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PubMed1997

Type-4 pilus-structure: outside to inside and top to bottom--a minireview.

We have recently proposed a computational model of the N. gonorrhoeae pilus fiber based on the high resolution X-ray crystal structure of the component protein pilin, combined with available biophysical and genetic data [Parge et al. (1995) Nature 378, 32-38]. In parallel, we have used anti-peptide antibodies to distinguish buried and exposed regions of pilin within the assembled fiber [Forest et al. (1996) Infect. Immun. 64, 644-652]. This mini-review addresses the properties of the current pilus model and the locations of end-exposed epitopes. The fiber forms a three-layered structure of coiled conserved alpha helices surrounded by beta-sheet, with the hypervariable region as the most highly exposed portion. Overall the pilus model developed from diffraction and antibody mapping is expected to be representative of type-4 pili with general implications for type-4 assembly, function, and interactions with other proteins and cell membranes.

Amino Acid Sequence↗
PubMed2000

The crystal structure of the Fab fragment of a rat monoclonal antibody against the main immunogenic region of the human muscle acetylcholine receptor.

The crystal structure of the Fab fragment of a rat monoclonal antibody, number 192, with a very high affinity (Kd = 0.05 nM) for the main immunogenic region of the human muscle acetylcholine receptor (AChR), has been determined and refined to 2.4 A resolution by X-ray crystallographic methods. The overall structure is similar to a Fab (NC6.8) from a murine antibody, used as a search model in molecular replacement. Structural comparisons with known antibody structures showed that the conformations of the hypervariable regions H1, H2, L1, L2, L3 of Fab192 adopt the canonical structures 1, 1, 2, 1, and 1, respectively. The surface of the antigen-binding site is relatively planar, as expected for an antibody against a large protein antigen, with an accessible area of 2865 A2. Analysis of the electrostatic surface potential of the antigen-binding site shows that the bottom of the cleft formed in the center of the site appears to be negatively charged. The structure will be useful in the rational design of very high affinity humanized mutants of Fab192, appropriate for therapeutic approaches of the model autoimmune disease myasthenia gravis.

Animals↗
PubMed2001

[Genomic structure and proteins sequence analysis of full-length of segment A of three infections bursal disease viruses].

The full-length of segment A of three infectious bursal disease viruses (IBDV), including an attenuated strain HZ2, an attenuated vaccine strain JD1 and a virulent field isolate ZJ2000, were cloned by long RT-PCR and sequenced respectively. All the three results revealed the identity of IB-DV with two overlapping open reading frames (ORF) flanked by 5'- and 3'-noncoding regions(NCR) in 3259 bp long. The strains shared high identity with each other at nucleotide or deduced amino acid level, and also had four unique sites H253, N279, T284, R330 which are common in other attenuated and some classic or highly virulent strains. The virulent strain ZJ2000 had several key amino acid mutations located in hypervariant region of VP2 and near the VP2-VP4 cleavage site of polypeptide, which is probably related to the virulence. Sequence comparison supported that VP2 is not the sole determinant of the virulence. The highly conservation in 5'- and 3'-NCR of different strains indicated the NCR may be not responsible for the virulence. But the same conservation appeared in VP5 revealed another complex relationship between VP5 and the virulence.

Amino Acid Sequence↗
PubMed2002

Enhancement of the immune response generated against hepatitis C virus envelope proteins after DNA vaccination with polyprotein-encoding plasmids.

