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.     

RNA recombination plays a major role in genomic change during circulation of coxsackie B viruses

RNA recombination has been shown to occur during circulation of enteroviruses, but most studies have focused on poliovirus. To examine the role of recombination in the evolution of the coxsackie B viruses (CVB), we determined the partial sequences of four genomic intervals for multiple clinical isolates of each of the six CVB serotypes isolated from 1970 to 1996. The regions sequenced were the 5'-nontranslated region (5'-NTR) (350 nucleotides [nt]), capsid (VP4-VP2, 416 nt, and VP1, approximately 320 nt), and polymerase (3D, 491 nt). Phylogenetic trees were constructed for each genome region, using the clinical isolate sequences and those of the prototype strains of all 65 enterovirus serotypes. The partial VP1 sequences of each CVB serotype were monophyletic with respect to serotype, as were the VP4-VP2 sequences, in agreement with previously published studies. In some cases, however, incongruent tree topologies suggested that intraserotypic recombination had occurred between the sequenced portions of VP2 and VP1. Outside the capsid region, however, isolates of the same serotype were not monophyletic, indicating that recombination had occurred between the 5'-NTR and capsid, the capsid and 3D, or both. Almost all clinical isolates were recombinant relative to the prototype strain of the same serotype. All of the recombination partners appear to be members of human enterovirus species B. These results suggest that recombination is a frequent event during enterovirus evolution but that there are genetic restrictions that may influence recombinational compatibility.     

Immunoglobulin heavy chains from anti-inulin myeloma proteins: evidence for a new heavy chain joining segment

Immunoglobulin heavy chains have been shown to be encoded by at least 3 widely separated genetic elements, designated variable (V), diversity (D), and joining (J), which undergo rearrangement during somatic differentiation to produce the active gene form. The D segment codes for a portion of the 3rd hypervariable region and thus potentially contributes significantly to structural diversity in this portion of the molecule. Heavy chains from anti-inulin proteins are unusual in that they essentially lack a 3rd hypervariable region. Thus, if a D segment exists in these proteins, it is extremely short, possibly 1 to 2 amino acids, and more likely serves a framework function rather than introduces structural diversity in the 3rd hypervariable region. We have completed the heavy chain variable region amino acid sequence from proteins AMPC1 and T957 bringing to 6 the number of complete sequences from this group. All of these proteins lack a 3rd hypervariable region. In addition, substitutions are found within the J segments of AMPC1 and T957, which are unlikely to be generated by the recombination event. The occurrence of Pro at position 105 in both of these J segments in contrast to the Gln found in all other heavy chains using this J segment suggests the possible existence of a previously unidentified J segment gene.     

Phylogenetic analysis of HDV isolates from HBsAg positive patients in Karachi, Pakistan

ABSTRACT: BACKGROUND: In spite of a high occurrence of Hepatitis Delta in the province of Sindh in Pakistan, no genetic study of Hepatitis Delta virus (HDV) isolates from this region was carried out. The aim of this study is to analyze the genetic proximity within local HDV strains, and relationship with other clades of HDV, using phylogenetic analysis. RESULTS: Phylogenetic analysis of nucleotide sequences of the Hepatitis Delta Antigen (HDAg) R0 region obtained in this study, showed considerable diversity among the local strains with a potential subgroup formation within clade I. The multiple sequence alignment of predicted amino acids within clade I showed many uncommon amino acid substitutions within some conserved regions that are crucial for replication and assembly of HDV. CONCLUSIONS: The studied strains showed a range of genetic diversity within HDV clade I. There is clustering of sequences into more than one group, along with formation of potential subgroup within clade I. Clustering shows the genetic closeness of strains and indicates a common origin of spread of HDV infection. Further phylogeny-based studies may provide more information about subgroup formation within clade I and may be used as an effective tool in checking and/or preventing the spread of hepatitis D virus infection in this region.     

丙型肝炎病毒高变区1中介导感染的关键氨基酸残基鉴定

丙型肝炎病毒( HCV)包膜E2蛋白氨基端的高变区1(HVR1)由27个氨基酸组成,是HCV蛋白中变异频率最高的肽段.HVR1含中和抗体表位,同时对HCV细胞侵入起重要作用,其结构与功能的关系目前尚不清楚.本研究对H77株包膜蛋白基因中的HVR1进行了一系列缺失突变,然后将突变体表达质粒与假病毒包装质粒共转染人胚肾(H...     

