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S Wain-Hobson

Publications and source records attributed to S Wain-Hobson.

At least 19 recordsLinked to original sources
PubMed1995

Human immunodeficiency virus type 1 reverse transcriptase tG:T mispair formation on RNA and DNA templates with mismatched primers: a kinetic and thermodynamic study.

The relationship between human immunodeficiency virus (HIV) type 1 reverse transcriptase tG:T mispair formation and base pair stability was investigated using DNA and RNA templates with 15 bp matched or mismatched DNA primers. tG:T mispair formation during primer elongation was undetectable on tDNA-DNA duplexes but occurred with a frequency of 10(-4) on matched tRNA-DNA duplexes. The frequency increased to 7.0 x 10(-4) and 1.3 x 10(-3) on tRNA-DNA duplexes with tG:T mismatches located 6 and 9 bp beyond the polymerization site. From Km values at 37 degrees C, the free energy change upon dissociation (delta G degrees 37) of the tG:T mispair increased from matched to mismatched tRNA-DNA duplexes by 0.36-1.21 kcal/mol. delta G degrees 37 for a correct tG:C pair decreased by 0.06-1.00 kcal/mol. In comparison with DNA-DNA duplexes, thermal melting measurements on RNA-DNA duplexes demonstrated smaller enthalpy (delta delta H degrees = -17.7 to -28.1 kcal/mol) and entropy (delta delta S degrees = -59.3 to -83.4 cal/mol/K) components. A strong entropy-enthalpy compensation resulted in small free energy differences (delta delta G degrees 37 = 0.8 to -2.2 kcal/mol). Thus, although DNA-DNA and RNA-DNA duplexes are of comparable stability in solution, the RNA-DNA duplex presents more facile base pair opening and higher conformational flexibility. The release of helical strain at constant helix stability in RNA-DNA duplexes may facilitate base mispairing during reverse transcription, particularly in the context of lentiviral G-->A hypermutation.

Base Composition↗
PubMed1995

Reverse transcriptase and substrate dependence of the RNA hypermutagenesis reaction.

G-->A hypermutation is a remarkable phenomenon resulting from retroviral reverse transcription in the presence of highly biased dNTP concentrations. Of the three reverse transcriptases (RTases) available, those of human immunodeficiency virus type 1 (HIV-1), avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MoMLV), the HIV-1 enzyme showed the greatest sensitivity to biased [dCTP]/[dTTP] ratios. The HIV-1 RTase was able to discriminate between dUTP, dITP and the four DNA precursors and was insensitive to pH. There was little preference for nucleotide contexts. A few exceptionally modified sequences were found presumably resulting from G-->A hypermutation and multiple strand transfer. This particular predilection of the HIV-1 and, by extrapolation, the lentiviral RTases towards G-->A hypermutation suggests that the phenomenon may have contributed to the remarkably elevated A content of these retroviral genomes.

Avian Myeloblastosis Virus↗
PubMed1995

Erratic G-->A hypermutation within a complete caprine arthritis-encephalitis virus (CAEV) provirus.

The complete nucleotide sequence of an integrated provirus of caprine arthritis-encephalitis virus (CAEV) has been determined. The provirus was defective due to extensive G-->A hypermutation. Rather than being a smooth phenomenon distributed throughout the genome it was highly erratic with hypermutated and normal regions being juxtaposed, probably reflecting local fluctuations in the intracellular dCTP pool during reverse transcription of the CAEV genome. The pattern of sequence variation within the surface glycoproteins differs subtly from that of the primate lentiviruses.

Adenine↗
PubMed1995

Solution structure of a homopyrimidine: homopurine dodecamer encoded by the HIV-1 envelope gene: NMR and molecular simulation studies.

The solution structure of the nonpalindromic dodecanucleotide homopyrimidine:homopurine, d(5'-TTTCTCCTTTCT):d(5'-AGAAAGGAGAAA), was determined by two-dimensional nuclear magnetic resonance spectroscopy combined with molecular simulation. The dodecamer sequence studied was found within the HIV-1 envelope sequence and had all four Gs substituted by A in two hypermutants. A set of low-energy B-DNA conformations satisfying the quantitative NOE data were obtained. These highly related structure had neither peculiar helical parameters for the base pairs nor axis curvature. Analysis of the dihedral angles (epsilon-zeta) suggests that the A stretches flanking the GpA dinucleotides were more flexible.

