Constrained genomic and conformational variability of the hypervariable region 1 of hepatitis C virus in chronically infected patients

We analysed the genomic and conformational variability of the hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) to evaluate the importance of its biological role. A total of 865 genotype 1b HVR1 subclones were collected from serially sampled sera in 11 patients with chronic hepatitis C, four of whom received interferon therapy. Consequently, 169 distinct sequences were examined for amino acid substitutions as well as hydrophilic or hydrophobic profile at each amino acid position within HVR1. Secondary structure of HVR1 was also predicted by the method of Robson in 90 distinct sequences from eight patients, including three interferon-treated patients. Some positions within the HVR1 were invariable or nearly so as to amino acid substitution. Hydrophilic or hydrophobic residues exclusively predominated at several positions. These constrained amino acid replacement and hydrophilic or hydrophobic profiles were conserved irrespective of interferon therapy, though the frequency of amino acid replacement was greater at almost all amino acid positions within the HVR1 in interferon-treated patients. The quasispecies of HCV showed various secondary structures of HVR1, but many sequences seemed to have common characteristics. β sheet conformations around both the N-terminus and position 20 (numbered from the NH₂ terminus of E2 envelope glycoprotein), and/or coil structures around the C-terminus of HVR1 could be identified. These results suggest that HVR1 amino acid replacements are strongly constrained by a well-ordered structure, in spite of being tolerant to amino acid substitutions, and imply an important biological role of the HVR1 protein in HCV replication.     

Bursts of nonsynonymous substitutions in HIV-1 evolution reveal instances of positive selection at conservative protein sites

The fixation of a new allele can be driven by Darwinian positive selection or can be due to random genetic drift. Identifying instances of positive selection is a difficult task, because its impact is routinely obscured by the action of negative selection. The nature of the genetic code dictates that positive selection in favor of an amino acid replacement should often cause a burst of two or three nucleotide substitutions at a single codon site, because a large fraction of amino acid replacements cannot be achieved after just one nucleotide substitution. Here, we study pairs of successive nonsynonymous substitutions at one codon in the course of evolution of HIV-1 genes within HIV-1 populations inhabiting infected individuals. Such pairs are more numerous and more clumped than expected if different substitutions were independent and than what is observed for pairs of successive synonymous substitutions. Bursts of nonsynonymous substitutions in HIV-1 evolution cannot be explained by mutational biases and must, therefore, be due to positive selection. Both reversals, exact or imprecise, of fixed deleterious mutations and acquisitions of amino acids with new properties are responsible for the bursts. Temporal clumping is strongest at codon sites with a low overall rate of nonsynonymous evolution, implying that a substantial fraction of replacements of conservative amino acids are driven by positive selection. We identified many conservative sites of HIV-1 proteins that occasionally experience positive selection.     

Multigene tracking of quasispecies in viral persistence and clearance of hepatitis C virus

AIM: To investigate the evaluation of hepatitis C virus (HCV) quasispecies in the envelope region and its relationship with the outcome of acute hepatitis C. METHODS: HCV quasispecies were characterized in specimens collected every 2-6 mo from a cohort of acutely HCV-infected subjects. We evaluated two individuals who spontaneously cleared viremia and three individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, 33 cloned cDNAs representing each specimen were assessed by a combined method of analysis of a single-stranded conformational polymorphism and heteroduplex analysis. The rates of both synonymous and nonsynonymous substitutions for the E1, HVR1 and E2 regions outside HVR1 were analyzed. RESULTS: Serum samples collected from chronic phase of infection had higher quasispecies complexity than those collected from acute phase of infection in all individuals examined. The genetic diversity (genetic distance) within HVR1 was consistently higher than that in the complete E1(0.0322±0.0068 vs-0.0020±0.0014, P<0.05) and E2 regions outside HVR1 (0.0322±0.0068 vs 0.0017±0.0011, P<0.05) in individuals with persistent viremia, but did not change markedly over time in those with clearance of viremia. For individuals with persistent viremia, the rate of nonsynonymous substitutions within the HVR1 region (2.76×10-3±1.51×10-3) predominated and gradually increased, as compared with that in the E1 and E2 regions outside HVR1 (0.23×10-3±0.15×10-3, 0.50×10-3±0.10×10-3). By contrast, the rates of both nonsynonymous and synonymous substitutions for the E1 and E2 regions including HVR1 were consistently lower in individuals with clearance of viremia. CONCLUSION: HCV persistence is associated with a complexity quasispecies and positive selection of HVR1 by the host immune system.     