Plasmids expressing variants of the hepatitis C virus (HCV) core, E1 and E2 proteins individually or as polyproteins were administered to BALB/c mice. All plasmids induced a detectable and specific antibody response. Antibody titres against core, E1 and E2 proteins, 19 weeks after primary immunization, ranged from 1:50 to 1:4500 depending on the inoculated plasmid and the HCV antigen evaluated. Constructs expressing HCV envelope proteins as polyprotein variants including the core amino acid region induced statistically stronger antibody responses than plasmids encoding individual E1 and E2 proteins. Particularly, the pIDKE2 plasmid, expressing the first 650 amino acids in the viral polyprotein, induced a potent and multispecific antibody and lymphoproliferative response against HCV core, E1 and E2 proteins. Anti-E2 antibodies generated by pIDKE2 immunization were cross-reactive to hypervariable region-1 peptides from different genotypes. Immunization with the pIDKE2 also generated a positive cellular immune response against the core antigen, determined by interferon-gamma enzyme-linked immunospot (ELISPOT) assay, and induced detectable levels of interferon-gamma but not interleukin-4 in vaccinated mice. The detection of both antibody and cytotoxic T-lymphocyte responses, potentially targeted to circulating or cell-infecting virions respectively, in mice vaccinated with the pIDKE2 plasmid is very attractive for the effective eradication of HCV infection.

Animals↗
PubMed2007

Targeting and localized signalling by small GTPases.

Polarized cellular responses, for example, cell migration, require the co-ordinated assembly of signalling complexes at a particular subcellular location, such as the leading edge of cells. Small GTPases of the Ras superfamily play central roles in many (polarized) responses to growth factors, chemokines or integrin ligands. These small GTPases are functionally distinct, yet remarkably homologous in their primary sequence and especially in their effector domains. Therefore it has long been unclear how GTPase signalling specificity is regulated. Small GTPases carry a lipid anchor, in the context of a hypervariable region, which mediates membrane association. However, whereas the lipid has long been proposed to be the critical regulator of subcellular GTPase targeting, there is now increasing evidence that specific protein-protein interactions are important as well. This review discusses recent findings on GTPase targeting and proposes a revised model for GTPase signalling. In this model, the hypervariable domain acts in conjunction with the lipid tail to target the GTPase to specific membrane-associated protein complexes. Here, local GTPase activation occurs, leading to subsequent exposure of the effector domain, binding to effector proteins and the initiation of downstream signalling.

Amino Acid Sequence↗
PubMed2026

Comprehensive plastome variation and RNA editing in Mentha: insights into phylogenetic relationships and candidate DNA barcodes.

INTRODUCTION: Mentha is an economically and medicinally important genus in Lamiaceae, but its taxonomy and species delimitation remain challenging because of frequent hybridization, polyploidy, and marked morphological plasticity. METHODS: In this study, we comparatively analyzed 12 plastomes representing major Mentha species, hybrid taxa, and unresolved accessions, including four newly assembled genomes, to characterize plastome structure, repeat composition, sequence divergence, phylogenetic relationships, and plastid RNA editing. The M. arvensis plastome and RNA-seq datasets originated from independent Swiss and Indian accessions, respectively. RESULTS: The plastomes were highly conserved in overall organization, ranging from 151,824 to 152,154 bp and displaying the typical quadripartite structure. Gene content and order were largely stable across taxa, with only minor variation likely associated with annotation differences at IR/SC boundary regions. Codon usage analysis revealed a clear bias toward A/U-ending synonymous codons, and most shared protein-coding genes showed low Ka/Ks ratios, indicating predominant purifying selection. Repeat analyses showed that simple sequence repeats were mainly composed of A/T-rich mononucleotide motifs, whereas long repeats were concentrated in the 30-40 bp size class. Comparative analyses identified six hypervariable regions, namely ccsA-ndhD, ycf1, ndhD, rpl32-trnL-UAG, rbcL-accD, and petA-psbJ, which represent promising candidate plastid markers for species discrimination. Phylogenetic analysis based on complete plastomes provided strong support for relationships among the sampled taxa and recovered a close affinity among M. aquatica, M. arvensis, and M. canadensis. In addition, RNA-seq analysis of M. arvensis identified 17 candidate plastid RNA editing sites, most of which were C-to-U conversions and nonsynonymous events. DISCUSSION: Together, these results expand plastid genomic resources for Mentha and provide a useful framework for phylogenetic inference, species identification, and future germplasm utilization.

