Characteristics of Nucleotide Substitution in the Hepatitis C Virus Genome: Constraints on Sequence Change in Coding Regions at Both Ends of the Genome

Comparison of complete genome sequences for different variants of hepatitis C virus (HCV) reveals several different constraints on sequence change. Synonymous changes are suppressed in coding regions at both 5′ and 3′ ends of the genome. No evidence was found for the existence of alternative reading frames or for a lower mutation frequency in these regions. Instead, suppression may be due to constraints imposed by RNA secondary structures identified within the core and NS5b genes. Nonsynonymous substitutions are less frequent than synonymous ones except in the hypervariable region of E2 and, to a lesser extent, in E1, NS2, and NS5b. Transitions are more frequent than transversions, particularly at the third position of codons where the bias is 16:1. In addition, nucleotide substitutions may not occur symmetrically since there is a bias toward G or C at the third position of codons, while T ↔ C transitions were twice as frequent as A ↔ G transitions. These different biases do not affect the phylogenetic analysis of HCV variants but need to be taken into account in interpreting sequence change in longitudinal studies. Received: 9 September 1996 / Accepted: 20 April 1997     

In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus

Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.     

Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy

We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 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, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1 dN/dS ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1 dN/dS ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.     

Molecular evolution of the major capsid protein VP1 of enterovirus 70.

Nucleotide sequences of the genome RNA encoding capsid protein VP1 (918 nucleotides) of 18 enterovirus 70 (EV70) isolates collected from various parts of the world in 1971 to 1981 were determined, and nucleotide substitutions among them were studied. The genetic distances between isolates were calculated by the pairwise comparison of nucleotide difference. Regression analysis of the genetic distances against time of isolation of the strains showed that the synonymous substitution rate was very high at 21.53 x 10(-3) substitution per nucleotide per year, while the nonsynonymous rate was extremely low at 0.32 x 10(-3) substitution per nucleotide per year. The rate estimated by the average value of synonymous and nonsynonymous substitutions (W.-H. Li, C.-C. Wu, and C.-C. Luo, Mol. Biol. Evol. 2:150-174, 1985) was 5.00 x 10(-3) substitution per nucleotide per year. Taking the average value of synonymous and nonsynonymous substitutions as genetic distances between isolates, the phylogenetic tree was inferred by the unweighted pairwise grouping method of arithmetic average and by the neighbor-joining method. The tree indicated that the virus had evolved from one focal place, and the time of emergence was estimated to be August 1967 +/- 15 months, 2 years before first recognition of the pandemic of acute hemorrhagic conjunctivitis. By superimposing every nucleotide substitution on the branches of the phylogenetic tree, we analyzed nucleotide substitution patterns of EV70 genome RNA. In synonymous substitutions, the proportion of transitions, i.e., C<==>U and G<==>A, was found to be extremely frequent in comparison with that reported on other viruses or pseudogenes. In addition, parallel substitutions (independent substitutions at the same nucleotide position on different branches, i.e., different isolates, of the tree) were frequently found in both synonymous and nonsynonymous substitutions. These frequent parallel substitutions and the low nonsynonymous substitution rate despite the very high synonymous substitution rate described above imply a strong restriction on nonsynonymous substitution sites of VP1, probably due to the requirement for maintaining the rigid icosahedral conformation of the virus.     

Nucleotide sequence of mouse Tcp-1a cDNA

We have isolated complete cDNA clones encoding the mouse t-complex polypeptides 1A and 1B (TCP-1A and TCP-1B) from t-haplotype and wild-type (wt) mice, respectively. The complete nucleotide (nt) sequence of the Tcp-1a cDNA was determined. The Tcp-1a cDNA has an open reading frame (ORF) encoding a 60-kDa protein of 556 amino acids (aa). A comparison of nt sequences between the Tcp-1a and Tcp-1b cDNAs revealed that the 1786-bp regions upstream from their polyadenylation signals differed by 17 substitutions and that Tcp-1a had different polyadenylation sites from Tcp-1b. In these ORFs, 15 bp were substituted between the two alleles, occurring in 14 codons and resulting in eleven single-aa substitutions. Among these 15 substitutions, twelve were nonsynonymous (aa change) and three were synonymous (no aa change). The aa substitution in TCP-1 has occurred at least 20 times faster between t-haplotype and wt than between mouse and human or mouse and Drosophila.     

