Cellular compartments of human immunodeficiency virus type 1 replication in vivo: determination by presence of virion-associated host proteins and impact of opportunistic infection

Abstract

Antigens derived from host cells are detectable in the envelope of human immunodeficiency virus type 1 (HIV-1) and result in a distinctive viral phenotype reflecting that of the host cell. An immunomagnetic capture assay targeting discriminatory host proteins was developed to differentiate between HIV-1 derived from macrophages and lymphocytes. HIV-1 propagated in macrophages or lymphocytes in vitro was selectively captured by monoclonal antibodies directed against the virally incorporated cell-type-specific host markers CD36 (macrophages) and CD26 (lymphocytes). Furthermore, by targeting these markers, virus of defined cellular origin was selectively captured from a mixed pool of in vitro-propagated viruses. This technique was further refined in order to determine the impact of opportunistic infection on HIV-1 expression from these cellular compartments in vivo. Analysis of cell-free virus purified from plasma of patients with HIV-1 infection suggested that in those with an opportunistic infection, viral replication occurred in activated lymphocytes. Interestingly, there was also significant replication in activated macrophages in those patients with untreated pulmonary tuberculosis. Thus, in addition to lymphocytes, the macrophage cellular pool may serve as an important source of cell-free HIV-1 in patients with opportunistic infections that lead to marked macrophage activation. This novel viral capture technique may allow researchers to address a wide range of important questions regarding virus-host dynamics.

FIG. 1

HIV-1 capture by antibodies targeted against highly expressed cell surface antigens selective for macrophages or CD4⁺ T lymphocytes. Antibodies against the macrophage-specific marker, CD36, but not the highly expressed complement receptors, CD32 and CD64, selectively captured HIV-1_Ba-L-MΦ (■). T-cell-derived HIV-1_f/s.8 () was captured selectively by use of anti-CD3, anti-CD25, and anti-CD26. Antibodies to antigens common to both T lymphocytes and macrophages (HLA-A/B/C, HLA-DR, and CD44) captured both viral stocks. Data are representative of three independent experiments with <20% variability in the magnitude of capture.

FIG. 2

(A) Comparison of viral capture of macrophage- and lymphocyte-derived HIV-1. The HIV-1_Ba-L-MΦ isolate was selectively captured by anti-CD36 (■). When HIV-1_Ba-L-MΦ was propagated in T lymphocytes, the virus obtained (HIV-1_Ba-L-CD4) was selectively captured by anti-CD26 () and not by anti-CD36, indicating a discriminating phenotype for identifying the cellular origin of viral replication. (B) HIV-1_Ba-L-MΦ and HIV-1_Ba-L-CD4 isolates were mixed at various ratios and then captured with both anti-CD36 (■) and anti-CD26 (). The amount of virus captured by each antibody was proportional to the input of each type of virus, further illustrating the selective capture of virus derived from diverse cell types. Data are representative of three independent experiments.

FIG. 3

To investigate the potential inhibitory role of normal serum proteins, HIV antibodies, and acute-phase proteins in HIV-1 capture, HIV-1_Ba-L-MΦ was first incubated in PBS, NHS, HIV antibody-positive serum (HIV+), and serum from a patient with acute hepatitis A infection (HEP.A). Virus was subsequently captured by using anti-CD36 antibody, and the experiment showed that both HIV+ and HEP.A sera were markedly inhibitory to virus capture. However, use of the virus purification algorithm substantially overcame the inhibitory effects of the HIV+ and HEP.A sera on capture. Data are representative of two independent experiments with <15% variability in the magnitude of capture.

FIG. 4

(A) Capture of HIV-1 purified from plasma samples of HIV-infected patients by using antibodies to lymphocyte- and macrophage-specific markers. Three patients had untreated smear-positive pulmonary TB (TB/HIV.1 to TB/HIV.3), another had a microbiologically undefined opportunistic infection (OI/HIV.4), and four other patients (HIV.1 to HIV.4) had no opportunistic infection. A signal-to-background ratio of ≥0.5 log₁₀ was taken as significant, and the data are representative of three independent experiments. HIV-1 from all samples was captured by anti-CD44 antibody, serving as a positive control. Virus from all four patients with an opportunistic infection was captured by antibody to the lymphocyte-specific marker (CD26), but only virus from those with TB was captured by antibodies to macrophage-specific markers (CD36 and CD14). Antibodies to CD26, CD14, and CD36 did not capture HIV-1 from patients with no opportunistic infection. (B) Plasma levels of acute-phase and immune activation markers expressed as a percentage of the maximum level of each seen in any patient. The maximum levels of TNF-α, TNF-R1, C-reactive protein, sCD14, and IL-6 were 22 pg/ml, 6,010 pg/ml, 220 μg/ml, 17.2 μg/ml, and 45 pg/ml, respectively.