丙型肝炎病毒感染自然过程中的准种变化

目的观察丙型肝炎病毒(HCV)持续感染者与自然阴转者外周血HCV准种构成的变化规律。方法应用基因扩增、分子克隆和测序的方法,对未接受过治疗的4例HCV持续感染者与4例自然阴转者前后间隔10年血清中HCV高变区1(HVR1)基因片段进行了序列分析及遗传进化关系比较。结果与持续感染者相比,自然阴转者外周血HCVHVR1区准种群体组内平均遗传距离、熵值较小。4例持续感染者中有3例10年前后血清HCVHVR1准种群体组内与组间遗传距离有明显差异。8例感染者中有7例血清HCV准种KA/KS值大于1。结论在丙型肝炎的自然病程中HCV准种遗传复杂度、变异度大小可能与丙型肝炎的转归相关;HCV准种构成可能发生改变。     

Hepatitis C Hypervariable Region 1: Association of Reduced Selection Pressure in African Americans with Treatment Failure

In a prospective therapeutic trial, features of the hepatitis C quasispecies were investigated as possible markers of therapeutic response. Individuals chronically infected with hepatitis C genotype 1 received antiviral therapy consisting of α-interferon plus ribavirin. The study targeted the most rapidly evolving segment of the viral genome, hypervariable region 1 within the envelope-2 gene. Among individuals failing to clear virus in response to therapy, significant differences were observed between quasispecies of African-American and Caucasian subjects. While distance measures for synonymous substitutions were similar between racial subgroups, measures of distance at the amino acid level (nonsynonymous substitutions) varied significantly. Taken together, the observed patterns of variability corresponded to reduced host selection pressure against hypervariable region 1 in African-American nonresponders. Reduced selection pressure was present at baseline and persisted through treatment and follow-up, suggesting population stratification of host factors that influence selection pressure on hepatitis C virus.     

Influence of specific CD4+ T cells and antibodies on evolution of hypervariable region 1 during acute HCV infection

BACKGROUND/AIMS: Several studies suggest that the evolutionary rate of HVR1 sequence in acute HCV hepatitis derives from the action of a continuous immune-driven positive selection. However, these studies have not been performed examining the relationship between HVR1 evolution and the development of specific immunity to autologous HVR1 sequences. METHODS: We performed a longitudinal analysis of HVR1 sequences and specific antibodies and CD4+ T cells in ten HCV acutely infected patients with different clinical outcomes (recovery versus persistence). RESULTS: We showed that although both recovered and chronically evolving individuals developed IFN-gamma+ T cells specific for Core and NS sequences, HVR1-specific CD4+ T cells were detected only in patients clearing the virus. On the contrary, all patients displayed anti-HVR1 antibodies that recognized sequences exclusively carried by autologous viruses. Measurements of genetic diversity and the number of non-synonymous per synonymous substitutions within HVR1 sequences before and after antibody appearance showed an increase of these parameters only in concomitance with the appearance of anti-HVR1 antibodies. CONCLUSIONS: The evidence that anti-HVR1 antibodies favor HVR1 variant selection suggests that viral complexity in chronically infected patients could represent a virus adaptive strategy to escape the continuous selective process mediated by anti-HVR1 antibodies.     