RNA editing↗
PubMed1990

Crystal structure of the antigen-binding fragment of the murine anti-arsonate monoclonal antibody 36-71 at 2.9-A resolution.

The structure of the antigen-binding fragment (Fab) of an anti-phenylarsonate monoclonal antibody (36-71) bearing a major crossreacting idiotype of A/J mice has been solved and refined to an R factor of 19.3% at a resolution of 2.9 A. An initial electron density map was obtained with phase information from a total of six isomorphous heavy-atom derivatives (from two different compounds) and a molecular replacement solution using the HED10 Fab crystal structure as a model. The structure of the McPC603 Fab was used to provide an initial set of atomic coordinates. The electron density maps are clear and easily interpretable for the entire sequence except for sections from two of the heavy-chain complementarity-determining regions totaling 21 residues. These residues have been left out of the refinement and are not represented in our current model. The antigen-combining site was located by means of a difference Fourier synthesis with one of the heavy-atom derivatives, which contained arsanilic acid. It lies in a small pocket formed by residues from the hypervariable regions of both the heavy and the light chains. Interactions with the hapten from framework residues are also possible.

Amino Acid Sequence↗
PubMed1998

Amino acid insertions and deletions contribute to diversify the human Ig repertoire.

The sequence analysis of Ig variable region genes transcribed within different B-cell subpopulations from human tonsil led us to identify a rare DNA sequence modification event consisting of bp insertions and/or deletions (I/D). Although these events were previously reported, they had never been formally associated with the somatic hypermutation process. I/D events share with more conventional somatic hypermutation events their localization within hypervariable regions and, most particularly, within DNA motifs known to be mutational hot spots. Repetitive DNA tracts or DNA elements capable of forming DNA loop intermediates seem to be the preferred substrate for I/D to occur. These characteristics suggest a model for somatic hypermutation reminiscent of the "polymerase slippage" model involved in replication and repair mutations in prokaryotes, yeast, and mammals.

Amino Acid Substitution↗
PubMed1999

Identification of Mycobacterium species by PCR-restriction fragment length polymorphism analyses using fluorescence capillary electrophoresis.

We developed a scheme for the rapid identification of Mycobacterium species based upon PCR amplification of polymorphic genetic regions with fluorescent primers followed by restriction and analysis by fluorescence capillary electrophoresis. Mycobacterium species were identified by restriction enzyme analysis of a 439-bp segment of the 65-kDa heat shock protein gene (labeled [both strands] at the 5' end with 4,7,2',7'-tetrachloro-6-carboxyfluorescein) using HaeIII and BstEII and of a 475-bp hypervariable region of the 16S rRNA gene (labeled [both strands] at the 5' end with 6-carboxyfluorescein) using HaeIII and CfoI. Samples were analyzed on an automated fluorescence capillary electrophoresis instrument, and labeled fragments were sized by comparison with an internal standard. DNA templates were prepared with pure cultures of type strains. In all, we analyzed 180 strains, representing 22 Mycobacterium species, and obtained distinctive restriction fragment length polymorphism (RFLP) patterns for 19 species. Three members of the Mycobacterium tuberculosis complex had a common RFLP pattern. A computerized algorithm which eliminates subjectivity from pattern interpretation and which is capable of identifying the species within a sample was developed. The convenience and short preparatory time of this assay make it comparable to conventional methodologies such as high-performance liquid chromatography and hybridization assays for identification of mycobacteria.

Bacterial Proteins↗
PubMedDate not supplied

Studies of a cross-reactive idiotype associated with anti-para-azophenylarsonate antibodies of A/J mice.

