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Radhakrishnan Padmanabhan - One of the best experts on this subject based on the ideXlab platform.
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modulation of the nucleoside triphosphatase rna helicase and 5 rna triphosphatase activities of dengue virus type 2 Nonstructural Protein 3 ns3 by interaction with ns5 the rna dependent rna polymerase
Journal of Biological Chemistry, 2005Co-Authors: Tadahisa Teramoto, Niklaus H Mueller, Jessica Phelan, Vannakambadi K Ganesh, Krishna H M Murthy, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 Nonstructural Protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5′-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5′-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.
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Modulation of the Nucleoside Triphosphatase/RNA Helicase and 5′-RNA Triphosphatase Activities of Dengue Virus Type 2 Nonstructural Protein 3 (NS3) by Interaction with NS5, the RNA-dependent RNA Polymerase
Journal of Biological Chemistry, 2005Co-Authors: Tadahisa Teramoto, Niklaus H Mueller, Jessica Phelan, Vannakambadi K Ganesh, Krishna H M Murthy, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family, is a re-emerging human pathogen of global significance. DEN2 Nonstructural Protein 3 (NS3) has a serine protease domain (NS3-pro) and requires the hydrophilic domain of NS2B (NS2BH) for activation. NS3 is also an RNA-stimulated nucleoside triphosphatase (NTPase)/RNA helicase and a 5′-RNA triphosphatase (RTPase). In this study the first biochemical and kinetic properties of full-length NS3 (NS3FL)-associated NTPase, RTPase, and RNA helicase are presented. The NS3FL showed an enhanced RNA helicase activity compared with the NS3-pro-minus NS3, which was further enhanced by the presence of the NS2BH (NS2BH-NS3FL). An active protease catalytic triad is not required for the stimulatory effect, suggesting that the overall folding of the N-terminal protease domain contributes to this enhancement. In DEN2-infected mammalian cells, NS3 and NS5, the viral 5′-RNA methyltransferase/polymerase, exist as a complex. Therefore, the effect of NS5 on the NS3 NTPase activity was examined. The results show that NS5 stimulated the NS3 NTPase and RTPase activities. The NS5 stimulation of NS3 NTPase was dose-dependent until an equimolar ratio was reached. Moreover, the conserved motif, 184RKRK, of NS3 played a crucial role in binding to RNA substrate and modulating the NTPase/RNA helicase and RTPase activities of NS3.
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expression purification and characterization of the rna 5 triphosphatase activity of dengue virus type 2 Nonstructural Protein 3
Virology, 2002Co-Authors: Greg Bartelma, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5′ end. The viral RNA is translated to produce a single polyProtein precursor that is processed to yield three virion Proteins and at least seven Nonstructural Proteins (NS) in the infected host. NS3 is a multifunctional Protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5′-RNA triphosphatase activity, suggesting its role in the synthesis of the 5′-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified Protein has 5′-RNA triphosphatase activity that cleaves the γ-phosphate moiety of the 5′-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 Protein indicate that the nucleoside triphosphatase and 5′-RNA triphosphatase activities of NS3 share a common active site.
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Expression, Purification, and Characterization of the RNA 5′-Triphosphatase Activity of Dengue Virus Type 2 Nonstructural Protein 3
Virology, 2002Co-Authors: Greg Bartelma, Radhakrishnan PadmanabhanAbstract:Abstract Dengue virus type 2 (DEN2), a member of the Flaviviridae family of positive-strand RNA viruses, contains a single RNA genome having a type I cap structure at the 5′ end. The viral RNA is translated to produce a single polyProtein precursor that is processed to yield three virion Proteins and at least seven Nonstructural Proteins (NS) in the infected host. NS3 is a multifunctional Protein having a serine protease catalytic triad within the N-terminal 180 amino acid residues which requires NS2B as a cofactor for activation of protease activity. The C-terminal portion of this catalytic triad has conserved motifs present in several nucleoside triphosphatases (NTPases)/RNA helicases. In addition, subtilisin-treated West Nile (WN) virus NS3 from infected cells was reported to have 5′-RNA triphosphatase activity, suggesting its role in the synthesis of the 5′-cap structure. In this study, full-length DEN2 NS3 was expressed with an N-terminal histidine tag in Escherichia coli and purified in a soluble form. The purified Protein has 5′-RNA triphosphatase activity that cleaves the γ-phosphate moiety of the 5′-triphosphorylated RNA substrate. Biochemical and mutational analyses of the NS3 Protein indicate that the nucleoside triphosphatase and 5′-RNA triphosphatase activities of NS3 share a common active site.