Pattern of nucleotide substitution in the overlapping nonstructural genes of influenza A virus and implication for the genetic diversity of the H5N1 subtype

In viruses under strong pressure to minimize genome size, overlapping genes represent a fine strategy to condense a maximum amount of information into short nucleotide sequences. Here, we investigated the evolution of the genes encoding the nonstructural proteins NS1 and NS2 of influenza A virus (IAV), which are one of the best characterized cases of gene overlap. By a detailed analysis of about four hundred sequences grouped into 11 IAV subtypes, we found that the overlapping coding region of the NS1 gene shows a significant increase of the rate of nonsynonymous change, with respect to its nonoverlapping counterpart. The same feature was observed in the overlapping coding region of the NS2 gene. Such a variation pattern, which implies the occurrence of several amino acid substitutions in the protein regions encoded by overlapping frames, is different from the pattern of constrained evolution typical of other viral overlapping-gene systems. Amino acid sequence analysis of the NS1 and NS2 proteins revealed that some nonsynonymous substitutions, located in the region of gene overlap, play a critical role in shaping the genetic diversity of the highly pathogenic subtype H5N1. Since both proteins contribute to disease pathogenesis by affecting many virus and host-cell processes, information provided by this study should be useful to highlight the impact of nonstructural gene variation on the pathogenicity of H5N1 viruses.     

Sequence Variation of the Hypervariable Region in HCV Carriers with Normal ALT Levels: A Comparison with Symptomatic Carriers

The clinical significance of the hypervariable region (HVR) in the N-terminus of the E2/NS1 region, which encodes the putative envelope glycoprotein (gp 70) of HCV, has not yet been elucidated. We studied the relation between HVR changes and elevation of the alanine aminotransaminase (ALT) level due to liver cell injury as well as the persistence of HCV infection. Three patients (carrier group) who were HCV RNA positive and had normal ALT levels for as long as five years and three patients with high ALT levels were studied. None of the six patients had a history of treatment. HCV RNA was extracted from serum obtained from each patient in 1990 and 1995. The E2/NS1 region, including HVR-1 and HVR-2, was amplified using the RT-PCR method. PCR products were cloned and nucleotide sequences were determined using the dideoxynucleotide chain termination method. No clear correlation was found between the ALT levels and the number of nucleotide substitutions in HVR-1. The number of nucleotide substitutions in HVR-1 during the five years was greater than in other regions. Furthermore, more nucleotide substitutions occurred in the 1st and 2nd codon positions of HVR-1 than in the control region, even in the carrier group. In conclusion, HVR-1 changes are probably a more important factor in persistent viral infection than liver cell injury.     

四份中国HCV阳性血清中包膜蛋白E1/E2准种基因的序列分析及新型插入突变的发现

为分析四份中国丙型肝炎病毒（HCV）阳性血清中包膜蛋白E1/E2基因的准种特征。本研究对从4份中国HCV阳性血清（1b亚型：274、366、383;2a亚型：283）中提取的HCV核酸,采用逆转录-聚合酶反应扩增编码全长E1/E2蛋白（191～764aa）的基因片段,随机挑取多个克隆测序。根据E1/E2基因核苷酸的序列与其他相关序列（来自于GenBank）构建亲缘性关系进化树,进行核苷酸与氨基酸同源性分析并对重要的基因位点进行分析。共获得阳性克隆序列43个（274株10个,283株12个,366株13个,383株8个）,发现高变区HVR1、HVR2的基因异质性高,而其他抗体中和表位及跨膜区I、II及N末端糖基化位点相对保守。并首次发现在HCV 2a亚型（283血清）中多个准种序列存在1279nt（E1区,313aa）处单碱基插入优势基因突变,导致HCV包膜蛋白编码突变与中断（E2区,398aa）。本研究对中国HCV代表株包膜蛋白E1/E2编码基因的准种多样性及一种新型插入突变进行了描述,可为进一步研究HCV免疫逃避与慢性化机制提供重要信息。关键词：丙型肝炎病毒;包膜蛋白;序列分析;准种;插入突变     

