Genetic variation in SH3-domain GRB2-like (endophilin)-interacting protein 1 has a major impact on fat mass.

OBJECTIVE: The SH3-domain GRB2-like (endophilin)-interacting protein 1 (SGIP1) gene has been shown to be differentially expressed in the hypothalamus of lean versus obese Israeli sand rats (Psammomys obesus), and is suspected of having a role in regulating food intake. The purpose of this study was to assess the role of genetic variation in SGIP1 in human disease. SUBJECTS: We performed single-nucleotide polymorphism (SNP) genotyping in a large family pedigree cohort from the island of Mauritius. The Mauritius Family Study (MFS) consists of 400 individuals from 24 Indo-Mauritian families recruited from the genetically homogeneous population of Mauritius. We measured markers of the metabolic syndrome, including diabetes and obesity-related phenotypes such as fasting plasma glucose, waist:hip ratio, body mass index and fat mass. RESULTS: Statistical genetic analysis revealed associations between SGIP1 polymorphisms and fat mass (in kilograms) as measured by bioimpedance. SNP genotyping identified associations between several genetic variants and fat mass, with the strongest association for rs2146905 (P=4.7 × 10(-5)). A strong allelic effect was noted for several SNPs where fat mass was reduced by up to 9.4% for individuals homozygous for the minor allele. CONCLUSIONS: Our results show association between genetic variants in SGIP1 and fat mass. We provide evidence that variation in SGIP1 is a potentially important determinant of obesity-related traits in humans.

Opposing effects of monomeric and pentameric C-reactive protein on endothelial progenitor cells.

C-reactive protein (CRP) has been linked to the pathogenesis of atherosclerosis. The dissociation of native, pentameric (p)CRP to monomeric (m)CRP on the cell membrane of activated platelets has recently been demonstrated. The dissociation of pCRP to mCRP may explain local pro-inflammatory reactions at the site of developing atherosclerotic plaques. As a biomarker, pCRP predicts cardiovascular adverse events and so do reduced levels and function of circulating endothelial progenitor cells (EPCs). We hypothesised that mCRP and pCRP exert a differential effect on EPC function and differentiation. EPCs were treated with mCRP or pCRP for 72 h, respectively. Phenotypical characterisation was done by flow cytometry and immunofluorescence microscopy, while the effect of mCRP and pCRP on gene expression was examined by whole-genome gene expression analysis. The functional capacity of EPCs was determined by colony forming unit (CFU) assay and endothelial tube formation assay. Double staining for acetylated LDL and ulex lectin significantly decreased in cells treated with pCRP. The length of tubuli in a matrigel assay with HUVECs decreased significantly in response to pCRP, but not to mCRP. The number of CFUs increased after pCRP treatment. RNA expression profiling demonstrated that mCRP and pCRP cause highly contradictory gene regulation. Interferon-responsive genes (IFI44L, IFI44, IFI27, IFI 6, MX1, OAS2) were among the highly up-regulated genes after mCRP, but not after pCRP treatment. In conclusion, EPC phenotype, genotype and function were differentially affected by mCRP and pCRP, strongly arguing for differential roles of these two CRP conformations. The up-regulation of interferon-inducible genes in response to mCRP may constitute a mechanism for the local regulation of EPC function.

Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance.

AIMS/HYPOTHESIS: The role of IL-6 in the development of obesity and hepatic insulin resistance is unclear and still the subject of controversy. We aimed to determine whether global deletion of Il6 in mice (Il6 (-/-)) results in standard chow-induced and high-fat diet (HFD)-induced obesity, hepatosteatosis, inflammation and insulin resistance. METHODS: Male, 8-week-old Il6 (-/-) and littermate control mice were fed a standard chow or HFD for 12 weeks and phenotyped accordingly. RESULTS: Il6 (-/-) mice displayed obesity, hepatosteatosis, liver inflammation and insulin resistance when compared with control mice on a standard chow diet. When fed a HFD, the Il6 (-/-) and control mice had marked, equivalent gains in body weight, fat mass and ectopic lipid deposition in the liver relative to chow-fed animals. Despite this normalisation, the greater liver inflammation, damage and insulin resistance observed in chow-fed Il6 (-/-) mice relative to control persisted when both were fed the HFD. Microarray analysis from livers of mice fed a HFD revealed that genes associated with oxidative phosphorylation, the electron transport chain and tricarboxylic acid cycle were uniformly decreased in Il6 (-/-) relative to control mice. This coincided with reduced maximal activity of the mitochondrial enzyme ß-hydroxyacyl-CoA-dehydrogenase and decreased levels of mitochondrial respiratory chain proteins. CONCLUSIONS/INTERPRETATION: Our data suggest that IL-6 deficiency exacerbates HFD-induced hepatic insulin resistance and inflammation, a process that appears to be related to defects in mitochondrial metabolism.

