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. 2006 Oct;80(19):9754-60.
doi: 10.1128/JVI.01052-06.

Proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse TRIM5 proteins

Jenny L Anderson  1 , Edward M Campbell, Xiaolu Wu, Nick Vandegraaff, Alan Engelman, Thomas J Hope
Affiliations

Proteasome inhibition reveals that a functional preintegration complex intermediate can be generated during restriction by diverse TRIM5 proteins

Jenny L Anderson et al. J Virol. 2006 Oct.

Abstract

The primate TRIM5 proteins constitute a class of restriction factors that prevent host cell infection by retroviruses from different species. The TRIM5 proteins act early after virion entry and prevent viral reverse transcription products from accumulating. We recently found that proteasome inhibitors altered the rhesus monkey TRIM5alpha restriction of human immunodeficiency virus type 1 (HIV-1), allowing reverse transcription products to accumulate even though viral infection remained blocked. To assess whether sensitivity to proteasome inhibitors was a common feature of primate TRIM5 proteins, we conducted a similar analysis of restriction mediated by owl monkey TRIM-cyclophilin A (CypA) or human TRIM5alpha. Similar to rhesus monkey TRIM5alpha restriction, proteasome inhibition prevented owl monkey TRIM-CypA restriction of HIV-1 reverse transcription, even though HIV-1 infection and the output of 2-LTR circles remained impaired. Likewise, proteasome inhibition alleviated human TRIM5alpha restriction of N-tropic murine leukemia virus reverse transcription. Finally, HIV-1 reverse transcription products escaping rhesus TRIM5alpha restriction by proteasome inhibition were fully competent for integration in vitro, demonstrating that TRIM5alpha likely prevents the viral cDNA from accessing chromosomal target DNA. Collectively, these data indicate that the diverse TRIM5 proteins inhibit retroviral infection in multiple ways and that inhibition of reverse transcription products is not necessary for TRIM5-mediated restriction of retroviral infection.

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Figures

FIG. 1.
FIG. 1.
The MG132 proteasome inhibitor rescues HIV-1 reverse transcription from owl monkey TRIM-CypA and rhTRIM5α restriction. HeLa (white bars), stable rhTRIM5α HeLa (black bars), or OMK (gray bars) cells were infected with 84 ng p24Gag/well of VSV-g-pseudotyped R7-GFPHIV-1 for 14 h overnight in the absence of drug, with MG132, with CsA, or with both MG132 and CsA. Drug was removed and cells were analyzed for infection using flow cytometry for GFP 48 h later (A), or cellular DNA was harvested and analyzed for the numbers of late RT products (B) and 2-LTR circles (C) formed per cell (β-actin) using real-time PCR. (A) The percentages of GFP-positive cells from a representative experiment are shown. (B and C) Error bars indicate the standard deviations of triplicate values. Nev, nevirapine.
FIG. 2.
FIG. 2.
MG132 rescues N-MLV reverse transcription from human TRIM5α restriction. (A) BHK, HeLa, and stable huTRIM5α HeLa cells were infected for 14 h with 3.6 × 104 infectious units of VSV-g-pseudotyped B-tropic (gray bars) or N-tropic (black bars) MLV vectors expressing GFP. Cells were analyzed 48 h later for infection by flow cytometry for GFP; the percentages of GFP-positive cells from a representative experiment are shown. (B) HeLa and stable huTRIM5α HeLa cells were infected with B-tropic (gray bars) or N-tropic (black bars) MLV vectors in the presence or absence of MG132 as indicated, and cellular DNA was harvested and then analyzed for the number of GFP RT products per cell 14 h after infection using real-time PCR.
FIG. 3.
FIG. 3.
HIV-1 late RT products rescued from rhTRIM5α restriction by MG132 are integration competent. HeLa cells (white bars) and stable rhTRIM5α HeLa cells (black bars) were infected with VSV-g-pseudotyped R7-GFPHIV-1 in the presence or absence of MG132 for 7 h. (A) Cells were then incubated an additional 48 h and analyzed for infection via flow cytometry for GFP. (B) Cell DNA was analyzed for late RT products by real-time PCR. (C) Relative PIC activity was determined by assaying PIC-containing cytoplasmic extracts for in vitro integration.
FIG. 4.
FIG. 4.
Model for retroviral restriction by TRIM5 proteins. Upon entry into the cytoplasm, determinants on the viral core are recognized and bound by TRIM5 proteins (1). In the absence of proteasome inhibition, the viral core is destroyed via a proteasome-dependent mechanism (2). However, in the presence of proteasome inhibition (e.g., MG132), the core stabilizes and viral cDNA reverse transcribes, unveiling an integration-competent PIC intermediate whose nuclear localization remains impaired analogous to Fv1 restriction (3).
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