Analysis of the complete open reading frame of genotype 2b hepatitis C virus in association with the response to peginterferon and ribavirin therapy

Background and Aims

Patients infected with genotype 2b hepatitis C virus (HCV) generally can achieve favorable responses to pegylated-interferon plus ribavirin therapy (PEG-IFN/RBV). However, a proportion of patients show poorer responses and the correlation between viral sequence variation and treatment outcome remains unclear.

Methods

The pretreatment complete open reading frame (ORF) sequences of genotype 2b HCV determined by direct sequencing were investigated for correlation with the final outcome in a total of 60 patients.

Results

In this study group, 87.5% (14/16) of non-sustained virological response (non-SVR) patients (n = 16) were relapsers. Compared to sustained virological response (SVR) patients (n = 44), non-SVR patients were older and could not achieve prompt viral clearance after the therapy induction. Comparing each viral protein between the two groups, viral sequences were more diverse in SVR patients and that diversity was found primarily in the E1, p7, and NS5A proteins. In searching for specific viral regions associated with the final outcome, several regions in E2, p7, NS2, NS5A, and NS5B were extracted. Among these regions, part of the interferon sensitivity determining region (ISDR) was included. In these regions, amino acid substitutions were associated with the final outcome in an incremental manner, depending upon the number of substitutions.

Conclusions

Viral sequences are more diverse in SVR patients than non-SVR patients receiving PEG-IFN/RBV therapy for genotype-2b HCV infection. Through systematic comparison of viral sequences, several specific regions, including part of the ISDR, were extracted as having significant correlation with the final outcome.     

Sequence evolution of the hypervariable region in the putative envelope region E2/NS1 of hepatitis C virus is correlated with specific humoral immune responses.

Sequence evolution of the hypervariable region 1 (HVR1) in the N terminus of E2/NS1 of hepatitis C virus (HCV) was studied retrospectively in six chimpanzees inoculated with the same genotype 1b strain, containing a unique predominant HVR1 sequence. Immediately after inoculation, all animals contained the same HVR predominant sequence. Two animals developed an acute self-limiting infection. Anti-HVR1 immunoglobulin G (IgG) was produced 40 to 60 days after inoculation and rapidly disappeared after normalization of transaminases. Another chimpanzee, previously infected with human immunodeficiency virus type 1, showed a delayed response to HVR1 epitopes after superinfection with HCV. No sequence variation of HVR1 was observed in these two animals during the transient viremia in the acute phase. Three other chimpanzees developed a chronic HCV infection. During follow up, sequence evolution occurred in two animals and their anti-HVR1 response remained at varying but detectable levels. The first mutations occurred immediately after the production of anti-HVR1 during the acute phase. However, IgM anti-HVR1 was not detectable. Remarkably, HVR1 sequences remained conserved for more than 6 years in another chronically infected animal. This correlated with the complete absence of detectable anti-HVR1 during this period. Seven years after inoculation, anti-HVR1 IgG was produced and coincided with an HVR1 alteration. These results strongly suggest the involvement of neutralizing anti-HVR antibodies in sequence evolution of HVR1 through immune selection.     

