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PubMed1995

T cell receptor V alpha bias can be determined by TCR-contact residues within an MHC-bound peptide.

Many antigen-specific T cell responses show profound V-region biases in one or both chains of the TCR alpha beta heterodimer. We have examined how changes in residues within an MHC-bound peptide can influence V-region selection. Single-chain TCR transgenic mice were derived using a beta-chain specific for the Kb-restricted peptide from ovalbumin, OVA257-264. Transgenic T cells were stimulated in vitro using OVA257-264 or a single residue variant having a lysine to aspartic acid substitution at determinant position 7 (7D). Recent crystallographic analyses have shown that this variant residue is involved in direct contact with the TCR. Sequence analysis of the T cell populations showed that the majority OVA257-264-specific T cells used V alpha 10-positive TCR while the majority of the 7D-specific TCR expressed the V alpha 4 element. In both peptide responses we found that some cell lines appeared to be made up of more than one clone expressing the same V alpha sequence but having limited variation at the V-J junction. These results show that the TCR contact residues within the target peptide can influence V-region bias and suggest that the first and second hypervariable regions of the TCR are directly or indirectly involved in determining peptide specificity.

Amino Acid Sequence↗

PubMed2004

Admixture, migrations, and dispersals in Central Asia: evidence from maternal DNA lineages.

Mitochondrial DNA (mtDNA) lineages of 232 individuals from 12 Central Asian populations were sequenced for both control region hypervariable segments, and additional informative sites in the coding region were also determined. Most of the mtDNA lineages belong to branches of the haplogroups with an eastern Eurasian (A, B, C, D, F, G, Y, and M haplogroups) or a western Eurasian (HV, JT, UK, I, W, and N haplogroups) origin, with a small fraction of Indian M lineages. This suggests that the extant genetic variation found in Central Asia is the result of admixture of already differentiated populations from eastern and western Eurasia. Nonetheless, two groups of lineages, D4c and G2a, seem to have expanded from Central Asia and might have their Y-chromosome counterpart in lineages belonging to haplotype P(xR1a). The present results suggest that the mtDNA found out of Africa might be the result of a maturation phase, presumably in the Middle East or eastern Africa, that led to haplogroups M and N, and subsequently expanded into Eurasia, yielding a geographically structured group of external branches of these two haplogroups in western and eastern Eurasia, Central Asia being a contact zone between two differentiated groups of peoples.

Africa↗

PubMed1992

Genomic characterization and mutation rate of hepatitis C virus isolated from a patient who contracted hepatitis during an epidemic of non-A, non-B hepatitis in Japan.

To investigate the genomic characterization of hepatitis C virus (HCV) isolated from patient who contracted hepatitis during an epidemic of non-A, non-B (NANB) hepatitis in Shimizu city, Japan, we have cloned the nucleotide sequence of the viral genome (HCV-KF) spanning the structural domain. When compared to other previously reported HCV isolates, HCV-KF showed an overall identity at the amino acid level of 90.0 to 92.1% with Japanese isolates and 80.9 to 82.1% with American-like isolates. The HCV-KF genome displays an insertion of three nucleotides in-frame (corresponding to one amino acid) found at the junction between the E1 and E2/NS1 region. The mutation rate of the HCV-KF genome was assessed by comparing the nucleotide and deduced amino acid sequences of the viral RNA obtained from the serum of the original patient with viral sequences derived from the serum of a chimpanzee inoculated with the same serum 9 years previously. The substitution rate of the viral genome was estimated at 0.9 x 10(-3) nucleotides per site per year for the HCV structural region. The highest mutation rate was found in the hypervariable region within the E2/NS1 domain. It is suggested that the outbreak in Shimizu city was caused by a strain of HCV closely related to the Japanese-like subgroup of isolates.