Base Sequence↗
PubMed1995

The tempo and mode of SIV quasispecies development in vivo calls for massive viral replication and clearance.

Simian immunodeficiency virus (SIV) quasispecies development was followed in four monkeys (Macacca fascicularis) infected by intramuscular inoculation of phage lambda-SIVmac239 DNA. Rooted phylogenetic trees were reconstructed and used to interpret the data. The rate of fixation of base substitutions varied within and between animals reaching 3.3 x 10(-2) per site per year. These data suggest that the tempo of quasispecies development requires both massive viral replication and efficient clearance of SIV. Despite this, no significant difference was found between the observed and expected ratio of synonymous/nonsynonymous substitutions, suggesting that there was little or no selection of antigenic variants in the V1 and V2 hypervariable regions of envelope.

Amino Acid Sequence↗
PubMed1995

Clonal expansion of T cells and HIV genotypes in microdissected splenic white pulps indicates viral replication in situ and infiltration of HIV-specific cytotoxic T lymphocytes.

Human immunodeficiency virus (HIV) replication and T cell proliferation was investigated in situ by a PCR based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation, rather than the infection of CD4+ T lymphoblasts by blood borne virus or immune complexes. HIV infected white pulps could be infiltrated by HIV specific cytotoxic T lymphocytes, so implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen driven T cell recruitment and activation, HIV replication and spread, with consequent destruction of the newly infected cells.

Amino Acid Sequence↗
PubMed1995

Clonal expansion of human T-cell leukemia virus type I-infected cells in asymptomatic and symptomatic carriers without malignancy.

Human T-cell leukemia virus type I (HTLV-I) is associated with adult T-cell leukemia/lymphoma and tropical spastic paraparesis/HTLV-associated myelopathy. Both diseases are usually preceded by a long clinically asymptomatic period. PCR amplification of the HTLV-I proviral integration sites shows that clonal expansion of HTLV-I-bearing T cells, rather than being an occasional phenomenon in nonmalignant disease, is the norm for both symptomatic and asymptomatic carriers. Sequencing of 100 molecular clones derived by PCR amplification of part of the envelope gene from two asymptomatic carriers revealed almost no genetic variation. Viral amplification via clonal expansion, rather than by reverse transcription, would explain this remarkable genetic stability.

Adult↗
PubMed1995

Infection frequency of dendritic cells and CD4+ T lymphocytes in spleens of human immunodeficiency virus-positive patients.

Dendritic cells (DC) are specialized antigen-presenting leukocytes that are responsible for the activation of naive as well as memory T lymphocytes. If infected by human immunodeficiency virus (HIV), DC may transfer virus to CD4+ lymphocytes. However, the question of whether DC are infected in vivo is controversial. As HIV infection is more active in secondary lymphoid organs than in blood, infection of splenic DC isolated from HIV-seropositive patients was investigated. Splenic DC were first enriched and characterized by flow cytometry from HIV- donors. After direct isolation, they were negative for monocyte and T- and B-lymphocyte markers, negative for CD1a, but positive for major histocompatibility complex class II and CD4. After in vitro maturation, major histocompatibility complex class II expression increased, while CD4 expression was lost. Extensive purification from the spleens of seven HIV+ patients was performed by fluorescence-activated cell sorting. The frequency of cells harboring HIV DNA in purified populations was quantified by limiting-dilution PCR. Directly isolated DC (average, 1/3,000; range, 1/720 to 1/18,000) were in each patient 10 to 100 times less infected than CD4+ T lymphocytes (average, 1/52; range, 1/17 to 1/190). On average, 1/1,450 (1/320 to 1/6,100) unseparated mononuclear splenocytes (containing 5% CD4+ lymphocytes) harbored HIV DNA. In conclusion, in these HIV+ patient spleens, DC seem to be infected, but HIV-DNA positive CD4+ T lymphocytes accounted for the vast majority of infected mononuclear splenocytes.

