Jennifer Stanton

Reply to: CCL3L1 and HIV/AIDS susceptibility

We read with interest the recent article in Nature Medicine describing the influence of variation in CCL3L1 copy number and CCR5 genotype on immune recovery during highly active antiretroviral therapy (HAART) in HIV-1–infected individuals1. The chemotactic cytokine CCL3L1 (encoding the macrophage inflammatory protein-1αP (MIP1αP) protein) is a potent ligand for the HIV-1 co-receptor CCR5, which is essential for viral entry into human host cells2. The recent study is part of a series that began in 2005 with a paper reporting effects of CCL3L1 copy number variation on HIV-1 acquisition, viral load and disease progression3, followed by several publications investigating clinically correlated phenotypes in a largely overlapping set of HIV-positive individuals1,4,5. Although these studies seem to generate considerable independent support for a role of CCL3L1 in viral control, many of the traits considered are at least partially correlated, and the studies include largely overlapping samples and presumably CCL3L1 assay data. For these reasons, we sought to reevaluate a core set of associations related to the effect of CCL3L1 copy number on viral control in a large group of HIV-infected individuals with known date of seroconversion enrolled in one of the nine cohorts of the Euro-CHAVI Consortium6 (n = 1,042), in an African-American cohort from the TACC (n = 277) or in the MACS (n = 451 HIV-positive, n = 195 high-risk seronegative (HRSN)). We assayed for CCL3L1 copy number using a previously described method3 (Supplementary Methods). A total of 1,855 subjects were successfully genotyped. Distributions of CCL3L1 copy numbers in individuals of European or African ancestry were similar to those reported elsewhere, with a median copy number of 2 or 4 in individuals of primarily European (range 0–9) or African (range 1–11) descent, respectively (Fig. 1a,b and Supplementary Figs. 1–4)1,3,4,7. We then tested for association of CCL3L1 copy number with HIV viral load at set point by linear regression after stratifying according to ethnicity and correcting for known covariates (gender, age at seroconversion and ancestry as determined by a principal components method described previously8), and found no evidence of association (European, P = 0.14; African, P = 0.27) (Fig. 1c,d). Dividing the sample into the previously described “high-risk” (CCL3L1low) and “low-risk” (CCL3L1high) genotype groups (where high risk versus low risk is defined as having copy number below versus equal to or above the population median, respectively)3, we again found no evidence of association, either within each population (European, P = 0.10; African, P = 0.41) or in the combined sample (P = 0.35) (Table 1). Furthermore, a model including known functional polymorphisms in the CCR5 receptor (CCR5∆32, CCR5*HHE) in a subset of n = 820 individuals of European descent for which CCR5 effects had been tested previously (J.F., D. Ge, K.V.S., S.C., B. Ledergerber et al., unpublished data) showed that although the CCR5 polymorphisms were strongly associated with viral load (CCR5∆32: β = –0.29 ± 0.08 log RNA copies, P = 0.001; CCR5*HHE: β = 0.14 ± 0.05 log RNA copies, P = 0.005), there remained no appreciable effect of CCL3L1 copy number (copy number, P = 0.24; genotype risk group, P = 0.12). CCL3L1 and HIV/AIDS susceptibility

Detection of HIV-1 p24 Gag in plasma by a nanoparticle-based bio-barcode-amplification method

BACKGROUND Detection of HIV-1 in patients is limited by the sensitivity and selectivity of available tests. The nanotechnology-based bio-barcode-amplification method offers an innovative approach to detect specific HIV-1 antigens from diverse HIV-1 subtypes. We evaluated the efficacy of this protein-detection method in detecting HIV-1 in men enrolled in the Chicago component of the Multicenter AIDS Cohort Study (MACS). METHODS The method relies on magnetic microparticles with antibodies that specifically bind the HIV-1 p24 Gag protein and nanoparticles that are encoded with DNA and antibodies that can sandwich the target protein captured by the microparticle-bound antibodies. The aggregate sandwich structures are magnetically separated from solution, and treated to remove the conjugated barcode DNA. The DNA barcodes (hundreds per target) were identified by a nanoparticle-based detection method that does not rely on PCR. RESULTS Of 112 plasma samples from HIV-1-infected subjects, 111 were positive for HIV-1 p24 Gag protein (range: 0.11-71.5 ng/ml of plasma) by the bio-barcode-amplification method. HIV-1 p24 Gag protein was detected in only 23 out of 112 men by the conventional ELISA. A total of 34 uninfected subjects were negative by both tests. Thus, the specificity of the bio-barcode-amplification method was 100% and the sensitivity 99%. The bio-barcode-amplification method detected HIV-1 p24 Gag protein in plasma from all study subjects with less than 200 CD4(+) T cells/microl of plasma (100%) and 19 out of 20 (95%) HIV-1-infected men who had less than 50 copies/ml of plasma of HIV-1 RNA. In a separate group of 60 diverse international isolates, representative of clades A, B, C and D and circulating recombinant forms CRF01_AE and CRF02_AG, the bio-barcode-amplification method identified the presence of virus correctly. CONCLUSIONS The bio-barcode-amplification method was superior to the conventional ELISA assay for the detection of HIV-1 p24 Gag protein in plasma with a breadth of coverage for diverse HIV-1 subtypes. Because the bio-barcode-amplification method does not require enzymatic amplification, this method could be translated into a robust point-of-care test.

A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles.

Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.