Americas↗

PubMed2003

Expression of multiple outer membrane protein sequence variants from a single genomic locus of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is the causative agent of an emerging tick-borne zoonosis in the United States and Europe. The organism causes a febrile illness accompanied by other nonspecific symptoms and can be fatal, especially if treatment is delayed. Persistence of A. phagocytophilum within mammalian reservoir hosts is important for ensuring continued disease transmission. In the related organism Anaplasma marginale, persistence is associated with antigenic variation of the immunoprotective outer membrane protein MSP2. Extensive diversity of MSP2 is achieved by combinatorial gene conversion of a genomic expression site by truncated pseudogenes. The major outer membrane protein of A. phagocytophilum, MSP2(P44), is homologous to MSP2 of A. marginale, has a similar organization of conserved and variable regions, and is also encoded by a multigene family containing some truncated gene copies. This suggests that the two organisms could use similar mechanisms to generate diversity in outer membrane proteins from their small genomes. We define here a genomic expression site for MSP2(P44) in A. phagocytophilum. As in A. marginale, the msp2(p44) gene in this expression site is polymorphic in all populations of organisms we have examined, whether organisms are obtained from in vitro culture in human HL-60 cells, from culture in the tick cell line ISE6, or from infected human blood. Changes in culture conditions were found to favor the growth and predominance of certain msp2(p44) variants. Insertions, deletions, and substitutions in the region of the genomic expression site encoding the central hypervariable region matched sequence polymorphisms in msp2(p44) mRNA. These data suggest that, similarly to A. marginale, A. phagocytophilum uses combinatorial mechanisms to generate a large array of outer membrane protein variants. Such gene polymorphism has profound implications for the design of vaccines, diagnostic tests, and therapy.

Amino Acid Sequence↗

PubMed1999

The capsid gene of feline calicivirus contains linear B-cell epitopes in both variable and conserved regions.

In order to map linear B-cell (LBC) epitopes in the major capsid protein of feline calicivirus (FCV), an expression library containing random, short (100- to 200-bp) fragments of the FCV F9 capsid gene was constructed. Analysis of this library showed it to be representative of the region of the capsid gene that encodes the mature capsid protein. The library was screened by using polyclonal antisera from a cat that had been challenged experimentally with F9 to identify immunoreactive clones containing LBC epitopes. Twenty-six clones that reacted positively to feline antisera in immunoblots were identified. FCV-derived sequence from these clones mapped to a region of the capsid that spanned 126 amino acids and included variable regions C and E. An overlapping set of biotinylated peptides corresponding to this region was used to further map LBC epitopes by using F9 antisera. Four principal regions of reactivity were identified. Two fell within the hypervariable region at the 5' end of region E (amino acids [aa] 445 to 451 [antigenic site (ags) 2] and aa 451 to 457 [ags 3]). However, the other two were in conserved regions (aa 415 to 421 [ags 1; region D] and aa 475 to 479 [ags 4; central region E]). The reactivity of the peptide set with antisera from 11 other cats infected with a range of FCV isolates was also determined. Ten of 11 antisera reacted to conserved ags 4, suggesting that this region may be useful for future recombinant vaccine design.

Amino Acid Sequence↗

PubMed2005

Wide range of quasispecies diversity during primary hepatitis C virus infection.

Hepatitis C virus (HCV) infections may be initiated by multiple infectious particles, resulting in a genetically heterogeneous viral population, or by a single particle, leading to a clonal population in the initial stage of infection. To determine which of these scenarios is most common, we evaluated the genetic diversity of HCV quasispecies in 12 seronegative subjects with primary infection following community exposures, six acutely infected recipients of HCV-seropositive blood transfusions and six seropositive individuals with infections of undetermined durations. RNA isolated from plasma and a region of the HCV envelope gene including the first hypervariable region (HVR-1) was reverse transcription-PCR amplified and subcloned, and multiple plasmid clones were sequenced. Phylogenetic analysis indicated that all HCV variants clustered by individuals. Genetic distances among HCV variants within recently infected subjects ranged from 1 to 7.8%. On the basis of the estimated mutation rate of HCV in vivo and the Taq polymerase error rate, primary infection viral quasispecies were classified as genetically heterogeneous when the maximum sequence divergence between genetic variants in the same person was >3%. Heterogeneous quasispecies were detected in 4 of 12 preseroconversion subjects, 1 of 6 transfusion recipients, and 4 of 6 seropositive subjects. The high level of viral quasispecies genetic diversity found in at least a third of recently infected individuals is consistent with the transmission of multiple infectious particles. Community-acquired HCV infection, predominantly the result of needle sharing by injection drug users, therefore appears to be frequently initiated by the successful transmission of multiple viral variants.

