Prospective characterization of full-length hepatitis C virus NS5A quasispecies during induction and combination antiviral therapy

The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR(237-276)) sequence associated with IFN resistance was not found, although the presence of Ala(245) within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P<0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P<0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P<0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.     

GB virus B and hepatitis C virus NS3 serine proteases share substrate specificity.

GB virus B (GBV-B) is a recently discovered virus responsible for hepatitis in tamarins (Saguinus species). GBV-B belongs to the Flaviviridae family and is closely related to the human pathogen hepatitis C virus (HCV). Nonstructural protein 3 (NS3) of HCV has been shown to encompass a serine protease domain required for viral maturation. GBV-B and HCV share only about 30% of the amino acid sequence within the NS3 protease domain. The catalytic triad is conserved, and the residue Phe-154, presumed to be a crucial amino acid for determining the S1 specificity pocket of the HCV NS3 protease, is also conserved. We have expressed a synthetic gene encoding the GBV-B NS3 protease domain in Escherichia coli and have characterized the purified recombinant protein for its activity on HCV substrates. We have shown that the NS3 region of the GBV-B genome actually encodes a serine protease that, despite the low sequence homology, shares substrate specificity with the HCV NS3 protease.     

Ⅲ型中国株HCVE2/NS1基因与已知分离株的同源性比较

利用逆转录套式ＰＣＲ扩增Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因片段,将其克隆到ｐｃＤＮＡ３载体上．采用双脱氧链终止法测定插入片段的核苷酸序列．并与已知分离株的相应区域进行同源性比较．首次克隆出Ⅲ型中国株ＨＣＶＥ２／ＮＳ１基因（ＨＣ－Ｗ１４）,其核苷酸序列与Ⅲ型日本株ＨＣＶ（ＨＣ－Ｊ６）该区域同源性为８８．３７％,其推定的氨基酸同源性为８９．２９％．而与已知的非Ⅲ型株ＨＣＶ该区域相比,核苷酸及氨基酸的同源性均相对较低．Ⅲ型中国株ＨＣＶ与Ⅱ型中国株ＨＣＶ在Ｅ２／ＮＳ１区域有较大的变异,揭示研制我国的ＨＣＶ疫苗应该考虑这种基因型之间的变异性．     

Is hepatitis C virus genotype 4 predominant in Saudi Arabia?

Hepatitis C virus (HCV) genotype 4 is believed to be predominant in the Middle East including Saudi Arabia (SA). We attempted to genotype 80 HCV isolates from different parts of SA by direct sequencing of a variable 222bp fragment from the NS5B region. The phylogenetic analysis of the NS5B sequences was complemented by direct sequence analysis of the conserved 5'-NCR region for HCV type-specific polymorphism. All 80 NS5B sequences separated into 3 clades which comprised 6 type 1b variants, 30 type 4 variants (24 of type 4a and 6 of type 4c or d) and 44 type 3 variants. Apart from two definitive type 3b variants the other 42 type 3 NS5B sequences formed 4 clusters with low similarity to type 3a-f HCV sequences from the database. The precise subtyping of these 42 type 3 variants awaits sequencing of longer HCV RNA stretches. Our results indicate that HCV type 4 may not be the only dominant genotype in SA.     

Similar Prevalence of Low-Abundance Drug-Resistant Variants in Treatment-Naive Patients with Genotype 1a and 1b Hepatitis C Virus Infections as Determined by Ultradeep Pyrosequencing

Background and Objectives

Hepatitis C virus (HCV) variants that confer resistance to direct-acting-antiviral agents (DAA) have been detected by standard sequencing technology in genotype (G) 1 viruses from DAA-naive patients. It has recently been shown that virological response rates are higher and breakthrough rates are lower in G1b infected patients than in G1a infected patients treated with certain classes of HCV DAAs. It is not known whether this corresponds to a difference in the composition of G1a and G1b HCV quasispecies in regards to the proportion of naturally occurring DAA-resistant variants before treatment.

Methods

We used ultradeep pyrosequencing to determine the prevalence of low-abundance (<25% of the sequence reads) DAA-resistant variants in 191 NS3 and 116 NS5B isolates from 208 DAA-naive G1-infected patients.