Chuyu Zhang - One of the best experts on this subject based on the ideXlab platform.
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characterization of classical swine fever virus csfv Nonstructural Protein 3 ns3 helicase activity and its modulation by csfv rna dependent rna polymerase
Virus Research, 2009Co-Authors: Yanping Shen, Chuyu ZhangAbstract:Abstract Classical swine fever virus (CSFV) Nonstructural Protein 3 (NS3) is believed to possess three enzyme activities that are likely to be essential for virus replication: a serine protease located in the N-terminus and NTPase as well as helicase activities located in the C-terminus. In this report, we expressed NS3 helicase domain (NS3h) in E. coli and characterized its helicase activity. The NS3h helicase activity was dependent on the presence of NTP and divalent cations, with a preference for ATP and Mn 2+ , and required the substrates possessing a 3′ un-base-paired region on the RNA template strand. The NS3h helicase activity was proportional to increasing lengths of the 3′ un-base-paired regions up to 16 nucleotides of the RNA substrates. We also investigated the modulation of NS3 NTPase/helicase activities by NS3 protease domain and NS5B, an RNA-dependent RNA polymerase (RdRp). Our data showed that the NS3 protease domain enhanced the helicase activity of NS3 but had no effect on its NTPase activity. For the truncated NS3 (helicase domain, NS3h), both NTPase and helicase activities were up-regulated by NS5B. However, for the full-length NS3 (NS3FL), the NTPase activity, but not the helicase activity, was stimulated by NS5B. Maltose-binding Protein (MBP) pull-down as well as enzyme-linked immunosorbent assays confirmed the specific interaction between NS3 and NS5B.
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identification and characterization of the ntpase activity of classical swine fever virus csfv Nonstructural Protein 3 ns3 expressed in bacteria
Archives of Virology, 2007Co-Authors: C Chen, Yi Wang, Chuyu ZhangAbstract:The Nonstructural Protein 3 (NS3) of members of the family Flaviviridae possesses multiple enzyme activities that are likely to be essential for viral replication. Here, we cloned and expressed full-length CSFV NS3 Protein (NS3FL) and its N-terminal truncated version (ntNS3) in E. coli. NTPase activities of the purified NS3FL and ntNS3 Proteins and their reaction conditions were investigated. The results showed that CSFV NS3FL and ntNS3 Proteins contained a specific polynucleotide-stimulated NTPase acitivity. Characterization of ntNS3 NTPase activity showed that optimal reaction conditions with respect to pH, MgCl2 and monovalent cations were similar to those of bovine viral diarrhea virus (BVDV) and hepatitis C virus (HCV). Site-directed mutagenesis analysis demonstrated that the GxGK 232T to GxGAT mutation in the conserved motif I abolished the NTPase activity of ntNS3, whereas substitution of TATPA 354 for TATPV in the motif III had no effect on the enzyme activity. Moreover, the kinetic properties (K m and k cat) of CSFV NS3 were more similar to those of BVDV. Our results provide insight into the structure-function relationship of CSFV NS3 and facilitate our understanding of its role in the replication cycle of CSFV.
Joonho Choe - One of the best experts on this subject based on the ideXlab platform.