Molecular evolution of hepatitis B virus over 25 years

Determining the longitudinal molecular evolution of hepatitis B virus (HBV) is difficult due to HBV's genomic complexity and the need to study paired samples collected over long periods of time. In this study, serial samples were collected from eight hepatitis B virus e antigen-negative asymptomatic carriers of HBV genotype B in 1979 and 2004, thus providing a 25-year period to document the long-term molecular evolution of HBV. The rate, nature, and distribution of mutations that emerged over 25 years were determined by phylogenetic and linear regression analysis of full-length HBV genome sequences. Nucleotide hypervariability was observed within the polymerase and pre-S/S overlap region and within the core gene. The calculated mean number of nucleotide substitutions/site/year (7.9 x 10(-5)) was slightly higher than published estimates (1.5 x 10(-5) to 5 x 10(-5)). Nucleotide changes in the quasispecies population did not significantly alter the molecular evolutionary rate, based on linear regression analysis of evolutionary distances among serial clone pre-S region sequences. Therefore, the directly amplified or dominant sequence was sufficient to estimate the putative molecular evolutionary rate for these long-term serial samples. On average, the ratio of synonymous (dS) to nonsynonymous (dN) substitutions was highest for the polymerase-coding region and lowest for the core-coding region. The low dS/dN ratios observed within the core suggest that selection occurs within this gene region, possibly as an immune evasion strategy. The results of this study suggest that HBV sequence divergence may occur more rapidly than previously estimated, in a host immune phase-dependent manner.     

Long-term follow-up of chimpanzees inoculated with the first infectious clone for hepatitis C virus

Two chimpanzees (Ch1535 and Ch1536) became infected with hepatitis C virus (HCV) following intrahepatic inoculation with RNA transcribed from a full-length cDNA clone of the virus. Both animals were persistently infected and have been followed for 60 weeks. They showed similar responses to infection, with transient liver enzyme elevations and liver inflammatory responses, which peaked at weeks 17 (Ch1535) and 12 (Ch1536) postinoculation (p.i.). Antibody responses to structural and nonstructural proteins were first detected at weeks 13 (Ch1535) and 10 (Ch1536) p.i. Serum RNA titers increased steadily during the first 10 to 13 weeks but decreased sharply in both animals following antibody and inflammatory responses. Despite direct evidence of humoral immune responses to multiple viral antigens, including hypervariable region 1 (HVR1), both animals remained chronically infected. Detailed sequence analysis of serum HCV RNA revealed no change in the majority HVR1 sequence in Ch1535 and a single-amino-acid mutation in Ch1536, with very little clonal variation in either animal. Full-length genome analysis at week 60 revealed several amino acid substitutions localized to antigens E1, E2, p7, NS3, and NS5. Of these, 55.6 and 40% were present as the majority sequence in serum RNA isolated at week 26 p.i. (Ch1535) and week 22 p.i. (Ch1536), respectively, and could represent immune escape mutations. Mutations accumulated at a rate of 1.57 x 10(-3) and 1.48 x 10(-3) nucleotide substitutions/site/year for Ch1535 and Ch1536, respectively. Taken together, these data indicate that establishment of a persistent HCV infection in these chimpanzees is not due to changes in HVR1; however, the possibility remains that mutations arising in other parts of the genome contributed to this persistence.     

Evolution of Envelope Sequences from the Genital Tract and Peripheral Blood of Women Infected with Clade A Human Immunodeficiency Virus Type 1

The development of viral diversity during the course of human immunodeficiency virus type 1 (HIV-1) infection may significantly influence viral pathogenesis. The paradigm for HIV-1 evolution is based primarily on studies of male cohorts in which individuals were presumably infected with a single virus variant of subtype B HIV-1. In this study, we evaluated virus evolution based on sequence information of the V1, V2, and V3 portions of HIV-1 clade A envelope genes obtained from peripheral blood and cervical secretions of three women with genetically heterogeneous viral populations near seroconversion. At the first sample following seroconversion, the number of nonsynonymous substitutions per potential nonsynonymous site (dn) significantly exceeded substitutions at potential synonymous sites (ds) in plasma viral sequences from all individuals. Generally, values of dn remained higher than values of ds as sequences from blood or mucosa evolved. Mutations affected each of the three variable regions of the envelope gene differently; insertions and deletions dominated changes in V1, substitutions involving charged amino acids occurred in V2, and sequential replacement of amino acids over time at a small subset of positions distinguished V3. The relationship among envelope nucleotide sequences obtained from peripheral blood mononuclear cells, plasma, and cervical secretions was evaluated for each individual by both phylogenetic and phenetic analyses. In all subjects, sequences from within each tissue compartment were more closely related to each other than to sequences from other tissues (phylogenetic tissue compartmentalization). At time points after seroconversion in two individuals, there was also greater genetic identity among sequences from the same tissue compartment than among sequences from different tissue compartments (phenetic tissue compartmentalization). Over time, temporal phylogenetic and phenetic structure was detectable in mucosal and plasma viral samples from all three women, suggesting a continual process of migration of one or a few infected cells into each compartment followed by localized expansion and evolution of that population.     

