Low copy number and limited variability of proviral DNA in alveolar macrophages from HIV-1-infected patients: evidence for genetic differences in HIV-1 between lung and blood macrophage populations.
BACKGROUND
We investigated the human immunodeficiency virus (HIV) proviral DNA sequence and copy number in alveolar macrophages (AM) and peripheral blood monocytes (PBM) from 10 HIV-positive patients without any active concurrent pulmonary disease to understand the nature of HIV-1 infection in vivo in the lung microenvironment.


METHODS
The 10 seropositive patients without active pulmonary disease were selected based on chest roentegenography and pathological/cytological test of bronchoalveolar (BAL) fluid. In order to determine accurate proviral copy numbers, AM and PBM were isolated to 99 and 94% purity, respectively, and quantitative polymerase chain reaction (PCR), with a sensitivity to detect three copies of HIV proviral DNA per 10(5) cells, was applied. For analysis of genetic variation in HIV-1, PCR-amplified HIV-1 DNA from AM and PBM of five patients were subcloned and 2-12 clones from each sample underwent DNA sequence analysis of HIV-1 gp120 V3-V5. Heteroduplex mobility assays were performed to confirm the results of the sequence analysis.


RESULTS
The proviral copy number in AM or PBM were less than 20 copies/10(5) cells in all patients, and five patients had less than the detection limit. There was no significant difference in HIV copy number between AM and PBM. No correlation was found between PBM/AM HIV copy number and CD4+ lymphocyte count in the peripheral blood. Sequence analysis revealed that the mean intrapatient genetic similarity in AM was 97.5 +/- 0.18% (n = 107), which was significantly higher than that in PBM (96.2 +/- 0.26% (n = 94), p < 0.001), suggesting that variability of HIV-1 DNA in AM was relatively limited. Divergence occurred when AM derived HIV-1 sequence was compared with PBM derived sequence from the same patient (95.8 +/- 0.17% (n = 223) p < 0.001). Phylogenetic analysis of DNA sequence demonstrated complete separation of HIV lineages from lung and blood in four of five patients.


CONCLUSIONS
The results suggest the HIV-1 infection in AM is restricted in vivo with low viral burden and homogenous genotype. We propose that the pulmonary microenvironment may limit the extent of HIV-1 infection.
Nakata
K
K
Division of Pulmonary and Critical Care Medicine, New York University Medical Center, New York, USA.
Weiden
M
M
Harkin
T
T
Ho
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Rom
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D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D001773:Blood Cells / Q000821:virology; D004279:DNA, Viral / Q000235:genetics; D015752:Genes, env; D014644:Genetic Variation; D006679:HIV Seropositivity / Q000821:virology*; D015497:HIV-1 / Q000145:classification / Q000235:genetics*; D006801:Humans; D016676:Macrophages, Alveolar / Q000821:virology*; D008875:Middle Aged; D008969:Molecular Sequence Data; D009000:Monocytes / Q000821:virology*; D009692:Nucleic Acid Heteroduplexes; D010802:Phylogeny; D011533:Proviruses / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes