Biomedical subjects

I M Jones

Publications and source records attributed to I M Jones.

At least 55 records · Page 3Linked to original sources

PubMed1996

Gag-Gag interactions in the C-terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for Gag particle assembly.

Seven internal deletions within the p24 domain of the human immunodeficiency virus type 1 Gag precursor have been assessed for their effect on Gag particle formation following their expression using recombinant baculoviruses. In addition, each deleted molecule was assessed for its ability to bind soluble p24 antigen in vitro. The mutants fell into three different phenotypic groups: (i) three mutants that had no effect on either p24 binding or Gag particle assembly, (ii) three mutants that abolished both features and (iii) one mutant that bound p24 in vitro but failed to assemble particles. Mutations that abolished both in vitro p24 binding and particle assembly mapped to the C terminus of p24 confirming this region as critical for virion assembly. We suggest the division of virion assembly into at least two distinct phases and suggest a model in which the critical sequences mapped to date are combined with available structural information.

Animals↗

PubMed1995

Crystal structure of SIV matrix antigen and implications for virus assembly.

Simian immunodeficiency virus (SIV) is closely related to human immunodeficiency virus (HIV), their matrix antigens (MAs) sharing some 50% sequence identity. MA is a component of Pr55Gag, the sole protein required for assembly of the virion shell. MA targets Pr55 to the plasma membrane, and facilitates incorporation of the virus envelope protein and assembly of the Pr55Gag shell. Cleavage of Pr55 by the viral protease produces the mature protein of relative molecular mass 17-18K, which underlies the host-derived membrane and is important in both virus entry and nuclear localization of the virion core. Here we report the crystal structure of SIV MA. The molecule forms a trimer consistent with oligomerization in vitro, the observed virion architecture, and various biological properties of MA.

Amino Acid Sequence↗

PubMed1995

Expression and purification of glutathione S-transferase-tagged HIV-1 gp120: no evidence of an interaction with CD26.

We describe the use of a new baculovirus expression vector to enable the secretion of the major surface glycoprotein of HIV-1 (gp120) fused to the carboxy-terminus of the widely used affinity tag glutathione S-transferase. The secreted protein can be purified in a single step with the minimum of denaturation on immobilised glutathione and is as active as the parental molecule in binding CD4. We use this molecule in a variety of assay formats to examine the gp120 interaction with CD26, a reported auxiliary molecule in the HIV entry process. We find no evidence of a CD26-gp120 interaction in the absence or presence of CD4.

Animals↗

PubMed1995

Impact of age and environment on somatic mutation at the hprt gene of T lymphocytes in humans.

Analysis of two human populations for dependence of somatic mutation on age has revealed both similarities and differences. The studies performed employed peripheral blood lymphocytes and measured the efficiency with which these cells form clones in vitro (cloning efficiency, CE) and the frequency of cells with inactivating mutations of the hypoxanthine phosphoribosyltransferase gene (mutant frequency, MF). The people studied were between 19 and 64 years of age. In one population, composed of 78 never smokers and 140 current smokers from the United States (US), both CE and MF were dependent on age: CE declined with age (p = 0.005); MF increased 0.15 per 10(6) cells per year of age for nonsmokers (p < 0.001) and at 1.3 times that rate for smokers (p = 0.01). In the second population, 80 people of unknown smoking status from Russia, the increase in MF per year was even greater, 2.5 times that of the US nonsmokers (p = 0.001) but the dependence of CE on age was the same as for the US population (p = 0.043). Because the increase of MF of the Russians with age is 2-fold greater than that of the US smokers, the intensity of smoking and/or other environmental exposures, or the susceptibility to these exposures, must account for the difference in age dependent MF increase, not the proportion of Russians that are smokers. Differences in the lymphocyte subpopulations that survived the longer transit from Russia may have contributed to the observed differences in MF. However, overall, the mutant frequency results suggest that the Russians were chronically exposed to higher levels of agents that induce somatic mutation and that, on an age adjusted basis, the Russia population studied is at increased risk for health consequences from accumulated genetic damage.