We have reviewed studies of a cross-reactive idiotype (CRI), associated with anti-para-azophenylarsonate (anti-Ar) antibodies of A/J mice, and have presented some recent data. H chains from molecules with the CRI are homogeneous through the first hypervariable region whereas L chains show heterogeneity in the first framework segment. The constancy of N-terminal H chain sequences from 10 individual mice indicates that the framework is encoded by a germ-line gene, and that mutations have little effect on the observed framework sequence. H chains lacking the CRI show great heterogeneity in the first hypervariable segment, indicating a direct relationship between sequence and idiotype. Adult A/J mice can be suppressed with respect to the production of the CRI by administration of rabbit antiidiotypic antibody. When immunized, such mice produce anti-Ar antibodies with "private" idiotypes present at extremely low concentrations, or absent, in anti-Ar antibodies of other A/J mice. These private idiotypes can be transferred adoptively to mildly irradiated (200 r) syngeneic recipients with lymphoid cells, and serial transfers can be carried out after immunization. The original CRI remains suppressed. This presents the possibility of structural studies of anti-Ar antibodies varying in idiotype from a single strain. The work should also provide information on the degree of diversity of anti-Ar antibodies within the strain.

Amino Acid Sequence↗
PubMed2004

Complete nucleotide sequence analysis of a vaccine strain and a field isolate of foot-and-mouth disease virus serotype Asia1 with an insertion in VP1 genomic region.

Complete nucleotide sequences except the poly (C) tract and poly (A) tail of a vaccine strain (IND 491/97) and an atypical field isolate (IND 321/01) of Foot-and-mouth disease virus (FMDV) serotype Asia1 are described. Amino acid (aa) sequence analysis of the VP1 protein of the field isolate revealed that the latter has 212 instead of 210 or 211 aa found in the so far available sequences of other FMDV isolates of Asia1 serotype. The insertion was localized in the hypervariable region of aa 130-160 of VP1 protein. Nucleotide sequencing of the entire genome was therefore carried out to detect changes in other parts of the genome, if any, besides VP1, which could contribute to its fitness. An 8.16 kb sequence of IND 491/97 and an 8.162 kb sequence of IND 321/01 were compared with each other and also with the known sequence of IND 63/72, another vaccine strain of serotype Asia1. Comparison of the entire polyprotein coding (L to 3D) region of IND 321/01 with those of the two Asia1 vaccine strains (IND 63/72 and IND 491/97) revealed no significant differences. A similar comparison of IND 491/97 with IND 63/72 revealed variability across the entire length of the genome. In addition to the capsid-coding region, sequence variability was also observed in non-structural proteins albeit to different extent. This study shows that in the gene pool of serotype Asia1 at least three groups of isolates/strains are present with respect to the length of VP1 protein.

Amino Acid Sequence↗
PubMed2003

Cell surface receptors for gammaretroviruses.

Evidence obtained during the last few years has greatly extended our understanding of the cell surface receptors that mediate infections of retroviruses and has provided many surprising insights. In contrast to other cell surface components such as lectins or proteoglycans that influence infections indirectly by enhancing virus adsorption onto specific cells, the true receptors induce conformational changes in the viral envelope glycoproteins that are essential for infection. One surprise is that all of the cell surface receptors for gamma-retroviruses are proteins that have multiple transmembrane (TM) sequences, compatible with their identification in known instances as transporters for important solutes. In striking contrast, almost all other animal viruses use receptors that exclusively have single TM sequences, with the sole proven exception we know of being the coreceptors used by lentiviruses. This evidence strongly suggests that virus genera have been prevented because of their previous evolutionary adaptations from switching their specificities between single-TM and multi-TM receptors. This evidence also implies that gamma-retroviruses formed by divergent evolution from a common origin millions of years ago and that individual viruses have occasionally jumped between species (zoonoses) while retaining their commitment to using the orthologous receptor of the new host. Another surprise is that many gamma-retroviruses use not just one receptor but pairs of closely related receptors as alternatives. This appears to have enhanced viral survival by severely limiting the likelihood of host escape mutations. All of the receptors used by gamma-retroviruses contain hypervariable regions that are often heavily glycosylated and that control the viral host range properties, consistent with the idea that these sequences are battlegrounds of virus-host coevolution. However, in contrast to previous assumptions, we propose that gamma-retroviruses have become adapted to recognize conserved sites that are important for the receptor's natural function and that the hypervariable sequences have been elaborated by the hosts as defense bulwarks that surround the conserved viral attachment sites. Previously, it was believed that binding to receptors directly triggers a series of conformational changes in the viral envelope glycoproteins that culminate in fusion of the viral and cellular membranes. However, new evidence suggests that gamma-retroviral association with receptors triggers an obligatory interaction or cross-talk between envelope glycoproteins on the viral surface. If this intermediate step is prevented, infection fails. Conversely, in several circumstances this cross-talk can be induced in the absence of a cell surface receptor for the virus, in which case infection can proceed efficiently. This new evidence strongly implies that the role of cell surface receptors in infections of gamma-retroviruses (and perhaps of other enveloped animal viruses) is more complex and interesting than was previously imagined. Recently, another gammaretroviral receptor with multiple transmembrane sequences was cloned. See Prassolov, Y., Zhang, D., Ivanov, D., Lohler, J., Ross, S.R., and Stocking, C. Sodium-dependent myo-inositol transporter 1 is a receptor for Mus cervicolor M813 murine leukemia virus.