Hepatitis C in human immunodeficiency virus-coinfected patients: Increased variability in the hypervariable envelope coding domain

Patients coinfected with the hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) were studied with regard to nucleotide sequence variability in the E2/NS1 first hypervariable region of the HCV genome. The nucleotide variability within individual patients was compared to patients infected only with HCV. The proportion of predicted synonymous and nonsynonymous amino acid changes, and the relationship to putative high-antigenicity sites, were evaluated in the hypervariable envelope domain. Ninety-one clones from 10 patients with HCV/HIV coinfection were sequenced, following polymerase chain reaction (PCR) amplification of the hypervariable region. The control HCV group included 53 clones from 7 patients. Sequence analysis encompassed the region coding for amino acids 384 to 414. Consensus sequences from each patient were used as the internal standard for nonsynonymous amino acid codon variability. Cumulative proportional comparison at each amino acid site revealed increased variability in HCV RNA from patients with HCV/HIV coinfection versus HCV alone (P < .05). The greatest variability was observed at amino acids 386, 397, 400, 402, 405, 407, and 414, with >l0 percent clonal variation at these sites. Jameson-Wolf plots were used to predict putative high-antigenicity domains. Nonsynonymous clonal variation resulted in alteration of putative antigenic sites within the hypervariable region. All clones had at least one high-probability site. Clones with unique predicted antigenic domains were observed more frequently in HIV/HCV coinfected patients, and, independent of viral titer, were consistent with increased sequence variability. These data suggest an accumulation of envelope variants in the HCV/HIV coinfected patients, which could be related to ineffective viral clearance, and may help explain prior reports of interferon (IFN) resistance in this patient group. (Hepatology 1996 Apr;23(4):688-94)     

Complexity and diversity of hepatitis C virus RNA in african americans and whites: analysis of the envelope-coding domain

African Americans infected with hepatitis C virus (HCV) are less responsive than whites to interferon-based therapy. HCV quasi species have been implicated. Quasi-species complexity and diversity were evaluated in matched African American and white individuals. Complexity and diversity in the first hypervariable region were similar in the 2 groups. Both the frequency of nonsynonymous amino acid substitutions and the mean ratio of nonsynonymous mutations to synonymous mutations were greater in clones derived from white patients. Racial differences in amino acid usage at otherwise conserved positions were observed. Differences in amino acid representation at key positions are suggestive of immunologic and functional selection along racial lines.     

Accumulation of slightly deleterious mutations in mitochondrial protein-coding genes of large versus small mammals

After the effective size of a population, N(e), declines, some slightly deleterious amino acid replacements which were initially suppressed by purifying selection become effectively neutral and can reach fixation. Here we investigate this phenomenon for a set of all 13 mitochondrial protein-coding genes from 110 mammalian species. By using body mass as a proxy for N(e), we show that large mammals (i.e., those with low N(e)) as compared with small ones (in our sample these are, on average, 369.5 kg and 275 g, respectively) have a 43% higher rate of accumulation of nonsynonymous nucleotide substitutions relative to synonymous substitutions, and an 8-40% higher rate of accumulation of radical amino acid substitutions relative to conservative substitutions, depending on the type of amino acid classification. These higher rates result in a 6% greater amino acid dissimilarity between modern species and their most recent reconstructed ancestors in large versus small mammals. Because nonsynonymous substitutions are likely to be more harmful than synonymous substitutions, and radical amino acid substitutions are likely to be more harmful than conservative ones, our results suggest that large mammals experience less efficient purifying selection than small mammals. Furthermore, because in the course of mammalian evolution body size tends to increase and, consequently, N(e) tends to decline, evolution of mammals toward large body size may involve accumulation of slightly deleterious mutations in mitochondrial protein-coding genes, which may contribute to decline or extinction of large mammals.     

Molecular Genetic Analysis of the ABO Blood Group System: 2. cis-AB Alleles

We have determined the nucleotide sequence of the coding region in the last two coding exons of ABO genes from two cis -AB individuals (genotype cis -AB/O) with no consanguinity. In this region, cis -AB alleles from these 2 individuals were identical to one another while different from the A¹ allele by two nucleotide substitutions. Both of these nucleotide substitutions result in amino acid substitutions. The first substitution is identical to the one previously found in the A² allele. The other substitution is found at the fourth position of the four amino acid substitutions which discriminate A¹ and B transferases.     

Molecular Genetic Analysis of the ABO Blood Group System: 2. cis-AB Alleles

We have determined the nucleotide sequence of the coding region in the last two coding exons of ABO genes from two cis -AB individuals (genotype cis -AB/O) with no consanguinity. In this region, cis -AB alleles from these 2 individuals were identical to one another while different from the A¹ allele by two nucleotide substitutions. Both of these nucleotide substitutions result in amino acid substitutions. The first substitution is identical to the one previously found in the A² allele. The other substitution is found at the fourth position of the four amino acid substitutions which discriminate A¹ and B transferases.     

Thymosin alpha1 treatment of chronic hepatitis B: results of a phase III multicentre, randomized, double-blind and placebo-controlled study

Previous clinical trials have suggested that thymosin α₁ (Tα₁), an immunomodulatory peptide, may be effective in the treatment of chronic hepatitis B (CHB). The aim of this study was to determine the efficacy of Tα₁ in a multicentre, placebo-controlled and double-blind study of 97 patients with serum hepatitis B virus (HBV) DNA- and hepatitis B e antigen (HBeAg)-positive CHB. Patients who had been hepatitis B surface antigen (HBsAg) positive for at least 12 months entered a 3-month screening period prior to randomization. Forty-nine patients received Tα₁ (1.6 mg) and 48 patients received placebo, twice weekly for 6 months, and were followed-up for an additional 6 months. At inclusion, both groups were comparable for age, gender, histological grading, and aminotransferase and HBV DNA levels. A complete response to treatment, defined as a sustained serum HBV DNA-negative status (two negative results at least 3 months apart) during the 12-month study, with negative HBV DNA and HBeAg values at month 12, was seen in seven (14%) patients given Tα₁ and in two (4%) patients treated with placebo ( P = 0.084). Five (10%) patients given Tα₁ and four (8%) patients given placebo exhibited a delayed response (defined as sustained serum HBV DNA negativity achieved after the 12-month study period with negative HBV DNA and HBeAg values at the last assessment). A total of 12 (25%) patients given Tα₁ and six (13%) patients given placebo showed a sustained loss of HBV DNA with a negative HBeAg value during or following the 12-month study period ( P < 0.11). These results do not confirm observations of treatment efficacy reported in other clinical studies.     

Generation and analysis of an IgG anti-platelet autoantibody reveals unusual molecular features

Although serum transfer studies implicate IgG anti-platelet autoantibodies in the premature platelet destruction of idiopathic thrombocytopenic purpura (ITP), many characteristics of these putative pathogenic autoantibodies remain unclear. The inability to obtain relevant monoclonal autoantibodies from patients has prevented their molecular, genetic and functional studies as a homogenous population. We have generated a monoclonal IgG anti-platelet α_IIbβ₃ autoantibody (termed G1) from an ITP patient. G1 binds human platelets (both resting and activated) and purified α_IIbβ₃ with a K _d of 1.57 × 10⁻⁸  M . G1 utilizes VH4 and Vλ2 genes. The G1 VH region apparently has a 30 nucleotide insertion in its second complementarity determining region (CDR). Notably, somatic CDR insertion in the VH region has been observed only in one IgG rheumatoid factor, and not in any characterized polyreactive human autoantibodies reported in the literature. Combined, these data suggest G1 may be a disease-relevant autoantibody. Further generation and study of monoclonal IgG anti-platelet antibodies are warranted to determine the significance of such unusual autoantibodies in the immunopathogenesis of chronic ITP.     