Cross-species replication of a resistin mRNA QTL, but not QTLs for circulating levels of resistin, in human and baboon.

Resistin has been associated with inflammation and risk for cardiovascular disease. We previously reported evidence of a QTL on chromosome 19p13 affecting the abundance of resistin (RETN) mRNA in the omental adipose tissue of baboons (L0D score 3.8). In this study, whole genome transcription levels were assessed in human lymphocyte samples from 1240 adults participating in the San Antonio Family Heart Study, using the Sentrix Human-6 Expression Beadchip. Lymphocytes were surveyed, as it has been proposed that their expression levels may reflect those in harder to ascertain tissues, such as adipose tissue, that are thought to be more directly relevant to disease procesn was conducted to detect loci affecting RETN mRNA levels. We obtained significant evidence for a QTL influencing the RETN expression (LOD score 10.7) on chromosome 19p. This region is orthologous/homologous to the region previously localized on baboon chromosome 19. The strongest positional candidate gene in this region is the structural gene for resistin, itself. We also found evidence for a QTL influencing resistin protein levels (LOD score 5.3) on chromosome 14q. This differs from our previously reported QTL on chromosome 18 in baboons. The different QTLs for circulating protein suggests that post-translational processing and turnover may be influenced by different or multiple genes in baboons and humans. The parallel findings of a cis-eQTL for RETN mRNA in baboon omental tissue and human lymphocytes lends support to the strategy of using lymphocyte gene expression levels as a surrogate for gene expression levels in other tissues.

Identification of two novel quantitative trait loci for pre-eclampsia susceptibility on chromosomes 5q and 13q using a variance components-based linkage approach.

Pre-eclampsia/eclampsia (PE/E) is a common and serious disorder of human pregnancy that is associated with substantial maternal and perinatal morbidity and mortality. The suspected aetiology of PE/E is complex, with susceptibility being attributable to multiple environmental factors and a large genetic component. By assuming that the underlying liability towards PE/E susceptibility is inherently quantitative, any PE/E susceptibility gene would represent a quantitative trait locus (QTL). This assumption enables a more refined and powerful variance components procedure using a threshold model for our PE/E statistical analysis. Using this more efficient linkage approach, we have now re-analysed our previously completed Australian/New Zealand genome scan data to identify two novel PE/E susceptibility QTLs on chromosomes 5q and 13q. We have obtained strong evidence of linkage on 5q with a peak logarithm-of-odds (LOD) score of 3.12 between D5S644 and D5S433 [at approximately 121 centimorgan (cM)] and strong evidence of linkage on 13q with a peak LOD score of 3.10 between D13S1265 and D13S173 (at approximately 123 cM). Objective identification and prioritization of positional candidate genes using the quantitative bioinformatics program GeneSniffer revealed highly plausible PE/E candidate genes encoding aminopeptidase enzymes and a placental peptide hormone on the 5q QTL and two type IV collagens on the 13q QTL regions, respectively.

Lentiviral delivery of HIV-1 Vpr protein induces apoptosis in transformed cells.

Most current anticancer therapies act by inducing tumor cell stasis followed by apoptosis. HIV-1 Vpr effectively induces apoptosis of T cells after arrest of cells at a G(2)/M checkpoint. Here, we investigated whether this property of Vpr could be exploited for use as a potential anticancer agent. As a potentially safer alternative to transfer of genes encoding Vpr, we developed a method to efficiently introduce Vpr protein directly into cells. Vpr packaged into HIV-1 virions lacking a genome induced efficient cell cycle arrest and apoptosis. Introduction of Vpr into tumor cell lines of various tissue origin, including those bearing predisposing mutations in p53, XPA, and hMLH1, induced cell cycle arrest and apoptosis with high efficiency. Significantly, apoptosis mediated by virion-associated Vpr was more effective on rapidly dividing cells compared with slow-growing cells, thus, in concept, providing a potential differential effect between some types of tumor cells and surrounding normal cells. This model system provides a rationale and proof of concept for the development of potential cancer therapeutic agents based on the growth-arresting and apoptotic properties of Vpr.