Animals↗
PubMed1998

Characterization of the proline-rich region of murine leukemia virus envelope protein.

Mammalian type C retroviral envelope proteins contain a variable proline-rich region (PRR), located between the N-terminal receptor-binding domain and the more highly conserved C-terminal portion of the surface (SU) subunit. We have investigated the role of the PRR in the function of murine leukemia virus (MuLV) envelope protein. In the MuLVs, the PRR contains a highly conserved N-terminal sequence and a hypervariable C-terminal sequence. Despite this variability, the amphotropic PRR could functionally substitute for the ecotropic PRR. The hypervariable region of the PRR was not absolutely required for envelope protein function. However, truncations in this region resulted in decreased levels of both the SU and TM proteins in viral particles and increased amounts of the uncleaved precursor protein, Pr85. In contrast, the N-terminal conserved region was essential for viral infectivity. Deletion of this region prevented the stable incorporation of envelope proteins into viral particles in spite of normal envelope protein processing, wild-type levels of cell surface expression, and a wild-type ability to induce syncytia in an XC cell cocultivation assay. However, higher levels of the SU protein were shed into the supernatant, suggesting a defect in SU-TM interactions. Our data are most consistent with a role for the PRR in stabilizing the overall structure of the protein, thereby affecting the proper processing of Pr85, SU-TM interactions, and the stable incorporation of envelope proteins into viral particles. In addition, we have demonstrated that the PRR can tolerate the insertion of a peptide-binding domain, making this a potentially useful site for constructing targetable retroviral vectors.

3T3 Cells↗
PubMed1995

Genetic heterogeneity of the hepatitis C virus.

Hepatitis C virus (HCV) is an important etiological agent in the development of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). The virus, identified only recently, contains a single-stranded RNA genome of positive polarity, is distantly related to pestiviruses and flaviviruses, and has been classified as the first member of a third genus within the family Flaviviridae. Extensive analysis of HCV genomic sequences demonstrated that this virus possesses significant genetic heterogeneity. Different regions of the viral genome demonstrate a varying degree of heterogeneity; the regions coding for the putative envelope proteins are the most variable sites between different isolates. Furthermore, HCV circulates as a quasispecies in the host. During the course of acute and chronic infection, the sequence composition of the HCV population in one patient has been found to change sequentially with an extremely high rate of nonconserved nucleotide changes in the hypervariable region I (HVR1) of HCV. Such sequence changes alter the antigenicity of the epitopes coded within HVR1 so that these are not always recognized by preexisting antibodies. It has been suggested that this could represent one mechanism by which HCV evades host immune surveillance and may account for the high rate of chronicity observed in such infections. Continuous viral replication may, in turn, lead to the development of chronic liver disease, including HCC, in infected individuals. To date, at least nine major genetic groups (genotypes 1-9) and more than 30 subgroups of HCV have been recognized based on genetic differences. A distinct difference has been observed in the genotype distribution in Africa compared with other continents. Recent data have suggested a difference in pathogenesis and in the outcome of interferon therapy in individuals infected with HCV of certain genotypes. For example, genotype 1b (II) seems to be associated with more severe liver disease, including HCC, and with a poorer response to interferon therapy. The extensive genetic heterogeneity of HCV may have serious implications for the diagnosis, treatment and prevention of hepatitis C as well as in understanding the biology of infection by this important human pathogen.