Adult↗

PubMed1995

Eukaryotic virus display: engineering the major surface glycoprotein of the Autographa californica nuclear polyhedrosis virus (AcNPV) for the presentation of foreign proteins on the virus surface.

We describe the development of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector for the display of distinct proteins on the viral surface in a manner that is analogous to the established bacterial "phage display" systems. As a model system, the marker gene encoding the 26kDa protein glutathione-S-transferase (GST) was used to construct several fusions with the major baculovirus glycoprotein gp64 gene. Following expression in Spodoptera frugiperda (Sf9) cells, the yield and cellular distribution of each GST-gp64 protein was assessed by Western blot of both cell and supernatant fractions. One fusion, in which GST was inserted between the leader peptide and the nature protein, was found to be efficiently secreted into the cell medium. In the context of expression of the full length gp64, the hybrid GST-gp64 was shown by immunogold labelling to be incorporated onto the virion surface. In addition, the affinity purification of the soluble transmembrane gp64-GST fusion protein resulted in the co-purification of wild type gp64 suggesting that co-oligomerization of the GST-tagged fusion and the wild type molecule was the basis for virion incorporation. The HIV major surface glycoprotein, gp120 was also efficiently displayed in functional form on the viral surface following fusion to the amino terminus of gp64. A general expression vector, pAcSurf-2, was constructed in which multiple cloning sites were positioned in-phase between the gp64 signal sequence and the sequence encoding the mature protein under the control of the polyhedrin promoter.

Amino Acid Sequence↗

PubMed1995

The glycosylation of human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) is important for the efficient intracellular transport of the envelope precursor gp160.

The role of the glycans of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) in the intracellular events of Env precursor (gp160) biosynthesis has been examined by the use of a mutant gp160 in which the cluster of conserved glycosylation sites within the gp41 domain (Asn-621, -630 and -642) has been mutated. Expression of the wild-type and mutant forms of gp160 in BHK-21 cells using recombinant vaccinia viruses has shown that the kinetics of the events occurring in the endoplasmic reticulum (ER) were normal: both Env proteins had similar kinetics of disulphide bond formation, as determined by the acquisition of CD4-binding capability, and both had similar kinetics of oligomer formation. However, in contrast to the parental molecule, mutated gp160 displayed relatively slow transport from the cis to the medial Golgi where it was retained in the oligomeric state. Transport to the trans Golgi was impaired, as determined by the sensitivity of gp160 to glycosidases. Cleavage of mutated gp160 at the gp120/gp41 junction was substantially reduced but this was apparently not due to the involvement of the gp41 glycosylation in the cleavage reaction by furin inasmuch as, in the baculovirus system, mutated gp160 could be cleaved when recombinant furin was co-expressed. The reduced cleavage in mammalian cells may thus reflect the impaired routing of mutated Env to the compartment where cleavage occurs. The glycan component of gp41 is, therefore, important for the efficient intracellular transport and processing of gp160. gp160 lacking gp41 carbohydrates is an additional example, among few others, of a protein lacking glycans that is arrested in the Golgi rather than the ER following its biosynthesis.

Amino Acid Sequence↗

PubMed1995

Regions required for CD4 binding in the external glycoprotein gp120 of simian immunodeficiency virus.

The external domain of the envelope glycoprotein, gp120, of simian immunodeficiency virus (SIV) has been expressed as a mature secreted product using recombinant baculoviruses and the expressed protein, which has an observed molecular mass of 110 kDa, was purified by monoclonal antibody (MAb) affinity chromatography. N-terminal sequence analysis showed a signal sequence cleavage identity similar to that of the gp120s of both human immunodeficiency virus type 1 (HIV-1) and HIV type 2. The expressed molecule bound to soluble CD4 with an affinity that was approximately 10-fold lower than that of gp120 from HIV-1. A screening of the ability of SIV envelope MAbs to inhibit CD4 binding revealed two groups of inhibitory MAbs. One group is dependent on conformation, while the second group maps to a discrete epitope near the amino terminus. The particular role of the V3 loop region of the molecule in CD4 binding was investigated by the construction of an SIV-HIV hybrid in which the V3 loop of SIV was precisely replaced with the equivalent domain from HIV-1 MN. The hybrid glycoprotein bound HIV-1 V3 loop MAbs and not SIV V3 MAbs but continued to bind conformational SIV MAbs and soluble CD4 as well as the parent molecule.