The presence of human immunodeficiency virus type 1 Vpr correlates with a decrease in the frequency of mutations in a plasmid shuttle vector.

The human immunodeficiency virus type 1 (HIV-1) Vpr protein induces cell cycle arrest at the border of G(2) and M similar to the arrest caused by agents which damage DNA. We determined whether the presence of Vpr would affect the ability of cells to repair DNA. We developed a shuttle vector system to analyze the effect of Vpr upon the repair of UV-damaged DNA. Our results demonstrated that the presence of Vpr decreased the rate of deletions in this system. Of note, cells arrested in G(2) by other genotoxic agents also increased the frequency of DNA repair of UV-damaged shuttle vectors. We did not observe any direct effect of Vpr upon the rate of double-strand break repair and/or nucleotide excision repair of genomic DNA in cells. Our results suggest a role for HIV-1 Vpr in altering the frequency of DNA repair, a property which may have importance for HIV-1 replication and pathogenesis.

Human immunodeficiency virus type 1 Vpr interacts with HHR23A, a cellular protein implicated in nucleotide excision DNA repair.

The human immunodeficiency virus type 1 (HIV-1) vpr gene is an evolutionarily conserved gene among the primate lentiviruses HIV-1, HIV-2, and simian immunodeficiency viruses. One of the unique functions attributed to the vpr gene product is the arrest of cells in the G2 phase of the cell cycle. Here we demonstrate that Vpr interacts physically with HHR23A, one member of an evolutionarily conserved gene family involved in nucleotide excision repair. Interaction of Vpr with HHR23A was initially identified through a yeast two-hybrid screen and was confirmed by the demonstration of direct binding between bacterially expressed recombinant and transiently expressed or chemically synthesized protein products. Visualization of HHR23A and Vpr by indirect immunofluorescence and confocal microscopy indicates that the two proteins colocalize at or about the nuclear membrane. We also map the Vpr-binding domain in HHR23A to a C-terminal 45-amino-acid region of the protein previously shown to have homology to members of the ubiquitination pathway. Overexpression of HHR23A and a truncated derivative which includes the Vpr-binding domain results in a partial alleviation of the G2 arrest induced by Vpr, suggesting that the interaction between Vpr and HHR23A is critical for cell cycle arrest induced by Vpr. These results provide further support for the hypothesis that Vpr interferes with the normal function of a protein or proteins involved in the DNA repair process and, thus, in the transmission of signals that allow cells to transit from the G2 to the M phase of the cell cycle.

Human immunodeficiency virus type 1 Vpr induces apoptosis following cell cycle arrest.

The human immunodeficiency virus type 1 (HIV-1) vpr gene encodes a protein which induces arrest of cells in the G2 phase of the cell cycle. Here, we demonstrate that following the arrest of cells in G2, Vpr induces apoptosis in human fibroblasts, T cells, and primary peripheral blood lymphocytes. Analysis of various mutations in the vpr gene revealed that the extent of Vpr-induced G2 arrest correlated with the levels of apoptosis. However, the alleviation of Vpr-induced G2 arrest by treatment with the drug pentoxifylline did not abrogate apoptosis. Together these studies indicate that induction of G2 arrest, but not necessarily continued arrest in G2, was required for Vpr-induced apoptosis to occur. Finally, Vpr-induced G2 arrest has previously been correlated with inactivation of the Cdc2 kinase. Some models of apoptosis have demonstrated a requirement for active Cdc2 kinase for apoptosis to occur. Here we show that accumulation of the hypophosphorylated or active form of the Cdc2 kinase is not required for Vpr-induced apoptosis. These studies indicate that Vpr is capable of inducing apoptosis, and we propose that both the initial arrest of cells and subsequent apoptosis may contribute to CD4 cell depletion in HIV-1 disease.

