In the course of the global pandemic, the human immunodeficiency virus type-1 (HIV-1) has established at least eight distinct genotypes in the main (M), or prevalent, group of isolates, a variety of rare outlier forms, and intergenotypic recombinants of group M viruses. This genotypic diversity has been documented, for the most part, by sequencing of subgenomic segments of the provirus. Using DNA from virus cultures on peripheral blood mononuclear cells (PBMC) and recent improvements of the PCR technique, we have amplified virtually full-length HIV-1 genomes from genetic subtypes A through G of group M viruses and molecularly cloned several of them. Resequencing of the complete genome of a prototype strain after long PCR amplification and cloning has established a PCR error rate of 0.14%. We also report the first complete PCR-derived sequence of a U.S. clinical isolate of genotype B expanded only in primary PBMC; this provirus harbors a uniquely truncated V3 loop.

CXCR4 is both a chemokine receptor and entry coreceptor 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.

Current understanding of human immunodeficiency virus type 1 (HIV-1) transcription is based on unidirectional expression of transcripts with positive-strand polarity from the 5' long terminal repeat. We now report HIV-1 transcripts with negative-strand polarity obtained from acutely and chronically infected cell lines by use of a template orientation-specific reverse transcriptase-PCR assay. These findings were confirmed in natural infection by analysis of RNA derived from peripheral blood mononuclear cell samples from 15 HIV-1-infected patients. A cDNA derived from a 2.3-kb polyadenylated HIV-1 RNA with negative-strand polarity which encodes a highly conserved 189-amino-acid open reading frame antiparallel to the envelope gene was isolated from acutely infected A3.01 cells. Through use of reporter gene constructions, we further found that a novel negative-strand promoter functions within the negative response element of the 3' long terminal repeat, which is downregulated by coexpression of Tat. Site-directed mutagenesis experiments demonstrated that NF-KB I and USF sites are crucial for negative-strand promoter activity. These data extend the coding capacity of HIV-1 and suggest a role for antisense regulation of the viral life cycle.

Key PointsDifferent pathogen-specific CD4 T cells manifest remarkable difference in susceptibility to HIV infection. Distinct gene-expression profiles of pathogen-specific CD4 T cells are associated with their susceptibilities to HIV infection.