Genetic drift in hypervariable region 1 of the viral genome in persistent hepatitis C virus infection.

The hypervariable region 1 (HVR1) of the putative second envelope glycoprotein (gp70) of hepatitis C virus (HCV) contains a sequence-specific immunological B-cell epitope that induces the production of antibodies restricted to the specific viral isolate, and anti-HVR1 antibodies are involved in the genetic drift of HVR1 driven by immunoselection (N. Kato, H. Sekiya, Y. Ootsuyama, T. Nakazawa, M. Hijikata, S. Ohkoshi, and K. Shimotohno, J. Virol. 67:3923-3930, 1993). We further investigated the sequence variability of the HCV genomic region that entirely encodes the envelope proteins (gp35 and gp70); these sequences were derived from virus isolated during the acute and chronic phases of hepatitis in one patient, and we found that HVR1 was a major site for genetic mutations in HCV after the onset of hepatitis. We carried out epitope-mapping experiments using the HVR1 sequence derived from the acute phase of hepatitis and identified two overlapping epitopes which are each composed of 11 amino acids (positions 394 to 404 and 397 to 407). The presence of two epitopes within HVR1 suggested that epitope shift happened during the course of hepatitis. Four of six amino acid substitutions detected in HVR1 were located within the two epitopes. We further examined the reactivities of anti-HVR1 antibodies to the substituted amino acid sequences within the two epitopes. HVR1 variants in both epitopes within the HVR1 escaped from anti-HVR1 antibodies that were preexisting in the patient's serum.     

Accelerated evolution in the protein-coding region of galectin cDNAs, congerin I and congerin II, from skin mucus of conger eel (Conger myriaster).

Two cDNAs encoding galectins named congerins I and II from the skin mucus of conger eel (Conger myriaster) were isolated and sequenced. Comparison of the nucleotide sequences of congerins I and II showed that the sequence similarities of the 5' and 3' untranslated regions (86 and 88%, respectively) were much higher than those of the protein-coding region (73%). The numbers of nucleotide substitutions per site (KN) for the untranslated regions are smaller than the numbers of nucleotide substitutions per synonymous site (KS) for the protein coding region. Furthermore, nonsynonymous nucleotide substitutions have accelerated more frequently than synonymous nucleotide substitutions in the protein coding region (KA/KS = 2.57). These results suggest that accelerated substitutions have occurred in the protein-coding regions of galectin genes to generate diverse galectins with different molecular properties. Northern blot analysis showed that both congerins were expressed not only in the skin tissues but also in the stomach of conger eel.     

SRY基因在人猿超科和旧大陆猴中具有不同的进化规律

通过ＰＣＲ扩增、测序,得到了白臀叶猴和红面猴的ＳＲＹ基因全序列。结合现有的灵长类其他物种序列进行分析,验证了ＨＭＧ盒的保守性。通过构建系统发育树,比较旧大陆猴和人猿超科两个类群内和类群间ＨＭＧ盒侧翼序列Ｋａ／Ｋｓ的比率。有趣的是,人猿超科两物种比较呈现较高的Ｋａ／Ｋｓ比值,但在旧大陆猴中及旧大陆猴与狨猴间的Ｋａ／Ｋｓ比值显著低于人猿超科的,呈现很不同的格局。同时,对于ＨＭＧ盒序列,Ｋａ／Ｋｓ比值在     

Molecular Evolution of the Plant R Regulatory Gene Family

Anthocyanin pigmentation patterns in different plant species are controlled in part by members of the myc-like R regulatory gene family. We have examined the molecular evolution of this gene family in seven plant species. Three regions of the R protein show sequence conservation between monocot and dicot R genes. These regions encode the basic helix-loop-helix domain, as well as conserved N-terminal and C-terminal domains; mean replacement rates for these conserved regions are 1.02 X 10(-9) nonsynonymous nucleotide substitutions per site per year. More than one-half of the protein, however, is diverging rapidly, with nonsynonymous substitution rates of 4.08 X 10(-9) substitutions per site per year. Detailed analysis of R homologs within the grasses (Poaceae) confirm that these variable regions are indeed evolving faster than the flanking conserved domains. Both nucleotide substitutions and small insertion/deletions contribute to the diversification of the variable regions within these regulatory genes. These results demonstrate that large tracts of sequence in these regulatory loci are evolving at a fairly rapid rate.     