Results

A total of 3.5 million high-quality reads of ≥200 nucleotides were generated. The median coverage depth was 4150x and 4470x per NS3 and NS5B amplicon, respectively. Both G1a and G1b populations showed Shannon entropy distributions, with no difference between G1a and G1b in NS3 or NS5B region at the nucleotide level. A higher number of substitutions that confer resistance to protease inhibitors were observed in G1a isolates (mainly at amino acid 80 of the NS3 region). The prevalence of amino acid substitutions that confer resistance to NS5B non-nucleoside inhibitors was similar in G1a and G1b isolates. The NS5B S282T variant, which confers resistance to the polymerase inhibitors mericitabine and sofosbuvir, was not detected in any sample.

Conclusion

The quasispecies genetic diversity and prevalence of DAA-resistant variants was similar in G1a and G1b isolates and in both NS3 and NS5B regions, suggesting that this is not a determinant for the higher level of DAA resistance observed across G1a HCV infected patients upon treatment.     

Sequence analysis of the 5' non coding region of Turkish HCV isolates: implications for PCR diagnosis

Hepatitis C virus (HCV) is a positive-strand RNA virus related to pestiviruses and flaviviruses. The 5' noncoding region (NCR) of the virus genome consists of 324-341 nucleotides and is generally highly conserved among different HCV isolates which has made this region the choice for primer selection in amplification of HCV sequences by polymerase chain reaction (PCR). In this study, we report the partial nucleotide sequences of the 5'-NCR from type 1a (n = 4), type 1b (n = 6) and type 4 (n = 1) Turkish HCV isolates. Sequence information was obtained by direct sequencing of RT-PCR product using biotinylated primers and single strands were sequenced using T7 DNA polymerase after binding to streptavidin coated magnetic beads. In comparison to prototype type 1a consensus sequence, all type 1b sequences had A-G substitution at position - 99. Nucleotid changes from the prototype 1a sequence were found in 12 of the 174 nucleotide positions. The most variable domain spans 51 nucleotides (positions - 167 to - 117) where nine polymorphic sites were identified. Although the nucleotide sequence of the 5'-noncoding region is highly conserved there are type-specific polymorphic sites within this region that has to be taken into consideration in the design of oligonucleotide primers for reliable amplification of sequences from different HCV genotypes.     

Genetic heterogeneity of hepatitis C virus (HCV) in clinical strains of HIV positive and HIV negative patients chronically infected with HCV genotype 3a

The clinical correlation between the degree of HCV variability and the response to anti-HCV treatment in HIV positive patients infected with HCV genotype 3a is unknown. In this study, 27 HIV positive and 5 HIV negative patients with HCV genotype 3a infection were treated with interferon-alpha-2b with or without ribavirin. Nine patients (5 HIV positive) achieved a sustained virological response (SR) and 23 (only one HIV negative) were non-responders (NR). Sequence analyses of the partial E2 domain and the non-structural 5A protein were performed at baseline in all patients, and before and during treatment in the HIV positive NRs. There was no difference in the mean number of amino acid mutations from HCV 3a prototype, within E2 region, between the HIV positive and HIV negative patients: 17 (range 11-25) vs 16 (range 14-17). The mean baseline number of mutations in E2 region, was similar in HIV positive SRs and NRs: 18 (range 14-25) vs 16 (range 11-19). Phylogenetic analysis of HCV paired serum samples at baseline and during treatment revealed identical E2 sequence in 5/21 HIV positive NR patients, whereas 6 other sequences were strictly related to baseline E2 domain and the remaining 10 were divergent. The mean number of amino acid mutations in the NS5A protein at baseline, was 1 (range 0-3) in HIV negative patients and 2 (range 0-4) in HIV positive ones. This region was highly conserved in all isolates of HIV positive NRs analysed during treatment. These results suggest that genetic variability at baseline within the E2 region and NS5A protein of HCV 3a strain obtained from HIV positive and HIV negative patients is not associated with treatment response. Furthermore, the anti-HCV treatment did not influence HCV heterogeneity within the E2 and NS5A domains in HIV positive patients infected with HCV genotype 3a.     