Structural basis for segmental gene conversion in generation of Anaplasma marginale outer membrane protein variants

Bacterial pathogens in the genus Anaplasma generate surface coat variants by gene conversion of chromosomal pseudogenes into single-expression sites. These pseudogenes encode unique surface-exposed hypervariable regions flanked by conserved domains, which are identical to the expression site flanking domains. In addition, Anaplasma marginale generates variants by recombination of oligonucleotide segments derived from the pseudogenes into the existing expression site copy, resulting in a combinatorial increase in variant diversity. Using the A. marginale genome sequence to track the origin of sequences recombined into the msp2 expression site, we demonstrated that the complexity of the expressed msp2 increases during infection, reflecting a shift from recombination of the complete hypervariable region of a given pseudogene to complex mosaics with segments derived from hypervariable regions of different pseudogenes. Examination of the complete set of 1183 variants with segmental changes revealed that 99% could be explained by one of the recombination sites occurring in the conserved flanking domains and the other within the hypervariable region. Consequently, we propose an 'anchoring' model for segmental gene conversion whereby the conserved flanking sequences tightly align and anchor the expression site sequence to the pseudogene. Associated with the recombination sites were deletions, insertions and substitutions; however, these are a relatively minor contribution to variant generation as these occurred in less than 2% of the variants. Importantly, the anchoring model, which can account for more variants than a strict segmental sequence identity mechanism, is consistent with the number of msp2 variants predicted and empirically identified during persistent infection.     

Heavy chain V region diversity in the duck-billed platypus (Ornithorhynchus anatinus): long and highly variable complementarity-determining region 3 compensates for limited germline diversity

In this work, to study the emergence of the H chain V region repertoire during mammalian evolution, we present an analysis of 25 independent H chain V regions from a monotreme, the Australian duck-billed platypus, Ornithorhynchus anatinus. All the sequences analyzed were found to form a single branch within the clan III of mammalian V region sequences in a distance tree. However, compared with a classical V gene family this branch was more diversified in sequence. Sequence analysis indicates that the apparent lack of diversity in germline V segments is well compensated for by relatively long and highly diversified D and N nucleotides. In addition, extensive sequence variation was observed in the framework region 3. Furthermore, at least five and possibly seven different J segments seem to be actively used in recombination. Interestingly, internal cysteine bridges in the complementarity-determining region (CDR)3 loop, or between the CDR2 and CDR3 loops, are found in approximately 36% of the platypus V(H) sequences. Such cysteine bridges have also been observed in cow, camel, and shark. Internal cysteine bridges may play a role in stabilizing long and diversified CDR3 and thereby have a role in increasing the affinity of the Ab-Ag interaction.     

An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH

We have determined the sequences of separate germline genetic elements which encode two parts of a mouse immunglobulin heavy chain variable region. These elements, termed gene segments, are heavy chain counterparts of the variable (V) and joining (J) gene segments of immunoglobulin light chains. The VH gene segment encodes amino acids 1-101 and the JH gene segment encodes amino acids 107-123 of the S107 phosphorylcholine-binding VH region. This JH gene segment and two other JH gene segments are located 5' to the mu constant region gene (Cmu) in germline DNA. We have also determined the sequence of a rearranged VH gene encoding a complete VH region, M603, which is closely related to S107. In addition, we have partially determined the VH coding sequences of the S107 and M167 heavy chain mRNAs. By comparing these sequences to the germline gene segments, we conclude that the germline VH and JH gene segments do not contain at least 13 nucleotides which are present in the rearranged VH genes. In S107, these nucleotides encode amino acids 102-106, which form part of the third hypervariable region and consequently influence the antigen-binding specificity of the immunoglobulin molecule. This portion of the variable region may be encoded by a separate germline gene segment which can be joined to the VH and JH gene segments. We term this postulated genetic element the D gene segment, referring to its role in the generation of heavy chain diversity. Essentially the same noncoding sequences are found 3' to the VH gene segment and as inverse complements 5' to two JH gene segments. These are the same conserved nucleotides previously found adjacent to light chain V and J gene segments. Each conserved sequence consists of blocks of seven and ten conserved nucleotides which are separated by a spacer of either 11 or 22 nonconserved nucleotides. The highly conserved spacing, corresponding to one or two turns of the DNA helix, maintains precise spatial orientations between blocks of conserved nucleotides. Gene segments which can join to one another (VK and JK, for example) always have spacers of different lengths. Based on these observations, we propose a model for variable region gene rearrangement mediated by proteins which recognize the same conserved sequences adjacent to both light and heavy chain immunoglobulin gene segments.     

