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Journal of Virology, February 2000, p. 1712-1717, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Diminished Human Immunodeficiency Virus Type 1 Reverse Transcription and Nuclear Transport in Primary Macrophages Arrested in Early G1 Phase of the Cell Cycle

Neeltje A. Kootstra, Bianca M. Zwart, and Hanneke Schuitemaker*

Department of Clinical Viral-Immunology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, and Laboratory for Experimental and Clinical Immunology, Academic Medical Center, University of Amsterdam, 1066 CX Amsterdam, The Netherlands

Received 5 April 1999/Accepted 10 November 1999

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 G1 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 G1 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 G2 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 G1 phase of the cell cycle may contribute to efficient reverse transcription and nuclear localization.

* Corresponding author. Mailing address: Dept. of Clinical Viral-Immunology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands. Phone: 31-20-5123317. Fax: 31-20-5123310. E-mail: J_Schuitemaker{at}CLB.NL.

Journal of Virology, February 2000, p. 1712-1717, Vol. 74, No. 4
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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