CD4-Positive T-Lymphocytes↗
PubMed1994

Hypermutagenesis of RNA using human immunodeficiency virus type 1 reverse transcriptase and biased dNTP concentrations.

The finding of G-->A hypermutated retroviral genomes in which up to 40% of guanines may be substituted by adenines was proposed to result from the depletion of the intracellular dCTP concentration and suggested a means to hypermutagenize nucleic acids. Using a RNA/reverse transcriptase ratio of approximately 1:30, comparable to that within the retroviral replication complex, G-->A hypermutants were produced in a simple in vitro reaction using highly biased dNTP concentrations--i.e., a low ratio of [dCTP]/[dTTP]. Up to 38% of G residues could be substituted, the proportion being inversely proportional to the concentration of dCTP. As G-->A hypermutation resulted from elongation beyond multiple rG.dT mismatches, U-->C hypermutants resulting from multiple rU.dG mismatches were sought, and found, during cDNA synthesis using low [dATP] and high [dGTP]. Mixed G-->A and U-->C hypermutants could also be produced under conditions of low [dCTP] plus low [dATP] and high [dTTP] plus high [dGTP]. Hypermutagenesis should allow jumping through, and subsequent exploration of, sequence space to a greater degree than heretofore and, in conjunction with genetic screening, might be of use in the search of proteins or ribozymes with novel or enhanced properties.

Adenine↗
PubMed1994

HIV and T cell expansion in splenic white pulps is accompanied by infiltration of HIV-specific cytotoxic T lymphocytes.

Human immunodeficiency virus (HIV) replication and T cell proliferation were investigated in situ by a PCR-based analysis of individual microdissected splenic white pulps. Founder effects, revealed by an exquisite compartmentalization of HIV genotypes and T cells, indicated the recruitment of latently infected CD4+ T cells through highly localized antigen presentation rather than the infection of CD4+ T lymphoblasts by blood-borne virus or immune complexes. HIV-infected white pulps could be infiltrated by HIV-specific cytotoxic T lymphocytes, thereby implicating them in CD4+ T cell destruction in vivo. Together these data describe an iterative and deleterious mechanism of antigen-driven T cell recruitment and activation, as well as HIV replication and spread, with consequent destruction of the newly infected cells.

Amino Acid Sequence↗
PubMed1994

G-->A hypermutation of the human immunodeficiency virus type 1 genome: evidence for dCTP pool imbalance during reverse transcription.

The quasispecies model for RNA viruses predicts the existence of a replication error threshold beyond which there is a melting or total loss of sequence information. Retroviral G-->A hypermutation is probably an example. Here it is shown that G-->A transitions may occur in both GpG and GpA dinucleotide contexts. Transitions in GpG preferentially occur via base mispairing at the ends of runs of G residues, whereas G-->A transitions within GpA may result from temporary dislocation of the primer and template strands by a single base. The two circumstances may be related by the local dCTP substrate concentration. An in vitro elongation assay shows that primer/template dislocation is more frequent for the human immunodeficiency virus type 1 reverse transcriptase than for murine or avian retroviral enzymes. Taken together these data suggest that G-->A hypermutation is an example of induced mutation whereby the viral reverse transcriptase is forced into making errors by imbalances in the intracellular dCTP concentration.

Base Composition↗
PubMed1994

Spatial discontinuities in human immunodeficiency virus type 1 quasispecies derived from epidermal Langerhans cells of a patient with AIDS and evidence for double infection.

A nonhomogeneous spatial distribution of human immunodeficiency virus type 1 quasispecies was observed for epidermal Langerhans cells purified from skin patches taken from a patient with AIDS soon after death. Each patch presented a unique collection of sequences, distinct from those of juxtaposed patches or those derived from the other leg. Infection of Langerhans cells by virus from underlying T cells in the dermis might explain this partition. The analysis revealed the presence of two distinct cocirculating viral strains, indicating double infection.

Acquired Immunodeficiency Syndrome↗
PubMed1994

RNA virus quasispecies: significance for viral disease and epidemiology.