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rna stimulated atpase and rna helicase activities and rna binding domain of hepatitis g virus Nonstructural Protein 3
Journal of Virology, 1999Co-Authors: Yousang Gwack, Inyoung Song, Joonho ChoeAbstract:Hepatitis G virus (HGV) Nonstructural Protein 3 (NS3) contains amino acid sequence motifs typical of ATPase and RNA helicase Proteins. In order to examine the RNA helicase activity of the HGV NS3 Protein, the NS3 region (amino acids 904 to 1580) was fused with maltose-binding Protein (MBP), and the fusion Protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 Protein possessed RNA-stimulated ATPase and RNA helicase activities. Characterization of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae viral NS3 Proteins. However, the kinetic analysis of NTPase activity showed that the MBP-HGV/NS3 Protein had several unique properties compared to the other Flaviviridae NS3 Proteins. The HGV NS3 helicase unwinds RNA-RNA duplexes in a 3′-to-5′ direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/NS3 helicase bound to RNA, RNA/DNA, and DNA duplexes with 5′ and 3′ overhangs but not to blunt-ended RNA duplexes. We also found that the conserved motif VI was important for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRTGRGRSGR. Our data showed that the MBP-HCV/NS3 Protein also contains the RNA binding domain in the similar domain.
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dna helicase activity of the hepatitis c virus Nonstructural Protein 3
FEBS Journal, 1997Co-Authors: Yousang Gwack, Joonho ChoeAbstract:Hepatitis C virus (HCV) Nonstructural Protein 3 (NS3) is a known RNA helicase, an enzyme that unwinds RNA · DNA and RNA · RNA duplexes. We have now deciphered the biochemical characteristics of the HCV NS3 DNA helicase activity. Recombinant NS3 was expressed in Escherichia coli, purified to near homogeneity, and tested for DNA helicase activity. The optimal conditions for DNA unwinding (for example, the preferred pH and magnesium ion concentration) were similar to those for RNA unwinding. The DNA helicase activity was very sensitive to potassium ion concentration, while DNA binding and DNA-stimulated ATPase activities were not. The direction of DNA unwinding was determined to be 3′ to 5′. All four ribonucleoside triphosphates (ATP, GTP, CTP, UTP) and deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) could serve as energy sources, but GTP and dGTP were less efficient than the others. When nucleotide analog inhibitors were added to the DNA helicase reaction, the overall order of inhibitory capacity was: adenosine 5′-O-(3-thiotriphosphate) > adenylyl-imidodiphosphate and adenylyl-(β,γ-methylene)-diphosphate > AMP. DNA helicase activity was inhibited strongly by ssDNA and ssRNA, but was little affected by dsDNA. The ATPase activity was stimulated greatly by ssDNA and ssRNA, but not by dsDNA. The NS3 Protein could unwind up to 500 base pairs of duplex DNA. The possible multifunctional nature of the NS3 Protein is discussed and compared with that of Simian virus 40 large T antigen.
Josep Quer - One of the best experts on this subject based on the ideXlab platform.
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Reversal of Nonstructural Protein 3-specific CD4 + T cell
2020Co-Authors: Silvia Sauleda, Natalia Casamitjana, Maria Piron, Josep Quer, Maria Cubero, L Puig, J Guardia, Isabel Campos-varela, J I EstebanAbstract:IL-7/IL-15-supplemented medium. Ex vivo bulk CD4 + T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11% vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-c. Antigen-free in vitro expansion of NS3-specific CD154 + cells from chronic patients restored IFN-c and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4 + T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.
