inhibitors.
43
Tornai, István. "A hepatitis C-vírus-bázispolimorfizmus jelentősége a kezelésben." Orvosi Hetilap 156, no. 21 (May 2015): 849–54. http://dx.doi.org/10.1556/650.2015.30180.
Full textAbstract:
The treatment of chronic hepatitis C has developed significantly during the last 25 years. In patients with genotype 1 infection 40–50% sustained virologic response could be achieved using pegylated interferon and ribavirin dual combination, which could be increased significantly with the introduction of direct acting antivirals. Three major groups of direct acting antivirals are known, which directly inhibit different phases of viral life cycle, by inhibiting the function of several non-structural proteins (NS3/4A protease, NS5A protein and NS5B polymerase). Due to the rapid replication rate of hepatitis C virus and the error-prone NS5B polymerase activity, mutant virions are generated, which might have reduced susceptibility to direct acting antiviral therapy. Since these resistance associated variants might exist before the antiviral therapy, they are still able to replicate during the direct acting antiviral treatment. Due to this selection pressure, the resistant virus will replace the wild type. This was especially detected during monotherapy, therefore, the first generation of direct acting antivirals have been combined with pegylated interferon and ribavirin, while recently interferon-free combinations are being developed including 2 or 3 direct acting antivirals. Using the first generation protease inhibitors boceprevir and telaprevir, it could have been seen, that the rate of resistance associated variants is higher and the therapeutic outcome is worse in patients with hepatitis C virus genotype 1a, than in 1b. Similar phenomenon was seen with the second generation of NS3/4A protease inhibitors as well as with NS5A or NS5B polymerase. This is due to the lower genetic barrier to resistance, ie. usually fewer mutations are enough for the emergence of resistance in genotype 1a. The selection of resistance associated variants is one of the most important challenges during the interferon-free therapy. Orv. Hetil., 2015, 156(21), 849–854.
44
Lee, Ki Jeong, Jinah Choi, Jing-hsiung Ou, and Michael M. C. Lai. "The C-Terminal Transmembrane Domain of Hepatitis C Virus (HCV) RNA Polymerase Is Essential for HCV Replication In Vivo." Journal of Virology 78, no. 7 (April 1, 2004): 3797–802. http://dx.doi.org/10.1128/jvi.78.7.3797-3802.2004.
Full textAbstract:
ABSTRACT Hepatitis C virus (HCV) RNA replication is dependent on the enzymatic activities of the viral RNA-dependent RNA polymerase NS5B, which is a membrane-anchored protein. Recombinant NS5B lacking the C-terminal transmembrane domain (21 amino acids) is enzymatically active. To address the role of this domain in HCV replication in vivo, we introduced a series of mutations into the NS5B of an HCV subgenomic replicon and examined the replication capabilities of the resultant mutants by a colony formation assay. Replicons lacking the transmembrane domain did not yield any colonies. Furthermore, when Huh-7 cells harboring the HCV subgenomic replicon were treated with a synthetic peptide consisting of the NS5B transmembrane domain fused to the antennapedia peptide, the membrane association of NS5B was completely disrupted. Correspondingly, the HCV RNA titer was reduced by approximately 50%. A scrambled peptide used as a control did not have any effects. These findings suggest that the membrane association of NS5B facilitates HCV RNA synthesis. However, a related transmembrane domain derived from bovine viral diarrhea virus could not replace the HCV NS5B transmembrane segment. This finding suggests that the C-terminal 21 amino acids not only have a membrane-anchoring function but also may perform additional functions for RNA synthesis in vivo.
45
Valutite, D. E., A. V. Semenov, Yu V. Ostankova, K. V. Kozlov, A. G. Borisov, V. D. Nazarov, and A. A. Totolian. "Detection of drug resistance mutations of hepatitis C virus in patients with failure of the treatment with direct acting antivirals." Journal of microbiology, epidemiology and immunobiology 98, no. 1 (March 4, 2021): 18–27. http://dx.doi.org/10.36233/0372-9311-47.
Full textAbstract:
Background. The development of direct acting antivirals (DAAs) has spurred a revolution in treatment of patients with chronic hepatitis C. However, there are cases showing no response to treatment. In 5% of cases, the viral breakthrough is most likely caused by DAA resistance mutations in the hepatitis C virus genome.The purpose of the study is to detect drug resistance mutations of hepatitis C virus in patients with DAA treatment failure.Materials and methods. The study was performed on plasma samples from 3 patients diagnosed with chronic hepatitis C virus infection and demonstrating DAA virological treatment failure. All isolates had genotype 1b. Drug resistance mutations were detected by using direct sequencing of NS3, NS5A, and NS5B genome regions. The detection technique was developed at the Pasteur Research Institute of Epidemiology and Microbiology.Results. Drug resistance mutations were detected in all cases. By using the Geno2pheno [hcv] 0.92 tool, nucleotide substitutions were detected in different viral genome regions and presumably caused resistance or decreased sensitivity to antivirals both present and absent in the sofosbuvir + daclatasvir combination therapy. Antiviral treatment failure in patients with chronic hepatitis C is caused by drug resistance mutations.Conclusions. The developed technique is efficient for detection of drug resistance mutations in NS3, NS5A, and NS5B regions in cases of virological failure of DAA treatment.