Genome, Viral↗
PubMed1992

Myeloid and lymphoid chimerism after T-cell-depleted bone marrow transplantation: evaluation of conditioning regimens using the polymerase chain reaction to amplify human minisatellite regions of genomic DNA.

Determining both myeloid and lymphoid chimerism after T-cell-depleted allogeneic bone marrow transplantation (BMT) could be helpful in the understanding of the biology of engraftment and could provide a rational method of assessing the ability of different conditioning regimens to promote engraftment. We prospectively investigated the role of different pretransplant conditioning regimens in 29 leukemic patients post-BMT by assessing myeloid and T-cell chimerism using a rapid and sensitive polymerase chain reaction (PCR) method. Minisatellites are hypervariable regions of DNA consisting of tandem repeats of a core nucleotide sequence, and allelic polymorphism results from differences in the number of the repeats. We used this variation to distinguish between donor and recipient cells post-BMT. Seventeen patients (9 sibling and 8 unrelated donors) received conditioning with hyperfractionated total body irradiation (TBI), thiotepa, and cyclophosphamide (Cy). Of the other 12 patients (all sibling donors), 11 received TBI plus Cy plus another agent: VP16, carboplatinum, or AZQ. One patient received TBI plus thiotepa plus VP16. All but one of the patients studied received marrow from HLA-identical donors. PCR analysis confirmed donor lymphoid engraftment within 8 days of transplant in six of six patients studied. All granulocyte DNA was of donor origin within the first 4 weeks of transplant, regardless of the conditioning regimen. The day +28 T cells were exclusively of donor origin in 14 of 17 patients who received TBI plus thiotepa plus Cy, but were mixed chimeric in 10 of 12 patients who received other conditioning regimens (P < .001). Early graft rejection was seen in one unrelated transplant recipient conditioned with TBI plus thiotepa plus Cy. Late graft failure was observed in 3 of 12 patients with mixed T-cell chimerism and in none of 16 patients with full donor chimerism at day +28. However, 5 of 16 patients who had complete T-cell chimerism at day +28 developed acute graft-versus-host disease (GVHD), whereas no patient with mixed chimerism had acute GVHD. Our results indicate that minisatellite PCR is a rapid and sensitive method for assessing chimerism post-BMT, that the donor T cells are important for consistent durable engraftment, and that TBI plus thiotepa plus Cy may be superior to the other regimens studied in inducing full donor chimerism. Larger numbers and longer follow-up are necessary to confirm these data and also to assess the relationship between complete donor T-cell chimerism and leukemia-free survival.

Adolescent↗
PubMed2005

Quasispecies heterogeneity within the E1/E2 region as a pretreatment variable during pegylated interferon therapy of chronic hepatitis C virus infection.