Amino Acid Sequence↗

PubMed1995

A molecular determinant of human immunodeficiency virus particle assembly located in matrix antigen p17.

We report single-point mutations that are located in the matrix protein domain of the gag gene of human immunodeficiency virus type 1 and that prevent Gag particle formation. We show that mutations of p17 that abolish human immunodeficiency virus particle assembly also prevent the dimerization of p17 protein, as measured directly by a protein-protein binding assay. In the three-dimensional structure of p17, mutations that abolish dimerization are located in a single alpha helix that forms part of a fingerlike projection from one side of the molecule. Peptides derived from this region of p17 also reduce the level of p17 dimer when they are added to p17-expressing cells and compete for p17 self-association when present in protein-protein binding assays. We propose that the dimerization of the Gag precursor that occurs by the interdigitation of alpha helices on adjacent matrix molecules is a key stage in virion assembly and that the prevention of such an interaction is the molecular basis of particle misassembly.

Base Sequence↗

PubMed1995

Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems.

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.

Animals↗

PubMed1995

Mapping the end points of large deletions affecting the hprt locus in human peripheral blood cells and cell lines.

We have examined the extent of HPRT- total gene deletions in three mutant collections: spontaneous and X-ray-induced deletions in TK6 human B lymphoblasts, and HPRT- deletions arising in vivo in T cells. A set of 13 Xq26 STS markers surrounding hprt and spanning approximately 3.3 Mb was used. Each marker used was observed to be missing in at least one of the hprt deletion mutants analyzed. The largest deletion observed encompassed at least 3 Mb. Nine deletions extended outside of the mapped region in the centromeric direction (> 1.7 Mb). In contrast, only two telomeric deletions extended to marker 342R (1.26 Mb), and both exhibited slowed or limited cell growth. These data suggest the existence of a gene, within the vicinity of 342R, which establishes the telomeric limit of recoverable deletions. Most (25/41) X-ray-induced total gene deletion mutants exhibited marker loss, but only 1/8 of the spontaneous deletions encompassed any Xq26 markers (P = 0.0187). Furthermore, nearly half (3/8) of the spontaneous 3' total deletion breakpoints were within 14 kb of the hprt coding sequence. In contrast, 40/41 X-ray-induced HPRT- total deletions extended beyond this point (P = 0.011). Although the overall representation of total gene deletions in the in vivo spectrum is low, 4/5 encompass Xq26 markers flanking hprt. This pattern differs significantly from spontaneous HPRT- large deletions occurring in vitro (P = 0.032) but resembles the spectrum of X-ray-induced deletions.

B-Lymphocytes↗

PubMed1994

Nonsense mutations in the vpr gene of HIV-1 during in vitro virus passage and in HIV-1 carrier-derived peripheral blood mononuclear cells.

Long-term, persistent infection by HIV-1 is a prerequisite for the development of AIDS. However, little is known of the determinants required for HIV-1 to cause persistence. We have reported previously that persistent infection of a T cell line by a cytopathogenic strain of HIV-1 became increasingly likely with in vitro serial passage of the virus. DNA sequencing of the persistent strains revealed a nonsense mutation in the vpr gene in all isolates tested. Here, we report the development and use of a semi-quantitative PCR method to detect the vpr nonsense mutation within populations of virus. Our results show that vpr mutants also arise in cells during acute infection and increase progressively with serial passage of the virus. In addition, HIV-1-seropositive individuals were examined and found to carry the same vpr nonsense mutation at high frequency in virus-infected PBMC. These data are consistent with a mechanism of HIV-1 persistence in vivo and in vitro in which virus cytopathogenic potential is lost by the build up of nonsense mutations in vpr.