The experimental evidence available for animal and plant RNA viruses, as well as other RNA genetic elements (viroids, satellites, retroelements, etc.), reinforces the view that many different types of genetic alterations may occur during RNA genome replication. This is fundamentally because of infidelity of genome replication and large population sizes. Homologous and heterologous recombination, as well as gene reassortments occur frequently during replication of retroviruses and most riboviruses, especially those that use enzymes with limited processivity. Following the generation of variant genomes, selection, which is dependent on environmental parameters in ways that are poorly understood, sorts out those genome fits enough to generate viable quasispecies. Chance events can also be destabilizing, as illustrated by recent results on fitness loss and other phenotypic changes accompanying bottleneck transmission. Variation, selection, and random sampling of genomes occur continuously and unavoidably during virus evolution. Evolution of RNA viruses is largely unpredictable because of the stochastic nature of mutation and recombination events, as well as the subtle effects of chance transmission events and host/environmental factors. Among environmental factors, alterations resulting from human intervention (deforestation, agricultural activities, global climatic changes, etc.) may alter dispersal patterns and provide new adaptive possibilities to viral quasispecies. Current understanding of RNA virus evolution suggests several strategies to control and diagnose viral diseases. The new generation of chemically defined vaccines and diagnostic reagents (monoclonal antibodies, peptide antigens, oligonucleotides for polymerase chain reaction amplification, etc.) may be adequate to prevent disease and detect some or even most of the circulating quasispecies of any given RNA pathogen. However, the dynamics of viral quasispecies mandate careful consideration of those reagents to be incorporated into diagnostic kits. Broadening diagnosis without jeopardizing specificity of detection will be challenging. There is a finite probability (impossible to quantify at present) that a defined vaccine may promote selection of escape mutants or a particular diagnostic kit may fail to detect a viral pathogen. Of particular concern are the potential long-term effects of weak selective pressures that may initially go unnoticed. Variant viruses resulting from evolutionary pressure imposed by vaccines or drugs may insidiously and gradually replace previous quasispecies. The great potential for variation and phenotypic diversity of some important RNA virus pathogens (human immunodeficiency virus, the hepatitis viruses, the newly recognized human hantaviruses, etc.) has become clear. Prevention and therapy should rely on multicomponent vaccines and antiviral agents to address the complexity of RNA quasispecies mutant spectra.(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Evolution↗
PubMed1993

The fastest genome evolution ever described: HIV variation in situ.

Human immunodeficiency virus is an RNA virus in which the degree of genetic variation observed is phenomenal--up to 20% within an infected individual. This is essentially due to remorseless cycles of viral replication, most probably due to chronic activation of the immune system. It can be estimated that the number of variants in existence worldwide must be in excess of 10(14)-10(18), and given the nature of RNA viruses even more novel variants should emerge.

Biological Evolution↗
PubMed1992

Strand specific PCR amplification of low copy number DNA.

A method for the amplification of a single DNA strand at low copy number is described. It is a wholly PCR based approach which involves an initial linear amplification of the target using a tagged strand specific primer. This is followed by classical PCR amplification of the progeny using a pair of primers, one specific for the sequence tagged onto the 5' end of the first round primer, the second specific for the target sequence. Given the protocol used the ratio of the two strands in the final amplification product was 50:1.

Base Sequence↗
PubMed1992

Cytotoxic T lymphocyte responses in the peripheral blood of children born to human immunodeficiency virus-1-infected mothers.

Cytotoxic T lymphocytes (CTL) are present at high activities in adult patients infected with the human immunodeficiency virus (HIV). In this report, CTL effectors were identified in peripheral blood mononuclear cells (PBMC) of children born to HIV-1-infected mothers. These CTL killed HLA-matched HIV-1-infected H9 target cells or doubly transfected P815-A2-env, gag or nef mouse tumor cells, which expressed the viral antigens in association with HLA-A1/A3 or HLA-A2, respectively. HIV-1-specific CTL were detected early after birth (less than 2 months) and remained present during the asymptomatic phase of the infection. As in HIV-1-infected adults, HIV-specific CTL declined with disease progression. Surprisingly, HIV-1-specific CTL were detected in the PBMC of three children who subsequently became seronegative.

Base Sequence↗