12th Conference on Retroviruses and Opportunistic Infections Boston, Massachusetts - February 22-25, 2005

Conf Retrovir Opportunistic Infect 2005 Feb 22-25;12:abstract no. 30

Ya-Lin Chiu, V Soros, K Stopak, J Kreisberg, W Yonemoto, J Neidleman, and W Greene
Gladstone Inst of Virology and Immunology, Univ of California, San Francisco, USA

BACKGROUND: APOBEC3G (A3G) is a host cytidine deaminase that displays potent antiretroviral activity. HIV Vif overcomes the antiviral effects of A3G by targeting this enzyme for accelerated proteasome-mediated degradation and decreased synthesis. In the absence of Vif, A3G is incorporated into virions where it mediates massive dC deamination of the HIV viral minus strand DNA generated by reverse transcription (RT). Despite rapid progress, many questions remain unanswered including how A3G is regulated in cells and why cytoplasmic forms of A3G are not active against incoming virions? To pursue these questions, we investigated whether A3G assembles with other host factors and whether the putative complexes differ in their antiviral activity.

METHODS: Potential A3G complexes in primary cells and laboratory-adapted cell lines were analyzed by fast performance liquid chromatography. Enzymatic activity of the A3G complexes was assessed using an in vitro cytidine deaminase assay. A3G expression in resting CD4 T-cells was “knocked down” using specific siRNA introduced by AMAXA nucleofection and HIV infection of these cells was studied using VSV-G pseudotyped NL4-3 HSA (heat stable antigen) reporter viruses.

RESULTS: Native A3G present in H9 T-cell lines, activated CD4 T-cells and monocyte-derived macrophages principally resides in a high molecular weight (HMW) ribonucleoprotein complex (> 700 kDa) that lacks cytidine deaminase activity. RNase treatment of this HMW complex promotes its disassembly yielding a low molecular weight form of A3G (46 to 100 kDa) that exhibits potent enzymatic activity. In contrast to the other cells studied, the low molecular weight form of A3G predominates in resting CD4 T cells and monocytes. Each of these cells fails to support HIV replication due in part to an early RT block. Activation of resting CD4 T cells or differentiation of monocytes into macrophages induces a shift of A3G into the inactive HMW complex. This change correlates with the full permissivity of these cells to HIV infection. Strikingly, when A3G expression is “knocked down” in resting CD4 T cells by the introduction of A3G-specific siRNA, the early block to HIV replication is relieved.

CONCLUSIONS: These findings reveal that low molecular weight form A3G functions as an effective post-entry restriction factor for HIV-1 in resting CD4 T cells. Accordingly, agents promoting high molecular weight form A3G disassembly might effectively block the growth of wild type HIV entering highly permissive cells.

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Copyright © 2005 - Foundation for Retrovirology and Human Health. Reproduction of this abstract (other than one copy for personal reference) must be cleared through the Foundation for Retrovirology and Human Health. Licensed (AIDSLINE) from National Library of Medicine.