46
Zhong, Weidong, Eric Ferrari, Charles A. Lesburg, David Maag, Saikat Kumar B. Ghosh, Craig E. Cameron, Johnson Y. N. Lau, and Zhi Hong. "Template/Primer Requirements and Single Nucleotide Incorporation by Hepatitis C Virus Nonstructural Protein 5B Polymerase." Journal of Virology 74, no. 19 (October 1, 2000): 9134–43. http://dx.doi.org/10.1128/jvi.74.19.9134-9143.2000.
Full textAbstract:
ABSTRACT Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3′ terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3′ terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3′ terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique β-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.
47
Di Stefano, Mariantonietta, Mona H. Ismail, Thomas Leitner, Giuseppina Faleo, Saada A. Elmnan Adem, Mohamed O. M. E. Elamin, Obeidi Eltreifi, Marwan J. Alwazzeh, Jose R. Fiore, and Teresa A. Santantonio. "Genetic Subtypes and Natural Resistance Mutations in HCV Genotype 4 Infected Saudi Arabian Patients." Viruses 13, no. 9 (September 14, 2021): 1832. http://dx.doi.org/10.3390/v13091832.
Full textAbstract:
This study aimed to characterize the HCV genetic subtypes variability and the presence of natural occurring resistance-associated substitutions (RASs) in Saudi Arabia patients. A total of 17 GT patients were analyzed. Sequence analysis of NS3, NS5A, and NS5B regions was performed by direct sequencing, and phylogenetic analyses were used to determine genetic subtypes, RAS, and polymorphisms. Nine patients were infected by GT 4a, two with GT 4o and three with GT 4d. Two patients were infected with apparent recombinant virus (4a/4o/4a in NS3/NS5A/NS5B), and one patient was infected with a previously unknown, unclassifiable, virus of GT 4. Natural RASs were found in six patients (35%), including three infected by GT 4a, two by GT 4a/GT 4o/GT 4a, and one patient infected by an unknown, unclassifiable, virus of GT 4. In particular, NS3-RAS V170I was demonstrated in three patients, while NS5A-RASs (L28M, L30R, L28M + M31L) were detected in the remaining three patients. All patients were treated with sofosbuvir plus daclatasvir; three patients were lost to follow-up, whereas 14 patients completed the treatment. A sustained virological response (SVR) was obtained in all but one patient carrying NS3-RAS V170I who later relapsed. GT 4a is the most common subtype in this small cohort of Saudi Arabia patients infected with hepatitis C infection. Natural RASs were observed in about one-third of patients, but only one of them showed a treatment failure.
48
Lévêque, Vincent J. P., Robert B. Johnson, Stephen Parsons, Jianxin Ren, Congping Xie, Faming Zhang, and Q. May Wang. "Identification of a C-Terminal Regulatory Motif in Hepatitis C Virus RNA-Dependent RNA Polymerase: Structural and Biochemical Analysis." Journal of Virology 77, no. 16 (August 15, 2003): 9020–28. http://dx.doi.org/10.1128/jvi.77.16.9020-9028.2003.
Full textAbstract:
ABSTRACT The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-Å resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to ∼50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.
49
Shimakami, Tetsuro, Masao Honda, Takashi Kusakawa, Takayuki Murata, Kunitada Shimotohno, Shuichi Kaneko, and Seishi Murakami. "Effect of Hepatitis C Virus (HCV) NS5B-Nucleolin Interaction on HCV Replication with HCV Subgenomic Replicon." Journal of Virology 80, no. 7 (April 1, 2006): 3332–40. http://dx.doi.org/10.1128/jvi.80.7.3332-3340.2006.
Full textAbstract:
ABSTRACT We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.
50
Li, Yan, Ruirui Wang, Xiaogang Du, Mingwang Zhang, and Meng Xie. "Genome-wide analysis for identification of adaptive diversification between hepatitis C virus subtypes 1a and 1b." Canadian Journal of Microbiology 62, no. 7 (July 2016): 608–16. http://dx.doi.org/10.1139/cjm-2016-0156.
Full textAbstract:
Hepatitis C virus (HCV) is a major cause of liver disease and has been estimated to infect approximately 2%–3% of the world’s population. HCV genotype 1 is the subject of intense research and clinical investigations because of its worldwide prevalence and poor access to treatment for patients in developing countries and marginalized populations. The predominant subtypes 1a and 1b of HCV genotype 1 present considerable differences in epidemiological features. However, the genetic signature underlying such phenotypic functional divergence is still an open question. Here, we performed a genome-wide evolutionary study on HCV subtypes 1a and 1b. The results show that adaptive selection has driven the diversification between these subtypes. Furthermore, the major adaptive divergence-related changes have occurred on proteins E1, NS4B, NS5A, and NS5B. Structurally, a number of adaptively selected sites cluster in functional regions potentially relevant to (i) membrane attachment and (ii) the interactions with viral and host cell factors and the genome template. These results might provide helpful hints about the molecular determinants of epidemiological divergence between HCV 1a and 1b.