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. 2006 Apr;80(8):4061-7.
doi: 10.1128/JVI.80.8.4061-4067.2006.

Trim-cyclophilin A fusion proteins can restrict human immunodeficiency virus type 1 infection at two distinct phases in the viral life cycle

Melvyn W Yap  1 , Mark P Dodding, Jonathan P Stoye
Affiliations

Trim-cyclophilin A fusion proteins can restrict human immunodeficiency virus type 1 infection at two distinct phases in the viral life cycle

Melvyn W Yap et al. J Virol. 2006 Apr.

Abstract

The Trim5alpha protein from several primates restricts retroviruses in a capsid (CA)-dependent manner. In owl monkeys, the B30.2 domain of Trim5 has been replaced by cyclophilin A (CypA) following a retrotransposition. Restriction of human immunodeficiency virus type 1 (HIV-1) by the resulting Trim5-CypA fusion protein depends on CA binding to CypA, suggesting both that the B30.2 domain might be involved in CA binding and that the tripartite RING motif, B-BOX, and coiled coil (RBCC) motif domain can function independently of the B30.2 domain in restriction. To investigate the potential of RBCCs from other Trims to participate in restricting HIV-1, CypA was fused to the RBCC of Trim1, Trim18, and Trim19 and tested for restriction. Despite low identity within the RBCC domain, all fusion proteins were found to restrict HIV-1 but not the nonbinding G89V mutant, indicating that the overall structure of RBCC and not its primary sequence was important for the restriction function. The critical interaction between CA and Trim-CypA appears to take place soon after viral entry. Quantitative PCR analysis of viral reverse transcriptase products revealed that the different fusion proteins block HIV-1 at two distinct stages of its life cycle, either prior to reverse transcription or just before integration. With Trim1 and Trim18, this timing is dependent on the length of the Trim component of the fusion protein. These observations suggest that restriction factor binding can have different mechanistic consequences.

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Figures

FIG. 1.
FIG. 1.
Restriction of HIV-1 by Trim-CypA fusion proteins. (A) A schematic representation of the fusion proteins is shown with the last amino acid position from the parental Trim indicated on top of each construct at the point of fusion. (B) Bar graph showing restriction. HT1080 cells were transduced with MLV-based vectors carrying the Trim-CypA gene as well as the YFP marker at an MOI of 1. Two days posttransduction, the cells were challenged with HIV-1 or the G89V CA mutant carrying the GFP marker. After 2 further days restriction was measured by FACS analyses. Restriction is measured as a ratio of the percentage of GFP-positive YFP-positive cells to that of GFP-positive YFP-negative cells. A ratio that was less than 0.3 was taken as positive restriction, while a ratio of 0.7 or more showed no restriction.
FIG. 2.
FIG. 2.
Abolition of restriction by CsA. HT1080 cells were transduced with MLV-based vectors carrying the Trim-CypA fusion gene and the YFP marker, and CsA (3 μg/ml) was added at different times following infection. Restriction was measured 2 days later by FACS analysis.
FIG. 3.
FIG. 3.
Trim-CypA fusion proteins block HIV-1 at different stages following entry. Cells were transduced with MLV-based vectors carrying the restricting gene. Two days posttransduction, the cells were challenged with HIV-1 that had been pretreated with DNase. Total DNA was isolated 7, 24, or 48 h following infection and early RT products, 2-LTR circles, and integrated proviruses were quantified, respectively. (A) Quantification of early HIV-1 RT products in TE671 cells. (B) Quantification of late HIV-1 2-LTR circles in TE671 cells. (C) Quantification of integrated proviruses in TE671 cells. (D) Western blot analysis of YFP expressed in Trim-CypA-containing cells. Total cellular protein was extracted from the cells, and 25 μg was separated on a 10% denaturing gel containing 2.5 M urea and blotted. Detection was performed using a monoclonal anti-GFP antibody.
FIG. 4.
FIG. 4.
Trim1 blocks N-MLV at reverse transcription. M. dunni cells were transduced with MLV-based vectors carrying the restricting gene. Two days after transduction, the cells were challenged with N-MLV that had been pretreated with DNase. Total DNA was isolated 7 h following infection and late RT products were quantified using a primer pair directed against the neomycin resistance gene carried by the challenge virus.
FIG. 5.
FIG. 5.
Expression of Trim-CypA HA-tagged fusion proteins in TE671 cells. Cells were transduced with MLV-based vectors carrying the restricting gene. Two days posttransduction, the cells were challenged with HIV-1 that had been pretreated with DNase. Total DNA was isolated 7 h following infection and early RT products were quantified. (A) Western blot analysis of HA-tagged Trim-CypA. Total cellular protein was extracted from the cells and 25 μg was separated on a 10% denaturing gel containing 2.5 M urea and blotted. Detection was performed using a monoclonal anti-HA antibody. (B) Quantification of early HIV-1 RT products in TE671 cells.
FIG. 6.
FIG. 6.
Model of action of restriction factors. Restriction factors bind early after entry but can lead to viral replication arrest at three different stages. Block I occurs prior to reverse transcription and is observed with Trim5α, T5C, T1CL, T1CM, and T18CL. Block II prevents the formation of 2-LTR circles as seen with Fv1 and most likely occurs just before nuclear entry, whereas block III interferes with integration and is observed with T1CS, T18CS, and T19C.
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