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reversal of Nonstructural Protein 3 specific cd4 t cell dysfunction in patients with persistent hepatitis c virus infection
Journal of Viral Hepatitis, 2012Co-Authors: Silvia Sauleda, Natalia Casamitjana, Maria Piron, Isabel Camposvarela, Josep Quer, Maria Cubero, L PuigAbstract:: Hepatitis C virus (HCV)-specific T cell responses are essential for HCV control, and chronic infection is characterized by functionally altered antigen-specific T cells. It has been proposed that the early inactivation of specific CD4(+) T cell responses may be involved in establishment of HCV persistence. We have investigated whether HCV-specific CD4(+) T cells dysfunction can be reversed in vitro. Nonstructural Protein 3 (NS3) and core-specific CD4(+) T cells from eight chronically infected and eight spontaneously resolved HCV individuals were selected through transient CD154 (CD40 ligand) expression, and their functional profile (IFN-γ, IL-2, TNF-α, IL-10 and IL-4 production by enzyme-linked immunospot assay, cytometric bead array and intracellular cytokine staining, and proliferation by carboxy-fluorescein diacetate succinimidyl ester dilution assay) was determined both ex vivo and after in vitro expansion of sorted CD154-expressing cells in the absence of specific antigen in IL-7/IL-15-supplemented medium. Ex vivo bulk CD4(+) T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11%vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-γ. Antigen-free in vitro expansion of NS3-specific CD154(+) cells from chronic patients restored IFN-γ and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4(+) T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.
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reversal of Nonstructural Protein 3 specific cd4 t cell
2012Co-Authors: Silvia Sauleda, Natalia Casamitjana, Maria Piron, Isabel Camposvarela, Josep Quer, Maria Cubero, L Puig, J Guardia, J I EstebanAbstract:IL-7/IL-15-supplemented medium. Ex vivo bulk CD4 + T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11% vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-c. Antigen-free in vitro expansion of NS3-specific CD154 + cells from chronic patients restored IFN-c and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4 + T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.
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effect of bottlenecking on evolution of the Nonstructural Protein 3 gene of hepatitis c virus during sexually transmitted acute resolving infection
Journal of Virology, 2005Co-Authors: Josep Quer, Silvia Sauleda, Maria Cubero, Juan Ignacio Esteban, Laura Ocana, Maria Martell, Teresa Otero, Eduard Palou, Pedro Murillo, Rafael EstebanAbstract:Sexual partners of patients infected with the hepatitis C virus (HCV) often have detectable HCV-specific T-cell responses in the absence of seroconversion, suggesting unapparent, spontaneously resolving infection. To determine whether differences in the evolutionary potential of bottlenecked inoculum may explain the low rate of HCV persistence after sexual exposure, we have investigated changes in the entire HCV Nonstructural 3 (NS3) gene over time in a chronic carrier and compared his viral quasispecies with that of the acute-phase isolate of his sexual partner, who developed acute resolving hepatitis C. The overall rate of accumulation of mutations, estimated by regression analysis of six consecutive consensus NS3 sequences over 8 years, was 1.5 × 10−3 mutations per site per year, with small intersample fluctuations related to changes in environmental conditions. Comparison of quasispecies parameters in one isolate of the chronic carrier with those of the acute-phase isolate of the infected partner revealed a higher heterogeneity and lower proportion of nonsynonymous mutations in the former. All NS3 sequences from the acute-phase isolate clustered with a single sequence from the chronic isolate, despite complete HLA mismatch between the patients, suggesting bottlenecking during transmission. The low risk of viral persistence after sexual exposure to HCV may be related to the selection of a limited number of viral particles carrying a particular combination of mutations which may further limit the potential of a relatively homogeneous quasispecies to rapidly diversify and overcome the immune response of the exposed host.
Maria Cubero - One of the best experts on this subject based on the ideXlab platform.
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Reversal of Nonstructural Protein 3-specific CD4 + T cell
2020Co-Authors: Silvia Sauleda, Natalia Casamitjana, Maria Piron, Josep Quer, Maria Cubero, L Puig, J Guardia, Isabel Campos-varela, J I EstebanAbstract:IL-7/IL-15-supplemented medium. Ex vivo bulk CD4 + T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11% vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-c. Antigen-free in vitro expansion of NS3-specific CD154 + cells from chronic patients restored IFN-c and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4 + T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.
