The role of CCR5 and CCR2 polymorphisms in HIV-1 transmission and disease progression.

Entry of human immunodeficiency virus type 1 (HIV-1) into target cells requires both CD4 (ref. 1, 2) and one of a growing number of G-protein-coupled seven-transmembrane receptors. Viruses predominantly use one, or occasionally both, of the major co-receptors CCR5 or CXCR4, although other receptors, including CCR2B and CCR3, function as minor co-receptors. CCR3 appears critical in central nervous system infection. A 32-base pair inactivating deletion in CCR5 (delta 32) common to Northern European populations has been associated with reduced, but not absolute, HIV-1 transmission risk and delayed disease progression. A more commonly distributed transition causing a valine to isoleucine switch in transmembrane domain I of CCR2B (64I) with unknown functional consequences was recently shown to delay disease progression but not reduce infection risk. Although we confirm the lack of association of CCR2B 64I with transmission, we cannot confirm the association with delayed progression. Although subjects with CCR5 delta 32 defects had significantly reduced median viral load at study entry, providing a plausible explanation for the association with delayed progression, this association was not seen with CCR2B 64I. Further studies are needed to define the role of CCR2B64I in HIV pathogenesis.

Genomic organization and functional characterization of the chemokine receptor CXCR4, a major entry co-receptor for human immunodeficiency virus type 1.

CXCR4 is both a chemokine receptor and entry co-receptor for T-cell line-adapted human immunodeficiency virus type 1. The genomic organization and promoter function for the entire transcription unit of CXCR4 were determined. The gene contains 2 exons of 103 and 1563 base pairs (bp) interrupted by a 2132-bp intron precisely between codons 5 and 6 of the coding sequences. A transcription start site was identified 88 bp upstream of the initiation codon, and a polyadenylate addition site was identified 22 bp 3' to a polyadenylation signal. Transient expression assays defined a minimal promoter at positions -114 to +43 relative to the transcription start site. This region contains a TATA box, a nuclear respiratory factor-1 (NRF-1) site, and two GC boxes. Specific factor binding to the NRF-1 site and GC boxes were demonstrated by gel mobility shifts and DNase I footprinting. Site-directed mutagenesis showed that the NRF-1 site is crucial for promoter activity providing the first evidence for the regulation of a signal transduction gene by NRF-1. Sequences between -691 and -191 repress CXCR4 promoter activity. Further study of these regulatory elements will be important to understanding how CXCR4 functions as both a chemokine receptor and human immunodeficiency virus type 1 entry co-receptor.

Comparison of serum and plasma viral RNA measurements in primary and chronic human immunodeficiency virus type 1 infection.

We sought to define the relation between serum and plasma HIV-1 viral RNA load in patients with primary and chronic HIV-1 disease. HIV-1 viral load was determined from 116 serum and plasma samples, including 33 matched pairs, from five patients with primary and three patients with chronic HIV disease using the Roche HIV Monitor assay. The mean +/- standard deviations of the serum and plasma viral RNA levels from the 33 matched pairs were 4.372 +/- 0.885 and 4.478 +/- 0.950 log10 (copies/ml), respectively. This -0.106 log difference between serum and plasma viral RNA levels, which equates to 21% of non-log-transformed values, was not statistically significant by the Wilcoxon sign rank test (p = 0.09). The distributions of serum and plasma viral load slopes, calculated from all available viral RNA load data for each patient, were also not statistically different (p = 0.07). The levels of HIV-1 RNA measured in the serum or plasma of HIV-seropositive patients yield equivalent biologic information.

Construction and biological characterization of infectious molecular clones of HIV-1 subtypes B and E (CRF01_AE) generated by the polymerase chain reaction.

We previously described the use of extended polymerase chain reaction (PCR) to amplify contiguous 9.2-kilobase (kb) single-long terminal repeat (LTR) proviral sequences from HIV-1 genetic subtypes A through G. We now extend these findings by describing a novel vector system to recover infectious molecular clones from long PCR amplicons. Directional ligation of 9.2-kb proviral amplicons into a recovery vector reconstitutes missing LTR sequences, providing candidate molecular clones for infectivity screening. We show that a previously characterized infectious molecular clone of HIV-1 retains its biological properties upon recovery with this strategy. Three additional infectious molecular clones generated, from primary isolates of subtype B (HIV-1(WR27)) and circulating recombinant form 01_AE (subtype E) (HIV-1(CM235)) by subtype-specific LTR reconstitution, displayed biological properties reflecting their cognate parental isolates. This represents the first report of infectious molecular clones from circulating recombinant form 01_AE (subtype E).

Dual infection with human immunodeficiency virus type 1 of distinct envelope subtypes in humans.

Multiple genetic subtypes of human immunodeficiency virus type 1 (HIV-1) have been identified among internationally collected isolates. The HIV-1 epidemic in Thailand is largely due to B and E subtypes of virus. Dual infection with distinct HIV-1 subtypes would suggest that antiviral immunity evoked by one subtype can be incompletely protective against a second. Polymerase chain reaction typing and serologic typing were used to screen a panel of specimens from HIV-1-infected subjects in Thailand. Two persons simultaneously harbored HIV-1 of env subtypes B and E, and this was confirmed by colony hybridization with subtype-specific probes and nucleotide sequence analysis of a 630-bp fragment of gp120 from multiple molecular clones. In addition, both subtypes were identified in cocultured peripheral blood mononuclear cells from 1 individual. These data provide the first evidence of dual HIV-1 infection in humans and reinforce the need for polyvalent vaccines.

Major histocompatibility complex genotype is associated with disease progression and virus load levels in a cohort of human immunodeficiency virus type 1-infected Caucasians and African Americans.

To assess the influence of HLA on AIDS-free survival, human immunodeficiency virus load, and CD4 cell counts, 91 Caucasian and 48 African-American seroprevalent men were typed for HLA classes I and II and TAP alleles. HLA associations with these markers were assessed by assigning sum integer scores based on 7 class I allele-TAP variants (+1) and 13 class I-class II-TAP combinations (-1) with different AIDS-free survival times found in a prior study. Subjects in both racial groups and combined with positive sum scores were less likely to have CD4 cell decline (P=.0004), to have increased virus burden (P=.014), and to develop AIDS (P=.034) in the follow-up period than were Caucasians and African Americans with scores of 0 or -1. These results confirm the reported associations of specific major histocompatibility complex genes with AIDS-free survival time in Caucasians and specifically extend them to African Americans and to two established markers of disease progression.