Human immunodeficiency virus type 1 vpr gene induces phenotypic effects similar to those of the DNA alkylating agent, nitrogen mustard.

The product of the human immunodeficiency virus type 1 (HIV-1) vpr gene induces cell cycle arrest in the G2 phase of the cell cycle and is characterized by an accumulation of the hyperphosphorylated form of cdc2 kinase. This phenotype is similar to the effect of DNA-damaging agents, which can also cause cells to arrest at G2. We previously reported that Vpr mimicked some of the effects of a DNA alkylating agent known as nitrogen mustard (HN2). Here we extend these earlier observations by further comparing the activation state of cdc2 kinase, the kinetics of G2 arrest, and the ability to reverse the arrest with chemical compounds known as methylxanthines. Infection of cells synchronized in the G1 phase of the cell cycle with a pseudotyped HIV-1 resulted in arrest at G2 within 12 h postinfection, before the first mitosis. Similar to that induced by HN2, Vpr-induced arrest led to a decrease in cdc2 kinase activity. Vpr-mediated G2 arrest was alleviated by methylxanthines at concentrations similar to those needed to reverse the G2 arrest induced by HN2, and cells proceeded apparently normally through at least one complete cell cycle. These results are consistent with the hypothesis that Vpr induces G2 arrest through pathways that are similar to those utilized by DNA-damaging agents.

Vpr-induced cell cycle arrest is conserved among primate lentiviruses.

We previously reported that expression of human immunodeficiency virus type 1 strain NL4-3 (HIV-1(NL4-3))vpr causes cells to arrest in the G2 phase of the cell cycle. We examined the induction of cell cycle arrest by other HIV-1 isolates and by primary lentiviruses other than HIV-1. We demonstrate that the vpr genes from tissue culture-adapted or primary isolates of HIV-1 are capable of inducing G2 arrest. In addition, we demonstrate that induction of cell cycle arrest is a conserved function of members of two other groups of primate lentiviruses, HIV-2/simian immunodeficiency virus strain sm (SIVsm)/SIVmac and SIVagm. vpr from HIV-1, HIV-2, and SIVmac induced cell cycle arrest when transfected in human (HeLa) and monkey (CV-1) cells. vpx from HIV-2 and SIVmac did not induce detectable cell cycle arrest in either cell type, and SIVagm vpx was capable of inducing arrest in CV-1 but not HeLa cells. These results indicate that induction of cell cycle perturbation is a general property of lentiviruses that infect primates. The conservation of this viral function throughout evolution suggests that it plays a key role in virus-host relationships, and elucidation of its mechanism may reveal important clues about pathology induced by primary lentiviruses.

The human immunodeficiency virus type 1 vpr gene arrests infected T cells in the G2 + M phase of the cell cycle.

Human immunodeficiency virus type 1 (HIV-1) infection causes profound immunological defects in afflicted patients. Various mechanisms have been proposed to account for the immune dysfunction in AIDS ultimately leading to loss of CD4+ T cells, including HIV-1 envelope-mediated syncytium formation, apoptosis, and cytokine modulation. Here we present results which suggest a novel hypothesis for T-cell dysfunction. We show, using HIV-1 bearing a novel cell surface reporter gene, that infected cells are unable to progress normally through the cell cycle and became arrested in the G2 + M phase. Furthermore, we identify the HIV-1 vpr gene product as being both necessary and sufficient for eliciting this cell cycle arrest. Cell cycle arrest induced by Vpr correlates with an increase in the hyperphosphorylated (inactive) form of the cyclin-dependent serine/threonine kinase CDC2, consistent with an arrest of cells at the boundary of G2 and M.

Fate of the human immunodeficiency virus type 1 provirus in infected cells: a role for vpr.

We investigated the fate of human immunodeficiency virus type 1 (HIV-1) viral DNA in infected peripheral blood lymphocytes and immortalized T-cell lines by using a replication-defective HIV-1. We observed that integrated HIV-1 DNA and viral gene expression decrease over time. A frameshift mutation in vpr resulted in maintenance of the HIV-1 provirus and stable persistence of viral expression. Transfection of vpr together with the neomycin resistance gene in the absence of other viral genes decreased the formation of geneticin-resistant colonies, indicating either a cytotoxic or a cytostatic effect upon cells. Therefore, maintenance of HIV-1 infection within an infected proliferating population is due to two competing processes, the rate of viral spread and the degree of cell growth inhibition and/or death induced by Vpr.