Intrahost human immunodeficiency virus type 1 evolution is related to length of the immunocompetent period.

The antigenic diversity threshold theory predicts that antigenic sites of human immunodeficiency virus type 1, such as the V3 region of the external glycoprotein gp120, evolve more rapidly during the symptom-free period in individuals progressing to AIDS than in those who remain asymptomatic for a long time. To test this hypothesis, genomic RNA sequences were obtained from the sera of 44 individuals at seroconversion and 5 years later. The mean number of nonsynonymous nucleotide substitutions in the V3 region of the viruses circulating in 31 nonprogressors (1.1 x 10(-2) +/- 0.1 x 10(-2) per site per year) was higher than the corresponding value for 13 progressors (0.66 x 10(-2) +/- 0.1 x 10(-2) per site per year) (P < 0.01), while no difference between the mean numbers of synonymous substitutions in the two groups was seen (0.37 x 10(-2) +/- 0.1 x 10(-2) and 0.51 x 10(-2) +/- 0.2 x 10(-2) per site per year for nonprogressors and progressors, respectively; P > 0.1). The mean ratios of synonymous nucleotide p distance to nonsynonymous p distance were 0.35 for nonprogressors and 0.62 for progressors. The number of nonsynonymous substitutions was not associated with virus load or virus phenotype, which are established predictors of disease progression, but correlated strongly with the duration of the immunocompetent period (r2 = 0.41; P = 0.001). This indicates that there is no causative relationship between intrahost evolution and CD4+ cell decline. Our data suggest that intrahost evolution in human immunodeficiency virus type 1 infection is driven by selective forces, the strength of which is related to the duration of the immunocompetent period.     

Evolution of glucagon genes

Statistical analyses of DNA sequences of the preproglucagon genes from bovine, human, hamster, and anglerfish suggest that a gene duplication creating two anglerfish genes (AF I and II) occurred about 160 Myr ago, long after the separation of fish and mammals. The analyses further suggest that the internal duplication producing the glucagon and glucagon-like peptide II (GLP-II) regions occurred about 1.2 billion years ago, which would indicate that the GLP-II region was present in the ancestral anglerfish sequence but was silenced or deleted before the gene duplication separating AF I and II. The glucagon-like peptide I (GLP-I) was derived from a duplication of the ancestral glucagon region about 800 Myr ago. The rate of synonymous substitution in these genes is approximately 4.3 x 10(-9) substitutions per year per synonymous site. The rate of nonsynonymous substitution in the signal peptide region is about 1.1 x 10(-9) substitutions per year per nonsynonymous site, a high rate comparable to that in the C-peptide region of preproinsulin. The rate of nonsynonymous substitution in the glicentin-related pancreatic polypeptide (GRPP) region is 0.63 x 10(-9) for the comparisons between mammalian species and 1.8 x 10(-9) for the comparisons between fish and mammals; the moderate rate in mammals suggests a physiological role for GRPP. The glucagon region is extremely conservative; no nonsynonymous substitution is observed in the mammalian genes, and a nonsynonymous rate of 0.18 x 10(-9) was obtained from the comparisons between fish and mammals. In the GLP-I region, the rate of nonsynonymous substitution was estimated to be 0.08 x 10(-9) for the comparisons between mammalian species and 0.30 x 10(- 9) for the comparisons between fish and mammals. In the GLP-II region, the rate was estimated to be 0.25 x 10(-9) for the comparisons between mammalian species. Thus, GLP-I and II are also very conservative, which suggests an important physiological role for these peptides.