THE ASSAY OF 1α, 25-DIHYDROXYVITAMIN D3: PHYSIOLOGIC AND PATHOLOGIC MODULATION OF CIRCULATING HORMONE LEVELS

A sensitive radioreceptor assay for 1α,25-dihydroxyvitamin D₃ (1α,25-(OH)₂D₃) is utilized to quantitate the circulating concentration of this sterol in experimental animals and humans. When weanling rats are grown for 2 weeks on low calcium or low phosphate diets, limited availability of either ion elicits a five-fold increase in the plasma level of 1α,25-(OH)₂D₃. The enhancement of 1α,25-(OH)₂D₃ in calcium deficiency is dependent upon the presence of the parathyroid and/or thyroid glands, which is consistent with parathyroid hormone (PTH) mediation of this effect. In contrast, the response to phosphate deficiency is independent of these glands and may result from a direct action of low phosphate on the renal synthesis of 1α,25-(OH)₂D₃. Studies in humans indicate that the normal level of 1α,25-(OH)₂D is 2.1-4.5 ng/100 ml plasma. Patients with chronic renal failure have markedly lower circulating 1α,25-(OH)₂D and this kidney hormone is undectectable in anephirc subjects, but returns to normal within 1 day after successful renal transplantation. Hypoparathyroidism and pseudohypoparatghyroidism are associated with reduced plasma 1α,25-(OH)₂D while patients with primary hyperparathyroidism have significantly elevated sterol hormone levels. Thus, from measurements in rats and humans, it appears that circulating 1α,25-(OH)₂D₃ is regulated by PTH and/or phosphate and that abnormal plasma 1α,25-(OH)₂D₃ is a part of the pathophysiology of renal osteodystrophy and parathyroid disorders.     

Conservation of hepatitis C virus nonstructural protein 3 amino acid sequence in viral isolates during liver transplantation

Summary. As a consequence of selective pressure exerted by the immune response during hepatitis C virus (HCV) infection, a high rate of nucleotide mutations in the viral genome is observed which leads to the emergence of viral escape mutants. The aim of this study was to evaluate the evolution of the amino acid (aa) sequence of the HCV nonstructural protein 3 (NS3) in viral isolates after liver transplantation. Six patients with HCV‐induced liver disease undergoing liver transplantation (LT) were followed up for sequence analysis. Hepatitis C recurrence was observed in all patients after LT. The rate of synonymous (dS) nucleotide substitutions was much higher than that of nonsynonymous (dN) ones in the NS3 encoding region. The high values of the dS/dN ratios suggest no sustained adaptive evolution selection pressure and, therefore, absence of specific NS3 viral populations. Clinical genotype assignments were supported by phylogenetic analysis. Serial samples from each patient showed lower mean nucleotide genetic distance when compared with samples of the same HCV genotype and subtype. The NS3 samples studied had an N‐terminal aa sequence with several differences as compared with reference ones, mainly in genotype 1b‐infected patients. After LT, as compared with the sequences before, a few reverted aa substitutions and several established aa substitutions were observed at the N‐terminal of NS3. Sites described to be involved in important functions of NS3, notably those of the catalytic triad and zinc binding, remained unaltered in terms of aa sequence. Rare or frequent aa substitutions occurred indiscriminately in different positions. Several cytotoxic T lymphocyte epitopes described for HCV were present in our 1b samples. Nevertheless, the deduced secondary structure of the NS3 protease showed a few alterations in samples from genotype 3a patients, but none were seen in 1b cases. Our data, obtained from patients under important selective pressure during LT, show that the NS3 protease remains well conserved, mainly in HCV 3a patients. It reinforces its potential use as an antigenic candidate for further studies aiming at the development of a protective immune response.     

Linkage of four polymorphisms on the αIIb gene

The subunits of the platelet integrin α_IIbβ₃ are encoded by two genes located on chromosome 17. Two pathologies are associated with structural modifications of this complex: Glanzmann's thrombasthenia and alloimmune thrombocytopenia. The former is a hereditary bleeding disorder, the latter is due to an immune response linked to the presence of specific epitopes defined by single amino acid substitutions called human platelet alloantigen (HPA) systems. Analysing the α_IIb gene from 112 independent chromosomes, we have defined two new silent polymorphisms in complete linkage disequilibrium. They are reciprocally linked to HPA-3 and a previously reported 9 pb deletion in intron 21. Linkage of these four DNA markers spanning a 5 kb fragment of genomic DNA provides a new tool for analysing α_IIb gene pathology and evolution.     