DNA, Complementary↗

PubMed1990

Self-binding antibodies (autobodies) form specific complexes in solution.

In this report we have shown that members of the murine self-binding antibody family, S107, form soluble complexes and precipitate under conditions in which non-self-binding antibodies remain in solution. Two approaches were used to demonstrate the self-association of autobodies: size-exclusion column chromatography and polyethylene glycol (PEG)-mediated precipitation assay. The anti-phosphorylcholine antibody T15 and two somatic variants, U4, which binds DNA, and U10, which has no identified specificity, produced larger precipitates in 10% PEG than other non-self-binding antibodies. The selectivity of PEG-mediated precipitation of self-binding antibodies is demonstrated by reduction of precipitation with specific haptens known to inhibit self-binding in solid-phase assays. Phosphorylcholine and nucleotides reduced precipitation of T15 and U4, respectively, but not U10. To rule out Fc-Fc mediated self-association in solution, we have also demonstrated self-complexing of F(ab')2 fragments of T15 using PEG. The self-binding locus was further dissected using peptides derived from V regions. A 24-residue peptide derived from the second hypervariable region of the VH of S107 specifically enhanced precipitation of T15, U4, and U10, but not other antibodies. These results provide evidence of a dormant potential of self-binding antibodies to precipitate under conditions that reduce the solubility of proteins. The implication of this potential is discussed with respect to pathological complex formation.

Animals↗

PubMed1989

[Immunogenetics of mesangial IgA glomerulonephritis and Schönlein- Henoch purpura].

We have recently examined polymorphisms of immunoglobuline heavy chain genes at S mu and S alpha 1 switch region loci by RFLP technique and described differences of genotype frequencies between healthy controls and patients with IgA-nephritis and Henoch-Schönlein-Purpura respectively. In the present study we further characterized the heavy chain constant region of IgG2 and IgG3 and, in addition, a hypervariable region (D 14 S 1) of unknown function localized downstream. In the present larger patient cohort we confirmed a higher frequency of S alpha 1 7.4 kb homozygotes in patients with IgA-nephritis (but not SHP), i.e. 62.0% vs 38.8% in controls, p less than 0.001. Compared to controls RFLP-frequencies of the constant region of IgG2 were also different in patients with IgA-nephritis, but not RFLP frequencies of D 14 S 1 or of the constant region of IgG3. Patients with IgA-nephritis homozygote for 7.4 kb in S alpha 1 had more adverse renal outcome and hypertension and more severe histological lesions (i.e. interstitial fibrosis, p less than 0.005).

Gene Frequency↗

PubMed1987

[Molecular organization of the N-terminal region of delta-endotoxin of Bacillus thuringiensis subspecies alesti].

The tryptic peptide sequences of the N-terminal domain ("true toxin") of delta-endotoxin of Bac. thuringiensis subspecies alesti carrying 282 amino acid residues were determined. A comparison of these sequences with the primary structures of delta-endotoxin of subspecies kurstaki (K-1, K-73) determined by an analysis of corresponding structural genes revealed a conservative region of "true toxin" (residues 29-346) and a hypervariable region (residues 347-617) carrying multiple (not less than 50%) substituents of amino acid residues. It is essential that the amino acid substituents in the variable region are distributed unevenly, being grouped into several highly variable sites carrying 7 to 31 residues. Besides, tryptic peptides of subspecies alesti delta-endotoxin were found to contain peptides having no homologs in the structures of subspecies kurstaki delta-endotoxins. It seems probable that such an uneven distribution of amino acid substituents in the structures of delta-endotoxins of subspecies alesti and kurstaki reflects the functional differences in the two halves of the N-terminal domain ("true toxin"), one of which (i. e., conservative) may be responsible for the toxic effect, while the other one (i. e., variable) seems to participate in toxin interactions with the appropriate receptors of larvae gut.