A series of 29 patients undergoing treatment for chronic hepatitis C virus (HCV) genotype 1 infection with pegylated alpha-2a interferon plus ribavirin were studied for patterns of response to antiviral therapy and viral quasispecies evolution. All patients were treatment naive and had chronic inflammation and fibrosis on biopsy. As part of an analysis of pretreatment variables that might affect the outcome of treatment, genetic heterogeneity within the viral E1-E2 glycoprotein region (nucleotides 851 to 2280) was assessed by sequencing 10 to 15 quasispecies clones per patient from serum-derived PCR products. Genetic parameters were examined with respect to response to therapy based on serum viral RNA loads at 12 weeks (early viral response) and at 24 weeks posttreatment (sustained viral response). Nucleotide and amino acid quasispecies complexities of the hypervariable region 1 (HVR-1) were less in the responder group in comparison to the nonresponder group at 12 weeks, and genetic diversity was also less both within and outside of the HVR-1, with the difference being most pronounced for the non-HVR-1 region of E2. However, these genetic parameters did not distinguish responders from nonresponders for sustained viral responses. Follow-up studies of genetic heterogeneity based on the HVR-1 in selected responders and nonresponders while on therapy revealed greater evolutionary drift in the responder subgroup. The pretreatment population sequences for the NS5A interferon sensitivity determinant region were also analyzed for all patients, but no correlations were found between treatment response and any distinct genetic markers. These findings support previous studies indicating a high level of genetic heterogeneity among chronically infected HCV patients. One interpretation of these data is that early viral responses are governed to some extent by viral factors, whereas sustained responses may be more influenced by host factors, in addition to effects of viral complexity and diversity.

Adolescent↗
PubMed1996

New types of primers (stair primers) for PCR amplification of the variable V3 region of the human immunodeficiency virus.

The aim of the study was to develop a reliable PCR method for the detection of viral genomes with frequent mutations like HIV and hepatitis C virus. A system of 'stair' primers is suggested which allows amplification of a genomic sequence despite the presence of mutations in the region of the primers. In this system, classical primers are replaced with primers composed of a mixture of equimolar oligonucleotides in which the 5' end remains constant (single sized fragment) and the 3' end is displaced base by base. By PCR, 'stair' primers (HIV set) were compared to single-sequence primers of 20 and 25 nucleotides chosen in the same hypervariable region of the HIV gp120 (on both sides of V3 region), as well as to classical primers chosen in the conserved pol (polV2) and gag (SK38-39) regions of the genome. Of 17 HIV isolates obtained by co-culture of lymphocytes from HIV-seropositive patients, 17/17 (100%) were amplified using stair primers, 14/17 (82%) with 25-nucleotide primers, and 12/17 (70%) with 20-nucleotide primers. Amplification occurred in 17/17 instances with polV2 primers and in 16/17 instances with SK38-39. In addition, 55 other isolates were tested comparatively using stair, polV2 and SK38-39 primers. All isolates were amplified using stair and SK38-39 primers and 54/55 isolates with polV2 primers. When applied to 22 extracts of patients' lymphocytes DNA, stair primers amplified all 22 extracts to the same degree as polV2 and SK38-39, whereas the 20 and 25 nucleotide primers chosen in the variable region were not as reliable. This new primer system allows reliable detection of variable genomic regions of the HIV genome and amplification of such regions directly in patient leukocytes. In addition, the contribution of this system to microbiology and human genetics in general may be important.

Base Sequence↗
PubMed2000

Identification of gp120 regions targeted by a highly potent neutralizing antiserum elicited in a chimpanzee inoculated with a primary human immunodeficiency virus type 1 isolate.

We have previously reported that a chimpanzee infected with a primary human immunodeficiency virus type 1 (HIV-1) isolate (HIV-1(DH12)) developed an extremely potent virus-neutralizing antibody. Immunoglobulin G purified from this animal conferred sterilizing immunity following passive transfer to macaques which were subsequently challenged with simian immunodeficiency virus/HIV-1 chimeric virus strain DH12. In addition to being highly strain specific, the chimpanzee antiserum did not bind to the V3 loop peptide of HIV-1(DH12), nor did it block the interaction of gp120 with the CD4 receptor. When neutralization was examined in the context of virus particles carrying chimeric envelope glycoproteins, the presence of all five hypervariable regions (V1 to V5) was required for optimal neutralization. Virions bearing chimeric gp120 containing the V1-V2 and V4 regions of HIV-1(DH12) could also be neutralized, but larger quantities of the chimpanzee antiserum were needed to block infection. These results indicate that the HIV-1 gp120 epitope(s) targeted by the chimpanzee antiserum is highly conformational, involving surface elements contributed by all of the hypervariable domains of the envelope glycoprotein.

Animals↗