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.

Requirement of human immunodeficiency virus type 1 nef for in vivo replication and pathogenicity.

The role of human immunodeficiency virus type 1 (HIV-1) accessory genes in pathogenesis has remained unclear because of the lack of a suitable in vivo model. The most controversial of these genes is nef. We investigated the requirement for Nef for in vivo replication and pathogenicity of two isolates of HIV-1 (HIV-1JR-CSF and HIV-1NL4-3) in human fetal thymus and liver implants in severe combined immunodeficient mice. HIV-1JR-CSF and HIV-1NL4-3 differ in their in vitro phenotypes in that HIV-1JR-CSF does not induce syncytia and is relatively noncytopathic, while HIV-1NL4-3 is highly cytopathic and readily induces syncytia. The nef mutants of both isolates grew with kinetics similar to those of parental virus strains in stimulated peripheral blood lymphocytes but demonstrated attenuated growth properties in vivo. HIV-1NL4-3 induced severe depletion of human thymocytes within 6 weeks of infection, whereas its nef mutant did not. Thus, HIV-1 Nef is required for efficient in vivo viral replication and pathogenicity.

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).

Recombinant baculovirus vectors expressing glutathione-S-transferase fusion proteins.

Recombinant baculoviruses are a popular means of producing heterologous protein in eukaryotic cells. Purification of recombinant proteins away from the insect cell background can, however, remain an obstacle for many developments. Recently, prokaryotic fusion protein expression systems have been developed allowing single-step purification of the heterologous protein and specific proteolytic cleavage of the affinity tag moiety from the desired antigen. Here we report the introduction of these attributes to the baculovirus system. "Baculo-GEX" vectors enable baculovirus production of fusion proteins with the above advantages, but in a eukaryotic post-translational processing environment. Glutathione-S-transferase (GST) fusions are stable cytoplasmic proteins in insect cells and may therefore be released by sonication alone, avoiding the solubility problems and detergent requirements of bacterial systems. Thus large amounts of authentic antigen may be purified in a single, non-denaturing step.

Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation.

The human immunodeficiency virus type 1 (HIV-1) gag gene product Pr55 self-assembles to form virus-like particles when expressed in Spodoptera frugiperda cells using recombinant baculoviruses. The particles resemble immature HIV and are released from the infected cell into the culture medium. Using this system we have progressively truncated the gag open reading frame from the C terminus and examined each deleted gag protein for its particle-producing capability. We show that deletion of Pr6 and deletions that progressively remove the distal region of the Pr7 domain, including one Cys-His box thought to function as an RNA capture signal, do not affect particle formation. However deletion of two Cys-His boxes causes production of slightly larger particles with altered sedimentation properties. Sequence-specific North-Western assays using an RNA probe representative of the HIV-1 packaging signal revealed specific RNA binding by all mutants that maintained both Cys-His boxes. However, deletion of one Cys-His box reduced RNA binding substantially and loss of two Cys-His boxes abolished binding entirely. We conclude that HIV-1 gag particle formation per se does not require viral RNA encapsidation, but that it may act as a cofactor in the condensation of the immature core. Further deletion of gag sequences upstream of the Cys-His boxes led to the abolition of particle-forming ability, and we show that one boundary of the gag sequence necessary for particle formation lies within eight amino acids spanning one of the known protease cleavage sites at the C terminus of Pr24.

Isolation of HIV from Australian patients with AIDS, AIDS related conditions and healthy antibody positive individuals.

The Fairfield Hospital experience with isolation of HIV from peripheral blood leucocytes, cerebrospinal fluid and semen is described. To date HIV has been isolated from single specimens of blood from 45% of patients with AIDS, 35% of patients with lymphadenopathy syndrome AIDS-related complex or ARC and 14% of asymptomatic antibody positive individuals. HIV was recovered from peripheral blood leucocytes in the presence of phytohemagglutinin and interleukin-2. The presence of virus in the supernatant fluid was detected using reverse transcriptase and immunofluorescence assays. Supernatants with borderline activity were confirmed by infection of a continuous cell line.