Amino Acid Sequence↗

PubMed1995

High resolution analysis of HIV-1 quasispecies in the brain.

OBJECTIVE: To characterize HIV-1 quasispecies at high resolution to determine the in vivo sequence heterogeneity of virus infecting the brain. METHODS: A 1 kilobase region of the envelope gene, which includes the five hypervariable regions, was amplified by polymerase chain reaction (PCR) using DNA obtained from brain tissue of an HIV-1-infected patient. PCR products were cloned and 50 clones sequenced. RESULTS: Thirty-nine unique nucleotide sequences producing 35 protein variants were found. A consensus sequence was identified along with three distinct subtypes, each present at a level of 12%. The sequence variation from the consensus was 0.1-2.1% at the nucleotide level with hypermutation and recombination responsible for the highest diversity. Sequence heterogeneity resulted in both the creation and the elimination of N-linked glycosylation sites. Only nine clones differed from the consensus sequence in the V3 loop. No inactivating mutations were found. CONCLUSIONS: HIV-1 proviruses found in the brain generally demonstrate a low level of genetic variability in env. However, genomes that vary considerably from the predominant species can be present at significant levels. This observation may be of importance for understanding viral pathogenesis in the central nervous system.

AIDS Dementia Complex↗

PubMed2006

Trypanosoma cruzi: sequence analysis of the variable region of kinetoplast minicircles.

The comparisons of 170 sequences of kinetoplast DNA minicircle hypervariable region obtained from 19 stocks of Trypanosoma cruzi and 2 stocks of Trypanosoma cruzi marenkellei showed that only 56% exhibited a significant homology one with other sequences. These sequences could be grouped into homology classes showing no significant sequence similarity with any other homology group. The 44% remaining sequences thus corresponded to unique sequences in our data set. In the DTU I ("Discrete Typing Units") 51% of the sequences were unique. In contrast, in the DTU IId, 87.5% of sequences were distributed into three classes. The results obtained for T. cruzi marinkellei, showed that all sequences were unique, without any similarity between them and T. cruzi sequences. Analysis of palindromes in all sequence sets show high frequency of the EcoRI site. Analysis of repetitive sequences suggested a common ancestral origin of the kDNA. The editing mechanism that occurs in kinetoplastidae is discussed.

Animals↗

PubMed1995

Variations in the core region of hepatitis C virus genomes in patients with chronic hepatitis.

In each infected patient, the population of hepatitis C virus is composed of quasispecies that differ in their nucleotide sequences. Among regions in hepatitis C virus genomes, nucleotide sequences of the hypervariable region have been shown to change quickly during the course of infection. It is not known, however, whether these variations exist in the core region that has recently been suggested to contain human lymphocyte antigen class 1 restricted sites for cytotoxic T cell recognition. To clarify this, RNA was extracted from the plasma of four patients with chronic hepatitis C. After cDNA synthesis, DNA fragments that contain the core region were amplified by the polymerase chain reaction and the diversity of the core region was analyzed by the single strand conformation polymorphism analysis. Using this method, single or multiple DNA bands were observed in each patient, and representative bands showed different nucleotide sequences. Comparison of single strand conformation polymorphism patterns revealed that the population of quasispecies changed during the course of chronic infection. These changes were more remarkable in patients with high serum alanine aminotransferase levels than those with low serum alanine aminotransferase levels. Thus, sequential variations exist in the core region of hepatitis C virus in same individuals, and the population of quasispecies as determined by the sequence of the core region changes during the course of infection, which might be related to cytopathic effects of hepatitis C virus.

Alanine Transaminase↗

PubMed1999

Hepatitis C--virology and future antiviral targets.