Structure and function of CC-chemokine receptor 5 homologues derived from representative primate species and subspecies of the taxonomic suborders Prosimii and Anthropoidea

ABSTRACT A chemokine receptor from the seven-transmembrane-domain G-protein-coupled receptor superfamily is an essential coreceptor for the cellular entry of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) strains. To investigate nonhuman primate CC-chemokine receptor 5 (CCR5) homologue structure and function, we amplified CCR5 DNA sequences from peripheral blood cells obtained from 24 representative species and subspecies of the primate suborders Prosimii (family Lemuridae) and Anthropoidea (families Cebidae, Callitrichidae, Cercopithecidae, Hylobatidae, and Pongidae) by PCR with primers flanking the coding region of the gene. Full-length CCR5 was inserted into pCDNA3.1, and multiple clones were sequenced to permit discrimination of both alleles. Compared to the human CCR5 sequence, the CCR5 sequences of the Lemuridae, Cebidae, and Cercopithecidae shared 87, 91 to 92, and 96 to 99% amino acid sequence homology, respectively. Amino acid substitutions tended to cluster in the amino and carboxy termini, the first transmembrane domain, and the second extracellular loop, with a pattern of species-specific changes that characterized CCR5 homologues from primates within a given family. At variance with humans, all primate species examined from the suborder Anthropoidea had amino acid substitutions at positions 13 (N to D) and 129 (V to I); the former change is critical for CD4-independent binding of SIV to CCR5. Within the Cebidae, Cercopithecidae, and Pongidae (including humans), CCR5 nucleotide similarities were 95.2 to 97.4, 98.0 to 99.5, and 98.3 to 99.3%, respectively. Despite this low genetic diversity, the phylogeny of the selected primate CCR5 homologue sequences agrees with present primate systematics, apart from some intermingling of species of the Cebidae and Cercopithecidae. Constructed HOS.CD4 cell lines expressing the entire CCR5 homologue protein from each of the Anthropoidea species and subspecies were tested for their ability to support HIV-1 and SIV entry and membrane fusion. Other than that of Cercopithecuspygerythrus, all CCR5 homologues tested were able to support both SIV and HIV-1 entry. Our results suggest that the shared structure and function of primate CCR5 homologue proteins would not impede the movement of primate immunodeficiency viruses between species.

Transcriptional Regulation of the Human CD3γ Gene: The TATA-Less CD3γ Promoter Functions via an Initiator and Contiguous Sp-Binding Elements

Growing evidence that the CD3γ gene is specifically targeted in some T cell diseases focused our attention on the need to identify and characterize the elusive elements involved in CD3γ transcriptional control. In this study, we show that while the human CD3γ and CD3δ genes are oriented head-to-head and separated by only 1.6 kb, the CD3γ gene is transcribed from an independent but weak, lymphoid-specific TATA-less proximal promoter. Using RNA ligase-mediated rapid amplification of cDNA ends, we demonstrate that a cluster of transcription initiation sites is present in the vicinity of the primary core promoter, and the major start site is situated in a classical initiator sequence. A GT box immediately upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these adjacent elements enhanced by the presence of a second GC box 10 nt further upstream. The primary core promoter is limited to a sequence that extends upstream to −15 and contains the initiator and GT box. An identical GT box located ∼50 nt from the initiator functions as a weak secondary core promoter and likely generates transcripts originating upstream from the +1. Finally, we show that two previously identified NFAT motifs in the proximal promoter positively (NFATγ1) or negatively (NFATγ1 and NFATγ2) regulate expression of the human CD3γ gene by their differential binding of NFATc1 plus NF-κB p50 or NFATc2 containing complexes, respectively. These data elucidate some of the mechanisms controlling expression of the CD3γ gene as a step toward furthering our understanding of how its transcription is targeted in human disease.

Characterization of a Downstream Positive Element Involved in Transcriptional Control of the Human CD3γ Gene Promoter

Experimental data from our laboratory has shown that TCR/CD3 surface receptors are downmodulated after HIV-1 and HIV-2 infection of CD4+ T cells due to a specific defect in CD3γ gene transcripts. In an effort to better understand the mechanism(s) involved, our laboratory has been investigating the critical elements responsible for regulating this gene. We have shown that the CD3 γ gene is transcribed from an independent but weak, lymphoidspecific TATA-less promoter and demonstrated that a cluster of transcription initiation sites is present in the vicinity of the principal core promoter with the major start site situated in a classical initiator sequence. A GT box upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these contiguous elements enhanced by a second Sp binding GC box ten nucleotides further upstream. We found that two previously identified NFAT motifs positively (NFAT γ 1) or negatively (NFAT γ 1 and NFAT γ 2) regulate expression of the CD3 γ gene by their differential binding of NFATc1 plus NF-κB p50 or NFATc2 containing complexes, respectively. Analysis of various mutant and deletion CD3 γ promoter constructs in a transient reporter assay revealed that a 53 bp region downstream from the major transcription start site is critical for positive gene expression. Deletion of ten nucleotides in this region results in a 50% decrease in promoter activity, while deletion of 39 nucleotides completely eliminates promoter activity. EMSA experiments using DNA or RNA probes covering the 53 bp region demonstrate that this element functions through an RNA rather than a DNA intermediate. At least three specific nuclear protein complexes bind to the RNA probe. Deletion of the U at position +9 and the U at +37 completely abrogate binding and promoter activity. Experiments are currently underway to determine whether the composition of the transcription factor(s) complexes bound to the CD3γ element contain components of P-TEFb, which binds to HIV TAR. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005