Human immunodeficiency virus type 1 (HIV-1) quasispecies at the sites of Mycobacterium tuberculosis infection contribute to systemic HIV-1 heterogeneity

Abstract

We have recently reported an increased heterogeneity in the human immunodeficiency virus type 1 (HIV-1) envelope gene (env) in HIV-1-infected patients with pulmonary tuberculosis (TB) compared to patients with HIV-1 alone. This increase may be a result of dissemination of lung-derived HIV-1 isolates from sites of Mycobacterium tuberculosis infection and/or the systemic activation of the immune system in response to TB. To distinguish between these two mechanisms, blood and pleural fluid samples were obtained from HIV-1-infected patients with active pleural TB in Kampala, Uganda (CD4 cell counts of 34 to 705 cells/microl, HIV-1 plasma loads of 2,400 to 280,000 RNA copies/ml, and HIV-1 pleural loads of 7,600 to 4,500,000 RNA copies/ml). The C2-C3 coding region of HIV-1 env was PCR amplified from lysed peripheral blood mononuclear cells and pleural fluid mononuclear cells and reverse transcriptase-PCR amplified from plasma and pleural fluid HIV-1 virions of eight HIV-1 patients with pleural TB. Phylogenetic and phenetic analyses revealed a compartmentalization of HIV-1 quasispecies between blood and pleural space in four of eight patients, with migration events between the compartments. There was a trend for a greater genetic heterogeneity in the pleural space, which may be the result of an M. tuberculosis-mediated increase in HIV-1 replication and/or selection pressure at the site of infection. Collectively, these findings suggest that HIV-1 quasispecies in the M. tuberculosis-infected pleural space may leak into the systemic circulation and lead to increased systemic HIV-1 heterogeneity during TB.

FIG. 1.

Phylogenetic tree of the PBMC average sequence patient samples for subtyping. Bootstrap resampling values of 90 to 100% and 70 to 90% are represented by ∗∗ and ∗, respectively. A 277-nt segment of the C2-C3 region was used to construct these trees by the neighbor-joining method. env genetic subtypes are indicated. GenBank accession numbers for the reference sequences are: SIVcpz, AF115393; group O isolate MVP5180, E16837; subtype C isolates 94UG114 (U88824), 84ZR085 (U88822), ELI (K03454), and NDK (A34828); subtype AE isolates TN235 (L03P55) and 90CF402 (U51188); subtype F isolates BZ163 (L22085) and FIN9363 (AF075703); subtype G isolates DRCBL (AF084936), HH8793 (AF061640), and SE6165 (AF061642); subtype A isolates Q2317 (AF004885), SE7253 (AF069670), 92UG037 (U51190), IBNG (L39106), and U455 (M62320); subtype H isolates VI557 (U09666) and VI991 (AF190127); subtype cpx isolates GR84 (AF119819) and GR11 (AF119820); subtype J isolates SE9173 (AF082395) and SE9280 (AF082394); subtype C isolates ETH2220 (U46016), 92BR025 (U52953), 21068 (AF067155), and 96BW05.02 (AF110967); and subtype B isolates WEAU1.60 (U21135), HX2BR (K03455), RF (M17451), JRFL (U63632), and KAL153 (AF193276).

FIG. 2.

Analysis of HIV-1 env quasispecies from the blood and pleural space of HIV-1-infected individuals with pleural TB. (A) Phylogenetic tree of all quasispecies. The letters preceding the name of the reference sequence indicate the subtype. The bootstrap values (percentages) separating each set of patient samples are indicated. The scale bar represents 0.1 substitution per nucleotide position (s/nt). The average nucleotide diversity of all HIV-1 env quasispecies within an individual was plotted against CD4 cell count (B) and plasma viral load (C) obtained from the same sample and time point.

FIG. 3.

Analysis of HIV-1 env quasispecies from two different tissue compartments of individual patients with pleural TB. Phylogenetic trees were derived from individual env sequences from the blood and pleural space of patients P50, P55, P56, P57, P59, P61, P63, and P64 (A to H, respectively). Branch lengths are drawn to scale. The scale bar represents 0.01 substitution per nucleotide position. The legend indicates bootstrap values greater than 50%. HIV-1 env sequences were obtained from 9 or 10 clones from each sample of PBMC, plasma, PFMC, and pleural fluids. HIV-1 env PCR products were cloned into the pCR2.1-Topo vector (Invitrogen) to select the quasispecies for sequencing and subsequent analyses.

FIG. 4.

Phylogenetic and phenetic evaluation of blood and pleural compartmentalization in HIV-1-infected Ugandans with pleural TB. (A) Determination of compartmental changes. The source of each sequence is determined by the compartment from which it was isolated (blood or pleural space). The ancestry of each branch was determined by the sources of the terminal quasispecies so as to minimize compartmental changes, which occur when the ancestry of a branch changes. The sum total of compartmental changes (s) was calculated for each tree. (B) Migration events between compartments for the 1,000 actual bootstrap trees and 1,000 randomly constructed trees were constructed using the neighbor-joining method. Tree topology for the actual bootstrap trees was based on nucleotide sequence data for the C2-C3 region of the env (see Fig. 3 and Materials and Methods). Thin and thick error bars indicate 1 and 2 standard deviations of s ratios of actual to random trees, respectively. s ratios that fall 2 standard deviations below 1 are considered significant and suggest compartmentalization. Patients with significant blood and pleural compartmentalization of env quasispecies (P < 0.01) as determined by Mantel's test are marked by asterisks.

FIG. 5.

Analysis of env genetic diversity in blood and pleural compartments before and after removal of migrant quasispecies. Quasispecies migrating from the blood to the pleural space (open arrow) or from the pleural space to the blood (solid arrow) are highlighted in the phylogenetic trees from patients P64 (A) and P65 (B). These migrants were then removed from the group of blood or pleural env quasispecies. Intracompartmental genetic distances were then calculated before and after the removal on migrant sequences and plotted in panel C. Error bars represent 1 standard error of the mean. Below each patient number is the number of sequences removed from the blood (B) and pleural space (P). Brackets indicate significant differences in the genetic distances between blood and pleural env populations. ∗, P < 0.05; ∗∗, P < 0.01.