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reversal of Nonstructural Protein 3 specific cd4 t cell dysfunction in patients with persistent hepatitis c virus infection
Journal of Viral Hepatitis, 2012Co-Authors: Silvia Sauleda, Natalia Casamitjana, Maria Piron, Isabel Camposvarela, Josep Quer, Maria Cubero, L PuigAbstract:: Hepatitis C virus (HCV)-specific T cell responses are essential for HCV control, and chronic infection is characterized by functionally altered antigen-specific T cells. It has been proposed that the early inactivation of specific CD4(+) T cell responses may be involved in establishment of HCV persistence. We have investigated whether HCV-specific CD4(+) T cells dysfunction can be reversed in vitro. Nonstructural Protein 3 (NS3) and core-specific CD4(+) T cells from eight chronically infected and eight spontaneously resolved HCV individuals were selected through transient CD154 (CD40 ligand) expression, and their functional profile (IFN-γ, IL-2, TNF-α, IL-10 and IL-4 production by enzyme-linked immunospot assay, cytometric bead array and intracellular cytokine staining, and proliferation by carboxy-fluorescein diacetate succinimidyl ester dilution assay) was determined both ex vivo and after in vitro expansion of sorted CD154-expressing cells in the absence of specific antigen in IL-7/IL-15-supplemented medium. Ex vivo bulk CD4(+) T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11%vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-γ. Antigen-free in vitro expansion of NS3-specific CD154(+) cells from chronic patients restored IFN-γ and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4(+) T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.
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reversal of Nonstructural Protein 3 specific cd4 t cell
2012Co-Authors: Silvia Sauleda, Natalia Casamitjana, Maria Piron, Isabel Camposvarela, Josep Quer, Maria Cubero, L Puig, J Guardia, J I EstebanAbstract:IL-7/IL-15-supplemented medium. Ex vivo bulk CD4 + T cells from chronic patients expressed CD154 in most cases, albeit at lower frequencies than those of resolved patients (0.11% vs 0.41%; P = 0.01), when stimulated with NS3, but not core, although they had a markedly impaired capacity to produce IL-2 and IFN-c. Antigen-free in vitro expansion of NS3-specific CD154 + cells from chronic patients restored IFN-c and IL-2 production and proliferation to levels similar to those of patients with spontaneously resolved infection. Hence, NS3-specific CD4 + T cell response can be rescued in most chronic HCV patients by in vitro expansion in the absence of HCV-specific antigen. These results might provide a rationale for adoptive immunotherapy.
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effect of bottlenecking on evolution of the Nonstructural Protein 3 gene of hepatitis c virus during sexually transmitted acute resolving infection
Journal of Virology, 2005Co-Authors: Josep Quer, Silvia Sauleda, Maria Cubero, Juan Ignacio Esteban, Laura Ocana, Maria Martell, Teresa Otero, Eduard Palou, Pedro Murillo, Rafael EstebanAbstract:Sexual partners of patients infected with the hepatitis C virus (HCV) often have detectable HCV-specific T-cell responses in the absence of seroconversion, suggesting unapparent, spontaneously resolving infection. To determine whether differences in the evolutionary potential of bottlenecked inoculum may explain the low rate of HCV persistence after sexual exposure, we have investigated changes in the entire HCV Nonstructural 3 (NS3) gene over time in a chronic carrier and compared his viral quasispecies with that of the acute-phase isolate of his sexual partner, who developed acute resolving hepatitis C. The overall rate of accumulation of mutations, estimated by regression analysis of six consecutive consensus NS3 sequences over 8 years, was 1.5 × 10−3 mutations per site per year, with small intersample fluctuations related to changes in environmental conditions. Comparison of quasispecies parameters in one isolate of the chronic carrier with those of the acute-phase isolate of the infected partner revealed a higher heterogeneity and lower proportion of nonsynonymous mutations in the former. All NS3 sequences from the acute-phase isolate clustered with a single sequence from the chronic isolate, despite complete HLA mismatch between the patients, suggesting bottlenecking during transmission. The low risk of viral persistence after sexual exposure to HCV may be related to the selection of a limited number of viral particles carrying a particular combination of mutations which may further limit the potential of a relatively homogeneous quasispecies to rapidly diversify and overcome the immune response of the exposed host.