The hepatitis C virus is a single-stranded RNA virus with a genome approximately 9,000 nucleotides in length. The genome consists of a single, large open reading frame (ORF) and 5' and 3' untranslated regions. The highly conserved 5' untranslated region is 341 nucleotides in length with a complex secondary structure and may function as an internal ribosomal entry site (IRES). The 3' untranslated region is approximately 500 nucleotides in length and contains a hypervariable region, followed by a poly(U) sequence and a highly conserved 98-nucleotide element with a stable secondary structure. The ORF codes form a single polyprotein that is processed into as many as 10 polypeptides, including a capsid protein (core), two envelope proteins (E1 and E2), and nonstructural proteins (NS2, NS3, NS4, and NS5). Potentially suitable antiviral targets include the IRES, protease, helicase, and RNA polymerase. In vitro studies show that antisense oligonucleotides can inhibit the production of structural HCV proteins and may be therapeutically useful if the problems of stability and delivery can be solved. The binding of HCV envelope proteins to CD81, a potential receptor for viral entry into hepatocytes, has recently been described and also raises the possibility of agents to block the binding to CD81 or the entry of the virus into cells.

Antiviral Agents↗

PubMed1998

Highly biased CDR3 usage in restricted sets of beta chain variable regions during viral superantigen 9 response.

Superantigens encoded by the mouse mammary tumor virus can stimulate a large proportion of T cells through interaction with germline-encoded regions of the T cell receptor beta chain like the hypervariable region 4 (HV4) loop. However, several lines of evidence suggest that somatically generated determinants in the CDR3 region might influence superantigen responses. We stimulated T cells from donors differing at the BV6S7 allele with vSAG9 to assess the nature and structure of the T cell receptor in amplified T cells and to evaluate the contribution of non-HV4 elements in vSAG recognition. This report demonstrates that vSAG9 stimulation caused the expansion of TCR BV6-expressing T cells, although to varying degrees depending on the BV6 subfamily. The BV6S7 subfamily was preferentially expanded in all donors, but in donors homozygous for the BV6S7*2 allele, a significant number of BV6S5 T cells were amplified and showed a highly biased beta chain junctional region (BJ) and CDR3 usage. As CDR3 regions are involved in major histocompatibility complex (MHC)-peptide interaction, such a selection is highly suggestive of an intimate MHC-TCR interaction and would imply that the topology of the MHC-vSAG-TCR complex is similar to the one occurring during conventional antigen recognition.

Amino Acid Sequence↗

PubMed1996

Detection of a cluster of hepatitis C infections in a renal transplant unit by analysis of sequence variation of the NS5a gene.

Hepatitis C virus (HCV) genotype 1 isolates from 31 patients on dialysis or with renal transplants were studied for evidence of patient-to-patient spread of infection. Nucleotide sequences from a variable region (NSSV) of the NSSa (nonstructural 5a) gene and the hypervariable region of the second envelope gene. (env2) were compared. Investigation of phylogenetically related isolates of HCV type 1a with identical derived amino acid sequences in the NS5V led to recognition of a cluster of 4 patients who had received renal transplants within an 8-day period in 1984. This demonstrates the value of molecular epidemiologic techniques in reconstructing routes of infection and adds to the evidence for nosocomial transmission of HCV by routes other than blood transfusion.

Amino Acid Sequence↗

PubMed2000

Construction and characterization of chimeric hepatitis C virus E2 glycoproteins: analysis of regions critical for glycoprotein aggregation and CD81 binding.

We compared the ability of two closely related truncated E2 glycoproteins (E2(660)) derived from hepatitis C virus (HCV) genotype 1a strains Glasgow (Gla) and H77c to bind a panel of conformation-dependent monoclonal antibodies (MAbs) and CD81. In contrast to H77c, Gla E2(660) formed disulfide-linked high molecular mass aggregates and failed to react with conformation-dependent MAbs and CD81. To delineate amino acid (aa) regions associated with protein aggregation and CD81 binding, several Gla-H77c E2(660) chimeric glycoproteins were constructed. Chimeras C1, C2 and C6, carrying aa 525-660 of Gla E2(660), produced disulfide-linked aggregates and failed to bind CD81 and conformation-dependent MAbs, suggesting that amino acids within this region are responsible for protein misfolding. The presence of Gla hypervariable region 1 (aa 384-406) on H77 E2(660), chimera C4, had no effect on protein folding or CD81 binding. Chimeras C3 and C5, carrying aa 384-524 or 407-524 of Gla E2(660), respectively, were recognized by conformation-dependent MAbs and yet failed to bind CD81, suggesting that amino acids in region 407-524 are important in modulating CD81 interaction without affecting antigen folding. Comparison of Gla and H77c E2(660) aa sequences with those of genotype 1a and divergent genotypes identified a number of variant amino acids, including two putative N-linked glycosylation sites at positions 476 and 532. However, introduction of G476N-G478S and/or D532N in Gla E2(660) had no effect on antigenicity or aggregation.

