Human immunodeficiency virus type 1 populations in blood and semen

Abstract

Transmission of human immunodeficiency virus type 1 (HIV-1) usually results in outgrowth of viruses with macrophage-tropic phenotype and consensus non-syncytium-inducing (NSI) V3 loop sequences, despite the presence of virus with broader host range and the syncytium-inducing (SI) phenotype in the blood of many donors. We examined proviruses in contemporaneous peripheral blood mononuclear cells (PBMC) and non-spermatozoal semen mononuclear cells (NSMC) of five HIV-1-infected individuals to determine if this preferential outgrowth could be due to compartmentalization and thus preferential transmission of viruses of the NSI phenotype from the male genital tract. Phylogenetic reconstructions of approximately 700-bp sequences covering the second constant region through the fifth variable region (C2 to V5) of the viral envelope gene revealed distinct variant populations in the blood versus the semen in two patients with AIDS and in one asymptomatic individual (patient 613), whereas similar variant populations were found in both compartments in two other asymptomatic individuals. Variants with amino acids in the V3 loop that predict the SI phenotype were found in both AIDS patients and in patient 613; however, the distribution of these variants between the two compartments was not consistent. SI variants were found only in the PBMC of one AIDS patient but only in the NSMC of the other, while they were found in both compartments in patient 613. It is therefore unlikely that restriction of SI variants from the male genital tract accounts for the observed NSI transmission bias. Furthermore, no evidence for a semen-specific signature amino acid sequence was detected.

FIG. 1

Distribution of divergences between pairwise comparisons of sequences. Provirus populations in the semen or PBMC were in each case sampled by PCR on multiple templates followed by cloning and sequencing of multiple plasmids (“clones”) or by endpoint dilution (“EPD”) followed by direct sequencing. For each set of sequences, the nucleotide differences between all possible pairwise comparisons were determined, collected into bins of 0.25% divergence intervals (uncorrected for back-mutation), and plotted.

FIG. 2

Neighbor-joining phylogenetic tree of proviral sequences from the blood and semen of five patients. Triangles represent sequences from NSMC; squares represent sequences from PBMC. Open symbols are from direct sequencing of molecular endpoints from PCR; filled symbols represent sequences from cloned PCR product. Cloned semen specimens from the JO sample patient were derived by PCR amplification, cloning, and sequencing of multiple templates from a single PCR initiated with >10 proviruses; those from PBMC were derived from PCR initiated with approximately 10 proviruses. Cloned NSMC and PBMC sequences from the PE sample patient were derived from single PCRs initiated with >10 and approximately 500 proviruses, respectively. Cloned PBMC specimens from the MA sample patient were derived from a PCR initiated with approximately 200 proviruses. Sequences with mutations predictive of a viral SI phenotype are indicated. Numbers at branch nodes refer to the number of bootstrap repetitions (of 1,000) at which the distal sequences grouped together; those occurring at greater than 80% frequency (corresponding to an approximate P value of 0.05) are shown.

FIG. 3

Deduced amino acid alignment over the C2-to-V5 region of HIV-1 env. For each patient, sequences are compared to one sequence from the PBMC. Dots are placed at positions at which individual sequences match that of the reference sequence. Dashes were introduced to maintain alignments. Asterisks indicate the presence of a stop codon. Question marks indicate an unknown amino acid due to an ambiguous base in the DNA sequence. The V3 loop is overlined and underlined at the top and bottom of each alignment, respectively.

FIG. 4

Distribution of divergences between pairwise comparisons of sequences. Proviral sequences from semen or PBMC derived by either endpoint dilution, cloning, or a combination of the two methods were grouped for analysis of compartments in each patient. For each set of sequences, the nucleotide differences between all possible pairwise comparisons were determined, collected into bins of 0.25% divergence intervals (uncorrected for back-mutation), and plotted.