Hepatitis C virus protease gene diversity in patients coinfected with human immunodeficiency virus

The clonal variability of the hepatitis C virus (HCV) protease gene in 24 individuals with HCV genotypes 1a, 1b, 2b, and 3a who were coinfected with the human immunodeficiency virus was evaluated. Within-genotype variability at the nucleotide and amino acid levels ranged from 6.5 to 8.6% and 2.2 to 3.8%, respectively. After adjustments were made for correlation of intrapatient clonal variation, mixed-model analysis indicated that nucleotide and amino acid variability among patients with different genotypes did not differ significantly. However, within individual patients, clonal variability differed by up to 5.3% and 5.8% at the nucleotide and amino acid levels, respectively, and genotype 1a had significantly greater nucleotide variability than other genotypes (P = 0.01). Significant variability exists within HCV protease gene variants at the patient level and could affect the effectiveness of HCV protease inhibitors.     

Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope ¹²³¹PTGSGKSTK¹²³⁹ (EP05) or core motif ¹³⁷³IPFYGKAI¹³⁸⁰ (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.     

Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4-18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance.     

Partial nucleotide sequencing of the NS3/helicase region of hepatitis G virus to prove vertical transmission

To study non-parental transmission of hepatitis G virus and/or GB virus C (HGV/GBV-C), we sequenced and compared the NS3/helicase region of the virus for five HGV/GBV-C RNA-positive mothers and their 11 children who had experienced neither blood transfusion nor overt hepatitis and were negative for HBV, HCV and HIV, except in one mother coinfected with HCV. The nucleotide sequences of the familial HGV/GBV-C isolates showed high similarity of 99-100% (mean 99.8%, 100% at the deduced amino acid level) between mother and her child(ren) in each family. These findings strongly suggest the spontaneous occurrence of mother-to-child transmission of HGV/GBV-C as reported previously. They also suggest that nucleotide sequence analysis on the NS3/helicase region of HGV/GBV-C may be a useful tool to study HGV/GBV-C transmission.     

丙型肝炎病毒丝氨酸蛋白酶特异性结合肽的筛选和鉴定

丙型肝炎病毒丝氨酸蛋白酶在病毒复制和包装中的重要作用使其成为特异性抗病毒药物研究的首选靶标。根据丝氨酸蛋白酶晶体结构特点,用柔性连接子连接NS3丝氨酸蛋白酶结构域和NS4A的核心序列,构建成单链丝氨酸蛋白酶基因并且在大肠杆菌中获得高水平的可溶性表达,纯化后的目的蛋白能够切割重组蛋白底物NS5ab。随后,以单链丝氨酸蛋白酶为靶分子对噬菌体展示的随机十二肽库进行了三轮淘筛,挑选的44个克隆中有37个克隆能够特异性地结合丝氨酸蛋白酶,并且这种结合作用为竞争性ELISA试验结果所支持。对13个克隆进行序列测定,得到6种序列,它们在氨基酸组成上存在明显偏性,富含组氨酸和色氨酸,缺乏酸性氨基酸;6种序列存在一个共有序列。     

Hepatitis C virus nonstructural 5A protein and interferon resistance: a new model for testing the reliability of mutational analyses

Presumably due to the capability of the hepatitis C virus (HCV) to evade the antiviral effects of alpha interferon, treatment is ineffective in more than half of chronically genotype HCV type 1 (HCV-1)-infected patients. Previous approaches to correlate the number of amino acid mutations within regions of HCV nonstructural (NS)-5A protein with virologic treatment response provided conflicting results. In the present study, we developed a new mathematical model to investigate NS5A sequences of HCV-1-infected patients. The mean number of all mutations within the complete NS5A protein was significantly higher in virologic responders compared to nonresponders (P = 0.008 and P = 0.0001 for amino acid residues predicted on the surface of the NS5A protein). Differences did not achieve statistical significance for NS5A regions that are currently assumed to be functionally relevant (e.g., the interferon sensitivity-determining region, the RNA-activated protein kinase-binding domain, etc.). Analyses of smoothed mutational frequencies showed that the number of mutations in other NS5A regions correlated with virologic response. Such a correlation was observed for both genuine and randomly generated NS5A sequences. The existence of local accumulations of mutations within genuine NS5A isolates that truly correlated with treatment response was defined by a refined test procedure. Upon considering the predicted residue accessibility, we identified the main focus of mutations correlating with treatment response to be the sequence from amino acids 2350 to 2370. Thus, evaluation of NS5A mutations in correlation with treatment response is improved by consideration of functional and predicted conformational amino acid properties. As shown by simulations with randomly generated sequences, multiple analyses of simple counts of local NS5A amino acid mutations and correlation with treatment response are insufficient. For improvement of mutational analysis, a refined specific functional data test procedure is proposed.     

Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells

Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.     

Efficient replication of hepatitis C virus genotype 1a RNAs in cell culture

Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.     

HCV genotyping using statistical classification approach

The genotype of Hepatitis C Virus (HCV) strains is an important determinant of the severity and aggressiveness of liver infection as well as patient response to antiviral therapy. Fast and accurate determination of viral genotype could provide direction in the clinical management of patients with chronic HCV infections. Using publicly available HCV nucleotide sequences, we built a global Position Weight Matrix (PWM) for the HCV genome. Based on the PWM, a set of genotype specific nucleotide sequence "signatures" were selected from the 5' NCR, CORE, E1, and NS5B regions of the HCV genome. We evaluated the predictive power of these signatures for predicting the most common HCV genotypes and subtypes. We observed that nucleotide sequence signatures selected from NS5B and E1 regions generally demonstrated stronger discriminant power in differentiating major HCV genotypes and subtypes than that from 5' NCR and CORE regions. Two discriminant methods were used to build predictive models. Through 10 fold cross validation, over 99% prediction accuracy was achieved using both support vector machine (SVM) and random forest based classification methods in a dataset of 1134 sequences for NS5B and 947 sequences for E1. Prediction accuracy for each genotype is also reported.     

中国人血清中丙型肝炎病毒聚合酶全长基因的克隆表达及鉴定

构建中国人丙型肝炎病毒(HCV)复制的RNA聚合酶原核表达载体pET30aNS5b,并在大肠杆菌中获得NS5B聚合酶蛋白的高效表达,为建立HCV NS5b聚合酶细胞外分子复制模型的方法创造条件。使用高保真Pfu DNA聚合酶进行反转录及套式PCR扩增,从我国HCV RNA阳性血清中扩增出HCV NS5b RNA多聚酶全基因序列,经BamHI和SalI酶切,将其克隆至同样酶切的pET-30a载体中;转化大肠杆菌BL21,IPTG诱导表达。用抗HCV NS5b单克隆抗体做Western-Blot进行鉴定。结果表明构建了原核表达载体,pET30aNS5bpET30aNS5b明显表达出12-His-NS5b聚合酶蛋白。测序结果表明,与已发表的相关HCV NS5b RNA聚合酶序列比较,其核苷酸和氨基酸的同源性分别在69％-92．7％及88．8％-96．8％之间。在最佳表达条件下,可高效诱导表达融合蛋白(65kDa),最高表达量占菌体蛋白18．9％。Western-Blot结果显示表达蛋白为HCV NS5b酶。HCV聚合酶蛋白全长基因可以成功地克隆在pET-30a载体上并有效表达出目的蛋白,为研究建立HCV NS5b聚合酶细胞外分子复制模型奠定了基础。     

Characterization of Hepatitis C Virus Genotypes by Direct Sequencing of HCV 5′UTR Region of Isolates from Saudi Arabia

The current study was designed to determine the Hepatitis C Virus (HCV) genotypes in a representative sample of HCV chronically infected patients in Saudi Arabia. All HCV isolates were genotyped by sequencing of the 5′UTR region and newly identified HCV isolates were identified. Specific universal primers targeting 5′UTR region were used for both amplification and sequencing of all isolates that resulted in 244 bp fragment which represent about 80% of 5′UTR region. Most of HCV isolates in this study were genotype 4 (76.4%) where only few isolates were recognized as genotype 1 (19.6%). All results were compared to HCV reference sequences from LOS ALAMOS HCV database, considering only the complete full genomes for the main phylogenetic analysis. Sequences that showed maximum identity (98% –100%) were selected. Most isolates were identical with HCV genotype 4 references. Some isolates were similar to different subtypes of HCV genotypes 4, 1 and 6. Phylogenetic analysis showed resemblance of most isolates to similar ones from the Far East, North America and Egypt. Using sequence Weblogo, Alignment analysis of isolated HCV genotypes 4 and 1 showed 92% and 95.5% nucleotide conservation, respectively. There was no predominant nucleotide in the varied sites, in both genotypes. All isolated sequences were submitted to GenBank database.