Adaptive diversification within a large family of recently duplicated, placentally expressed genes

The pregnancy-associated glycoproteins (PAG) are putative peptide-binding proteins and products of a large family of genes whose expression is localized to the placental surface epithelium of artiodactyl species. We have tested the hypothesis that natural selection has favored diversification of these genes by examining patterns of nucleotide substitution in a sample of 28 closely related bovine, caprine, and ovine family members that are expressed only in trophoblast binucleate cells. Three observations were made. First, in codons encoding highly variable domains of the proteins, there was a greater accumulation of both synonymous and nonsynonymous mutations than in the more conserved regions of the genes. Second, in the variable regions, the mean number of nonsynonymous nucleotide substitutions per site was significantly greater than the mean number of synonymous substitutions per site. Third, nonsynonymous changes affecting amino acid charge occurred more frequently than expected under random substitution. This unusual pattern of nucleotide substitution implies that natural selection has acted to diversify these PAG molecules at the amino acid level, which in turn suggests that these molecules have undergone functional diversification. We estimate that the binucleate cell-expressed PAG originated 52 +/- 6 million years ago, soon after the divergence of the ruminant lineage. Thus, rapid functional diversification of PAG expressed in trophoblast binucleate cells seems to have been associated with the origin of this unique placental adaptation.     

Modeling the site-specific variation of selection patterns along lineages

The unambiguous footprint of positive Darwinian selection in protein-coding DNA sequences is revealed by an excess of nonsynonymous substitutions over synonymous substitutions compared with the neutral expectation. Methods for analyzing the patterns of nonsynonymous and synonymous substitutions usually rely on stochastic models in which the selection regime may vary across the sequence but remains constant across lineages for any amino acid position. Despite some work that has relaxed the constraint that selection patterns remain constant over time, no model provides a strong statistical framework to deal with switches between selection processes at individual sites during the course of evolution. This paper describes an approach that allows the site-specific selection process to vary along lineages of a phylogenetic tree. The parameters of the switching model of codon substitution are estimated by using maximum likelihood. The analysis of eight HIV-1 env homologous sequence data sets shows that this model provides a significantly better fit to the data than one that does not take into account switches between selection patterns in the phylogeny at individual sites. We also provide strong evidence that the strength and the frequency of occurrence of selection might not be estimated accurately when the site-specific variation of selection regimes is ignored.     

HCV selection and HVR1 evolution in a chimpanzee chronically infected with HCV-1 over 12 years.

Aim: To study hepatitis C virus (HCV) selection and hypervariable region-1 (HVR1) evolution in a chimpanzee chronically infected with HCV-1 over 12 years after inoculation with a human factor VIII concentrate contaminated with HCV. Methods: From the inoculum, the earliest chimpanzee plasma and 12 annual plasma samples, HCV fragments including HVR1 were amplified followed by cloning and sequencing. Results: Five HCV subtypes - 1a, 1b, 2a, 2b, 3a - and multiple 1a strains were identified in the inoculum. Two 1a strains were found in the earliest chimpanzee sample, while a single HCV-1 strain was detected in the 12 annual samples. None of the chimpanzee sequences were identical to those found in the inoculum. Over 12 years, HVR1 patterns changed irregularly, but a few patterns showed identical nucleotide or amino acid sequences. In the last three years, the variety of HVR1 patterns decreased, while the proportion of major patterns increased. These corresponded to a higher virus load and a lower number of amino acid substitutions. Simultaneously, the HVR1 sequences became more similar to the consensus sequence of the 1a subtype. Conclusion: HCV selection was observed from the inoculum to the inoculated chimpanzee and from the early acute hepatitis to the persistent chronic infection. The selection occurred at three levels: among subtypes after transmission, among isolates during acute hepatitis and among quasispecies in chronic infection.