Conservation of the conformation and positive charges of hepatitis C virus E2 envelope glycoprotein hypervariable region 1 points to a role in cell attachment

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease. The HCV polyprotein contains a hypervariable region (HVR1) located at the N terminus of the second envelope glycoprotein E2. The strong variability of this 27-amino-acid region is due to its apparent tolerance of amino acid substitutions together with strong selection pressures exerted by anti-HCV immune responses. No specific function has so far been attributed to HVR1. However, its presence at the surface of the viral particle suggests that it might be involved in viral entry. This would imply that HVR1 is not randomly variable. We sequenced 460 HVR1 clones isolated at various times from six HCV-infected patients receiving alpha interferon therapy (which exerts strong pressure towards quasispecies genetic evolution) and analyzed their amino acid sequences together with those of 1,382 nonredundant HVR1 sequences collected from the EMBL database. We found that (i) despite strong amino acid sequence variability related to strong pressures towards change, the chemicophysical properties and conformation of HVR1 were highly conserved, and (ii) HVR1 is a globally basic stretch, with the basic residues located at specific sequence positions. This conservation of positively charged residues indicates that HVR1 is involved in interactions with negatively charged molecules such as lipids, proteins, or glycosaminoglycans (GAGs). As with many other viruses, possible interaction with GAGs probably plays a role in host cell recognition and attachment.     

Hypervariable region IV of Salmonella gene fliCd encodes a dominant surface epitope and a stabilizing factor for functional flagella.

To identify the major antigenic determinant of native Salmonella flagella of antigenic type d, we constructed a series of mutated fliCd genes with deletions and amino acid alterations in hypervariable region IV and in region of putative epitopes as suggested by epitope mapping with synthetic octameric peptides (T.M. Joys and F. Schödel, Infect. Immun. 59:3330-3332, 1991). The expressed product of most of the mutant genes, with deletions of up to 92 amino acids in region IV, assembled into functional flagella and conferred motility on flagellin-deficient hosts. Serological analysis of these flagella with different anti-d antibodies revealed that the peptide sequence centered at amino acids 229 to 230 of flagellin was a dominant B-cell epitope at the surface of d flagella, because replacement of these two amino acids alone or together with their flanking sequence by a tripeptide specified by a linker sequence eliminated most reactivity with antisera against wild-type d flagella as tested by enzyme-linked immunosorbent assay or by Western immunoblot. Functional analysis of the mutated flagellin genes with or without an insert suggested that amino acids 180 to 214 in the 5' part of hypervariable region IV (residues 181 to 307 of the total of 505) is important to the function of flagella. The hybrid proteins formed by insertion of peptide sequence pre-S1 12-47 of hepatitis B virus surface antigen into the deleted flagellins assembled into functional flagella, and antibody to the pre-S1 sequence was detected after immunization of mice with the hybrid protein. This suggests that such mutant flagellins containing heterologous epitopes have potential as vaccines.     

Contrasting Roles of Interallelic Recombination at the Hla-a and Hla-B Loci

A statistical study of DNA sequences of alleles at the highly polymorphic class I MHC loci of humans, HLA-A and HLA-B, showed evidence of both large-scale recombination events (involving recombination of exons 1-2 of one allele with exons 3-8 of another) and small-scale recombination events (involving apparent exchange of short DNA segments). The latter events occurred disproportionately in the region of the gene encoding the antigen recognition site (ARS) of the class I molecule. Furthermore, they involved the ARS codons which are under the strongest selection favoring allelic diversity at the amino acid level. Thus, the frequency of recombinant alleles appears to have been increased by some form of balancing selection (such as overdominant selection) favoring heterozygosity in the ARS. These analyses also revealed a striking difference between the A and B loci. Recombination events appear to have occurred about twice as frequently at the B locus, and recombinants at the B locus were significantly more likely to affect polymorphic sites in the ARS. At the A locus, there are well-defined allelic lineages that have persisted since prior to the human-chimpanzee divergence; but at the B locus, there is no evidence for such long-lasting allelic lineages. Thus, relatively frequent interallelic recombination has apparently been a feature of the long-term evolution of the B locus but not of the A locus.     

Hypervariable regions in the putative glycoprotein of hepatitis C virus.