Amino Acid Sequence↗

PubMed1997

Expression of Aleutian mink disease parvovirus capsid proteins in defined segments: localization of immunoreactive sites and neutralizing epitopes to specific regions.

The capsid proteins of the ADV-G isolate of Aleutian mink disease parvovirus (ADV) were expressed in 10 nonoverlapping segments as fusions with maltose-binding protein in pMAL-C2 (pVP1, pVP2a through pVP2i). The constructs were designed to capture the VP1 unique sequence and the portions analogous to the four variable surface loops of canine parvovirus (CPV) in individual fragments (pVP2b, pVP2d, pVP2e, and pVP2g, respectively). The panel of fusion proteins was immunoblotted with sera from mink infected with ADV. Seropositive mink infected with either ADV-TR, ADV-Utah, or ADV-Pullman reacted preferentially against certain segments, regardless of mink genotype or virus inoculum. The most consistently immunoreactive regions were pVP2g, pVP2e, and pVP2f, the segments that encompassed the analogs of CPV surface loops 3 and 4. The VP1 unique region was also consistently immunoreactive. These findings indicated that infected mink recognize linear epitopes that localized to certain regions of the capsid protein sequence. The segment containing the hypervariable region (pVP2d), corresponding to CPV loop 2, was also expressed from ADV-Utah. An anti-ADV-G monoclonal antibody and a rabbit anti-ADV-G capsid antibody reacted exclusively with the ADV-G pVP2d segment but not with the corresponding segment from ADV-Utah. Mink infected with ADV-TR or ADV-Utah also preferentially reacted with the pVP2d sequence characteristic of that virus. These results suggested that the loop 2 region may contain a type-specific linear epitope and that the epitope may also be specifically recognized by infected mink. Heterologous antisera were prepared against the VP1 unique region and the four segments capturing the variable surface loops of CPV. The antisera against the proteins containing loop 3 or loop 4, as well as the anticapsid antibody, neutralized ADV-G infectivity in vitro and bound to capsids in immune electron microscopy. These results suggested that regions of the ADV capsid proteins corresponding to surface loops 3 and 4 of CPV contain linear epitopes that are located on the external surface of the ADV capsid. Furthermore, these linear epitopes contain neutralizing determinants. Computer comparisons with the CPV crystal structure suggest that these sequences may be adjacent to the threefold axis of symmetry of the viral particle.

Aleutian Mink Disease Virus↗

PubMed2002

Molecular analysis of the complete set of length mutations found in the plastomes of Triticum-Aegilops species.

Precise location and nature of each of 14 length mutations detected among chloroplast DNAs of Triticum-Aegilops species by RFLP analysis were determined at the nucleotide sequence level. Each mutation was compared with at least three non-mutated wild-type plastomes as standards. These 14 length mutations were classified into 4 duplications and 10 deletions. One duplication occurred in the small single-copy region close to the border of the inverted repeat, and the remaining 13 length mutations took place in the large single-copy region. All length mutations occurred in the intergenic regions, suggesting that these length mutations do not affect plastid gene expression. Saltatory replication was the cause of all duplications, whereas intramolecular recombination mediated by short direct repeats played a substantial role in the deletions. Recurrent occurrences of certain deletion events were found in some AT-rich regions, which constituted hot spots for deletion. Out of four hypervariable regions detected among the grass plastomes, two (downstream of rbcL and a tRNA gene accumulated region) were still active after differentiation of Triticum and Aegilops complex.

Base Sequence↗