AIDS-Related Complex↗

PubMed1994

Functional consequences of mutations in HIV-1 Gag p55 selected by CTL pressure.

Amino acid changes in the CA domain of the p55 Gag protein of HIV-1 have been observed during the course of an infection that appear to correlate with escape from cytotoxic T cell surveillance (Phillips et al., Nature 354, 453-459, 1991). A corollary of this observation is that all such changes should be functionally silent but, as the changes were observed in populations of virus, this has not been formally demonstrated. We have introduced the amino acid changes representative of those observed to occur in vivo into the Gag p55 gene cloned in the baculovirus expression system where the wild-type gene product produces virus-like particles (VLP). We show that none of these mutations affect particle formation as judged by VLP morphology and density despite their location within a sequence of the Gag open reading frame known to be important for assembly. These data add tacit support to the hypothesis that CTL pressure can drive virus evolution in HIV and add to the fine mapping of sequences involved in Gag subunit interactions.

Amino Acid Sequence↗

PubMed1994

Fullerene-like organization of HIV gag-protein shell in virus-like particles produced by recombinant baculovirus.

Virus-like particles produced by a recombinant baculovirus containing the HIV gag gene were examined by negative staining after delipidization. This technique demonstrated that the gag-protein shell consisted of radially arranged short rods which formed a network of ring-like structures. Similar structures were observed at the plasma membrane of infected cells which had been opened by wet-cleaving. Occasionally five or six subunits were observed forming a ring. These findings suggest that the gag-encoded precursor (pr55) is a rod-like molecule about 34 A in diameter and 85 A in length. A protein cylinder of such dimensions would have a molecular weight of 56K. The center-to-center distance of two neighboring rings formed by the rods was 66 +/- 8 A (N = 200) by direct measurements and 65 A as obtained from averaged images. This morphology and these dimensions indicate that the virus-like particles contain the gag precursor in the form of a near-spherical "fullerene-like" icosahedral shell. Our data indicate that the triangulation number of the rings equals 63. However, since one rod of pr55 is shared by two rings, the number of copies of the precursor will be 1890 as opposed to 2522 if the molecules were closely packed. The particle diameter of 102 nm deduced from the proposed model was close to the diameter obtained from thin sections of low-temperature-embedded specimens (103-108 nm).

Animals↗

PubMed1994

Complementation of human immunodeficiency virus (HIV-1) gag particle formation.

The human immunodeficiency virus gag precursor protein Pr55Gag exhibits the ability of particle assembly when expressed using recombinant baculoviruses. In order to delineate the sequences required for particle formation, two mutants of Gag (D1 and D2) were constructed in which 10 amino acids within the CA domain were deleted. Both mutants yielded stable high levels of Gag antigen following expression in Spodoptera frugiperda insect cells. Electron microscopy of sections through infected cells revealed that neither mutant was able to assemble particles although targeting of the protein to the plasma membrane still occurred. The Gag antigen that accumulated beneath the plasma membrane exhibited distinctive morphologies when compared to each other and to parental (Pr46Gag) particles. Particle assembly was rescued when S. frugiperda cells were coinfected with both AcD1 and AcD2 viruses, or with AcD1 and a carboxyl-terminal deletion of Gag (Pr41.5) which was previously shown not to form particles (J.B.M., D.J. Hockley, M.V. Nermot, and I.M. Jones, 1992, J. Gen. Virol. 73, 3079-3086). The genetic complementation of Gag-driven assembly is discussed.

Amino Acid Sequence↗

PubMed1994

Multiple effects of CD4 CDR3-related peptide derivatives showing anti-HIV-1 activity on HIV-1 gp120 functions.

