Diminished human immunodeficiency virus type 1 reverse transcription and nuclear transport in primary macrophages arrested in early G(1) phase of the cell cycle

Abstract

Previously, we and others have demonstrated that the process of reverse transcription of human immunodeficiency virus type 1 (HIV-1) is disturbed in nondividing macrophages and quiescent T lymphocytes. Here we analyzed which phase of the cell cycle in macrophages is crucial for early steps in the HIV-1 replication cycle. HIV-1 Ba-L-inoculated macrophages arrested early in the G(1) phase by n-butyrate contained incomplete products of reverse transcription. In gamma-irradiated macrophages, reverse transcription was successfully completed but proviral integration could not be detected. In these cells, nuclear import was disturbed as reflected by the absence of two-long-terminal-repeat circles. In macrophages arrested late in G(1) phase by aphidicolin or 5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), reverse transcription was unaffected. Proviral integration occurred efficiently in DRB-treated macrophages, whereas integrated proviral DNA could not be detected after aphidicolin treatment. Arrest at G(2) phase of the cell cycle by nocodazole did not affect reverse transcription or proviral integration. Treatment of macrophages with hydroxyurea (HU), which reduces the intracellular deoxynucleoside triphosphate (dNTP) pool by blocking the de novo synthesis of dNTP, resulted in a dose-dependent inhibition of HIV-1 reverse transcription. This could partially be restored by the addition of nucleoside precursors. Addition of nucleoside precursors enhanced both reverse transcription and cell proliferation. However, the disturbed reverse transcription observed in the nonproliferating and n-butyrate-treated macrophages could not be restored by addition of nucleoside precursors. Similar to observations in quiescent T lymphocytes, incomplete proviral DNA species were arrested in the cytoplasm of the macrophages. Our results indicate that also in primary macrophages the intracellular nucleotide pools and other cellular factors that coincide with late G(1) phase of the cell cycle may contribute to efficient reverse transcription and nuclear localization.

FIG. 1

Analysis of reverse transcription and proviral integration in growth-arrested MDM. MDM were treated with the following cell cycle inhibitors starting 24 h before inoculation: n-butyrate (1 to 10 mM), aphidicolin (0.5 to 5 μg/ml), DRB (0.5 to 2.5 μg/ml), and nocodazole (0.5 to 5 μg/ml). Alternatively, cells were subjected to gamma irradiation (3,000 rads) performed either directly after isolation (day 0) or 24 h before inoculation (day 5). MDM were inoculated at day 6 after isolation with 500 TCID₅₀s of DNase-treated HIV-1 Ba-L. As a control for the efficacy of the DNase treatment, zidovudine (2.5 to 25 μM)-treated MDM were inoculated with Ba-L and analyzed for the presence of proviral DNA. DNA was extracted 48 h after inoculation and was subjected to PCR analysis. To specifically detect integrated proviral DNA, a nested HIV-1 Alu PCR with primers specific for ubiquitous repeats found in the human genome and HIV-1 was used. As a control for the general efficiency of PCR amplification of the DNAs, all DNAs were subjected to PCR analysis amplifying part of the human β-globin gene. The results are representative of four independent experiments. Serial dilutions of in vitro-, HIV-1-infected PHA-stimulated PBMC in carrier DNA were used as DNA standards.

FIG. 2

Analysis of reverse transcription and proviral integration in HU-treated arrested MDM in the presence or absence of dN. MDM were treated with HU (0.05 to 1 mM) in combination with dN (10 to 100 μM) starting 24 h before inoculation. For further details on the PCR analysis, see the legend to Fig. 1. An additional PCR was performed to detect the R/PBS region. The results are representative of four independent experiments.

FIG. 3

Analysis of reverse transcription in nonproliferating MDM and n-butyrate-treated MDM in the presence of dN. (A) MDM proliferation was analyzed by BrdU incorporation in the presence or absence of dN. (B) MDM were sorted into BrdU-negative (gate R3) and BrdU-positive (gate R2) populations, and these cell fractions were analyzed for the presence of HIV-1 proviral DNA. (C) MDM were treated with n-butyrate (1 and 2.5 mM) in the presence or absence of dN (10 μM) starting 24 h before inoculation. Proviral DNA synthesis was analyzed by R/U5 and pol PCR. Serial dilutions of in vitro-, HIV-1-infected PHA-stimulated PBMC in carrier DNA were used as DNA standards.

FIG. 4

Analysis of proviral DNA in nuclear and cytoplasmic fractions of MDM. Cell fractions were isolated from MDM 48 h after inoculation. DNA extracted from these fractions was analyzed for the presence of HIV-1 proviral DNA. Serial dilutions of in vitro-, HIV-1-infected PHA-stimulated PBMC in carrier DNA were used as DNA standards.