Characterization of the early steps of infection of primary blood monocytes by human immunodeficiency virus type 1

Abstract

Blood-circulating monocytes migrate in tissues in response to danger stimuli and differentiate there into two major actors of the immune system: macrophages and dendritic cells. Given their migratory behavior and their pivotal role in the orchestration of immune responses, it is not surprising that cells of the monocyte lineage are the target of several viruses, including human immunodeficiency virus type 1 (HIV-1). HIV-1 replicates in monocytoid cells to an extent that is influenced by their differentiation status and modulated by exogenous stimulations. Unstimulated monocytes display a relative resistance to HIV infection mostly exerted during the early steps of the viral life cycle. Despite intensive studies, the identity of the affected step remains controversial, although it is generally assumed to take place after viral entry. We reexamine here the early steps of viral infection of unstimulated monocytes using vesicular stomatitis virus G protein-pseudotyped HIV-1 virions. Our data indicate that a first block to the early steps of infection of monocytes with these particles occurs at the level of viral entry. After entry, reverse transcription and integration proceed with extremely slow kinetics rather than being blocked. Once completed, viral DNA molecules delay entry into the nucleus and integration for up to 5 to 6 days. The inefficacy of these steps accounts for the resistance of monocytes to HIV-1 during the early steps of infection.

FIG. 1.

Infection of monocytes with HIV-1. (A) Purified primary blood monocytes and GM-CSF-differentiated macrophages derived from the same donor were infected with VSVg-pseudotyped HIV-1 vectors coding gfp. Vectors were produced by transient transfection of 293T cells and purified over a 25% sucrose cushion by ultracentrifugation. Their infectious titer was measured on target HeLaP4 or 293T cells. (B) Cells were infected at an MOI of 10 for 2 h prior to virus removal by cell washing and cell seeding. The percentage of infected GFP-positive cells was measured by flow cytometry every day over a period of 10 days postinfection. The graph presents values obtained with three different donors.

FIG. 2.

HIV-1 infects bona fide monocytes. To determine the identity of the small fraction of GFP⁺ cells, monocytes were infected with HIV-1 vectors at an MOI of 10 as described above and seeded in the absence or presence of GM-CSF or M-CSF. The expression of CD14, a specific marker of monocytes/macrophages, was examined by flow cytometry at 4 and 10 days postinfection. We and others have already shown that GM-CSF and M-CSF either decrease or increase the amount of CD14 at the cell surface (19). The results from a representative experiment out of four are shown here.

FIG. 3.

Monocytes display diminished intracellular CA signal after infection with VSVg-pseudotyped HIV. Monocytes were infected with increasing viral inputs for 2 h, along with HeLaP4 cells and GM-CSF-differentiated macrophages. Loosely bound and noninternalized virion particles were removed by trypsin treatment. Cells were then permeabilized and examined for their CA content by flow cytometry using a specific anti-CA antibody. The figure presents data obtained with three to four different donors showing the percentage of CA⁺ cells (A) and the MFIs of positive cells at an MOI of 20, normalized to the value obtained in HeLaP4 cells (B). ***, P = 0,009 (Student t test).

FIG. 4.

Monocytes display an entry defect after a Vpr-BLAM entry assay. Vpr-Blam was incorporated into virion particles by transfection of 293T cells during vector production. Cells were infected with an equal number of virion particles for 2 h at an MOI of 3. After a washing step, cells were loaded with the CCF2 dye, and the Vpr-BLAM activity was revealed after 18 h as a fluorescence shift in CCF2-positive cells by flow cytometry. The graph presents the percentages of Vpr-BLAM-positive cells obtained for the different cell types examined (n = 3).

FIG. 5.

Intracellular CA staining after confocal microscopy. Monocytes (A) and macrophages (B) were infected at an MOI of 10 for 2 h, washed, and analyzed by confocal microscopy. Cells were first labeled with a specific anti-CA antibody and then with the DNA dye TOTO-3 to mark their nuclei. The results of representative experiments are shown here.

FIG. 6.

The kinetics of reverse transcription and integration are extremely slow in monocytes. Monocytes were infected for 2 h with HIV-1 vectors at an MOI of 10 prior to virus removal. Reverse transcription and integration were arrested at the indicated time points by the addition of nevirapine and of the dichetoacid IN inhibitor L-731,988 (at 10 and 20 μg/ml, respectively). The percentages of GFP⁺ cells obtained for each time point were determined at 10 days postinfection. The graph presents data obtained from three different donors.

FIG. 7.

The kinetics of total viral DNA production were assessed by PCR. Monocytes were infected as described in the text, and cells were lysed at the indicated time points. Semiquantitative PCR analysis was conducted on serial fivefold dilutions of samples with primers that amplify specifically FL and 2LTR forms (i.e., FL and 2LTRs, respectively). PCR products were transferred onto a nylon membrane, hybridized with a specific ³²P-labeled probe, and quantified by phosphorimager analysis. The graph presents these results after normalization of the different samples for actin DNA.

FIG. 8.

Behavior of R5-tropic Env HIV-1 virion particles. Virion particles were pseudotyped with the JR-FL Env upon cotransfection of 293T cells, titers were determined on target P4P5 HeLa cells (expressing CD4 and CCR5), and the particles were evaluated on monocytes and macrophages in an intracellular p24 staining assay (A) or to determine the percentage of infected cells over time (B). For intracellular staining, cells were infected with increasing amounts of viral particles for 2 h prior to cell washing, permeabilization, and p24 staining. Cells were then assayed by flow cytometry as described in the legend to Fig. 3. To determine viral infectivity, cells were infected at an MOI of 3 for 2 h, plated, and then analyzed at 3 and 7 days postinfection. The graphs present results obtained from three independent experiments.