The interaction of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 with CD4 CDR3-related peptide derivatives showing anti-HIV-1 activity has been studied. Conformational changes in gp120, which could affect its interaction with CD4 and its shedding from virions, were detected by fluorescence spectrum analysis of tryptophan residues after addition of peptide representative of the CD4 CDR3-related region, but not the CD4 CDR2-related region. Interestingly, the addition of scrambled peptide, S1 (with altered amino acid sequence compared with the native CDR3-related peptide but unaltered overall composition), which we recently showed to have stronger anti-HIV-1 activity than the original CDR3-related peptide, had no effects on the conformational change in gp120 or on its interaction with CD4 and its shedding from HIV-1 virions. However, all of the CDR3-related peptides, including S1, showed blocking effects on the binding of antibodies against gp120 V3 loop and C-terminus regions. Thus, we concluded that there were at least two separable activities of the CDR3-related peptides in anti-HIV-1 activity, i.e. induction of conformational changes in gp120, which could affect its binding to CD4 and to gp41 (as observed in native CDR3-related peptides), and inactivation of V3 loop and C-terminus regions in gp120 (as observed in all of the CDR3-related peptides, including S1).

Amino Acid Sequence↗

PubMed1994

Functions of HIV envelope glycans.

The unusually highly glycosylated state of the major envelope glycoprotein (gp160) of the human immunodeficiency virus has offered a challenge to both glycobiologists and virologists. What is the functional significance of such a mass of glycans and how might they be manipulated to disadvantage virus pathogenesis? Some answers to each of these questions have already been obtained: N-linked glycans are necessary for the creation, but not the maintenance, of a bioactive conformation, and drug-induced alteration of the glycosylation pattern can lead to impaired virus infectivity. As a model for studying glycan function and as a target for antiviral therapy, gp160 represents a unique candidate.

Animals↗

PubMed1994

Comparative morphology of Gag protein structures produced by mutants of the gag gene of human immunodeficiency virus type 1.

Six mutants that differ in the extent of their carboxyterminal sequences and two deletion mutants of the gag gene of HIV-1 have been characterized morphologically following their expression in Spodoptera frugiperda cells using recombinant baculoviruses. Electron microscopy has revealed distinct morphological forms of the Gag protein that can be classified as either (i) particulate, three-dimensional, spherical or tubular shells or (ii) non-particulate, two-dimensional, flat, curved or convoluted sheets. Progressive truncation of the carboxy terminus of Gag was accompanied by changes in the morphology and formation of spherical particles from predominantly C-type assembly and budding at the plasma membrane, through B-type intracytoplasmic assembly, to A-type assembly with budding mainly into cytoplasmic vacuoles. Deletions within the Pr24 CA domain of Gag abolished particle formation but retained association of the protein with the plasma membrane. All of the observed morphologies of the mutant Gag proteins could be accommodated within an icosahedral model for the organization of spherical particles and a basic hexagonal arrangement of assembled Gag protein monomers.

Animals↗

PubMed1993

Characterization of in vivo somatic mutations at the hypoxanthine phosphoribosyltransferase gene of a human control population.

The ability to recognize a change in mutation spectrum after an exposure to a toxic substance and then relate that exposure to health risk depends on the knowledge of mutations that occur in the absence of exposure. Toward this end, we have been studying both the frequency and molecular nature of mutations of the hypoxanthine phosphoribosyltransferase (hprt) gene in peripheral blood lymphocytes as surrogate reporters of genetic damage. We have analyzed mutants, one per donor to ensure independence, from a control population in which the quantitative effects of smoking and age on mutant frequency have been well defined. Analyses of cDNA and genomic DNA by polymerase chain reaction and sequencing have identified the mutations in 63 mutants, 45 from males and 18 from females, of which 34 were smokers and 29 were nonsmokers. Slightly less than half of the mutations were base substitutions; they were predominantly at GC base pairs. Different mutations at the same site indicated that there are features of the hprt polypeptide that affect the mutation spectrum. Two pairs of identical mutations indicated that there may also be hot spots. Mutations not previously reported have been detected, indicating that the mutation spectrum is only partly defined. The remainder of the mutations were deletions or insertions/duplications; deletions ranged from one base pair to complete loss of the locus. Despite a small average increase in mutant frequency for smokers, an increased proportion of base substitutions at AT base pairs in smokers (p = 0.2) hinted at a smoking-associated shift in the mutation spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