A comparison of the sequences of the putative glycoprotein region in three independent cDNA clones of hepatitis C virus and of sequences of four other clones revealed extensive genetic variation clustered and interspersed with highly conserved amino acid sequences. We obtained evidence for two hypervariable regions in the putative envelope glycoprotein, one region was assumed to be a potential antigenic site, as deduced from the hydrophilicity and analyses of secondary structures. These observations suggest the existence of a large pool of antigenic variants of hepatitis C virus, in Japan.     

Genetic changes in hepatitis delta virus from acutely and chronically infected woodchucks

A woodchuck-derived hepatitis delta virus (HDV) inoculum was created by transfection of a genotype I HDV cDNA clone directly into the liver of a woodchuck that was chronically infected with woodchuck hepatitis virus. All woodchucks receiving this inoculum became positive for HDV RNA in serum, and 67% became chronically infected, similar to the rate of chronic HDV infection in humans. Analysis of HDV sequences obtained at 73 weeks postinfection indicated that changes had occurred at a rate of 0.5% per year; many of these modifications were consistent with editing by host RNA adenosine deaminase. The appearance of sequence changes, which were not evenly distributed on the genome, was correlated with the course of HDV infection. A limited number of modifications occurred in the consensus sequence of the viral genome that altered the sequence of the hepatitis delta antigen (HDAg). All chronically infected animals examined exhibited these changes 73 weeks following infection, but at earlier times, only one of the HDV carriers exhibited consensus sequence substitutions. On the other hand, sequence modifications in animals that eventually recovered from HDV infection were apparent after 27 weeks. The data are consistent with a model in which HDV sequence changes are selected by host immune responses. Chronic HDV infection in woodchucks may result from a delayed and weak immune response that is limited to a small number of epitopes on HDAg.     

Recombination and population mosaic of a multifunctional viral gene, adeno-associated virus cap

Homologous recombination is a dominant force in evolution and results in genetic mosaics. To detect evidence of recombination events and assess the biological significance of genetic mosaics, genome sequences for various viral populations of reasonably large size are now available in the GenBank. We studied a multi-functional viral gene, the adeno-associated virus (AAV) cap gene, which codes for three capsid proteins, VP1, VP2 and VP3. VP1-3 share a common C-terminal domain corresponding to VP3, which forms the viral core structure, while the VP1 unique N-terminal part contains an enzymatic domain with phospholipase A2 activity. Our recombinant detection program (RecI) revealed five novel recombination events, four of which have their cross-over points in the N-terminal, VP1 and VP2 unique region. Comparison of phylogenetic trees for different cap gene regions confirmed discordant phylogenies for the recombinant sequences. Furthermore, differences in the phylogenetic tree structures for the VP1 unique (VP1u) region and the rest of cap highlighted the mosaic nature of cap gene in the AAV population: two dominant forms of VP1u sequences were identified and these forms are linked to diverse sequences in the rest of cap gene. This observation together with the finding of frequent recombination in the VP1 and 2 unique regions suggests that this region is a recombination hot spot. Recombination events in this region preserve protein blocks of distinctive functions and contribute to convergence in VP1u and divergence of the rest of cap. Additionally the possible biological significance of two dominant VP1u forms is inferred.     

Characterization of hypervariable regions in the putative envelope protein of hepatitis C virus.

We previously identified two hypervariable regions [HVR1 (27 amino acids) and HVR2 (7 amino acids)] in the putative envelope glycoprotein (gp70) by comparison of the amino acid sequences of many isolates of the HCV-II genotype. To understand the functional features of these HVRs, using the polymerase chain reaction we analyzed the rate of actual sequence variability in the region including HVR1 and HVR2 of HCV isolated successively at intervals of several months from two patients with chronic C-type hepatitis. In both patients, the amino acid sequence of HVR1, but not HVR2, was found to change dramatically during the observation period (about one amino acid per month). However, no alteration of the amino acid sequence of HVR1 of HCV was observed in a patient in the acute phase of chronic hepatitis. Restriction digestion analysis of sequence diversity showed that a HCV genome with a newly introduced mutation in HVR1 often became the predominant population at the next time of examination. Alterations of amino acids in HVR1 occurred sequentially in the two patients in the chronic phase. These findings suggest that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection.     

The V-region sequence of the H chain from a third rabbit anti-pneumococcal antibody.

The amino acid sequence of the V (variable) region of the heavy (H) chain of rabbit antibody BS-1, raised against type III pneumococcal vaccine, is reported. Together with the sequence data of the V region of the light (L) chain previously determined [Jaton (1974a) Biochem. J. 141, 1-13], the present work completes the analysis of the V domain of the homogeneous antibody BS-1. The V domains (VL + VH regions) of this antibody are compared with those of two other anti-(type III) pneumococcal antibodies BS-5 and K-25 [Jaton (1975) Biochem. J. 147, 235-247]. Except for the second hypervariable section of the L chains, these antibodies have very different sequences in the hypervariable segments of the V domains. Within the third hypervariable region of the H chain, each antibody has a different length: BS-1 is three amino acids shorter than K-25 and two amino acids shorter than BS-5. When the sequences in that section are aligned for maximal homology, only two residues, glycine-97 and leucine-101, are common to the three antibodies. On the basis of the amino acid sequences of these three anti-pneumococcal antibodies, the results do not support the concept of a simple correlation between primary structure in the hypervariable sections (known to determine the shape of the combining site) and antigen-binding specificity.     

Random mutations to evaluate the role of bases at two important single-stranded regions of genomic HDV ribozyme.

In elucidating function of two important single-stranded regions [SSrA (726-731 nt) and SSrB (762-766 nt)] derived mainly from three secondary structure models in genomic hepatitis delta virus (HDV) ribozyme possessing self-cleavage activity, we have constructed several random mutants at those two regions on the HDV88 molecule (683-770 nt) by oligonucleotide-directed mutagenesis. When self-cleavage activities were compared among mutants, at the region SSrA, G726 was found to play an important role during cleavage reaction since substitutions of the base to A (mutant A20) or C (mutant A16) or U (mutant A23), reduced the ribozyme activity to very low levels suggesting the importance of G726 position. C763 at SSrB region was found to play a more significant role during catalysis than G726 (at region SSrA) since any substitutions at C763 completely inactivated the ribozyme. Other bases located in these two regions could be substituted to other bases at the expense of some self-cleavage activity. The results presented here together with our previous deletion analysis indicate that these two regions may play an important role during cleavage process.     

天津市致病毒性脑炎柯萨奇病毒B组5型全基因组序列分析

肠道病毒是我国病毒性脑炎(Viral encephalitis,VE)的主要病原体。本文研究对4株引起VE的天津柯萨奇病毒B组5型(Coxsackievirus B5,CV-B5)分离株进行Illumina MiniSeq高通量测序,并对其全基因组特征、进化及重组特点进行分析。结果提示,4株CV-B5天津分离株的全基因组核苷酸和氨基酸序列同源性分别为84.5%~100.0%和98.1%~100.0%,与国内流行株的全基因组核苷酸序列同源性为83.2%~96.5%,氨基酸序列同源性为96.4%~99.4%。基于全基因组的系统进化分析将CV-B5流行株分为A-D四个基因型,其中天津与国内流行株均属于C基因型。C基因型进一步分为3个进化分支,而天津分离株处在两个不同的分支上。基于基因组各区段序列的系统进化与SimPlot重组分析结果显示,天津分离株15-39N、15-41N与埃可病毒30型(Echovirus 30,E-30)原型株在P3区3B、3C、3D区域均检测到重组信号。本研究有助于了解CV-B5的全基因组特点和重组规律,为相关疾病的防控提供依据。     

Homeologous recombination in Solanum lycopersicoides introgression lines of cultivated tomato

A library of "introgression lines" containing Solanum lycopersicoides chromosome segments in the genetic background of cultivated tomato (Lycopersicon esculentum) was used to study factors affecting homeologous recombination. Recombination rates were estimated in progeny of 43 heterozygous introgressions and whole-chromosome substitution lines, together representing 11 of the 12 tomato chromosomes. Recombination within homeologous segments was reduced to as little as 0-10% of expected frequencies. Relative recombination rates were positively correlated with the length of introgressed segments on the tomato map. The highest recombination (up to 40-50% of normal) was observed in long introgressions or substitution lines. Double-introgression lines containing two homeologous segments on opposite chromosome arms were synthesized to increase their combined length. Recombination was higher in the double than in the single segment lines, despite a preference for crossovers in the region of homology between segments. A greater increase in homeologous recombination was obtained by crossing the S. lycopersicoides introgression lines to L. pennellii--a phylogenetically intermediate species--or to L. esculentum lines containing single L. pennellii segments on the same chromosome. Recombination rates were highest in regions of overlap between S. lycopersicoides and L. pennellii segments. The potential application of these results to breeding with introgression lines is discussed.