The human and African green monkey TRIM5alpha genes encode Ref1 and Lv1 retroviral restriction factor activities

Abstract

The rhesus macaque tripartite motif containing protein TRIM5alpha specifically restricts HIV-1 infection at an early post-entry step before reverse transcription [Stremlau, M., Owens, C. M., Perron, M. J., Kiessling, M., Autissier, P. & Sodroski, J. (2004) Nature 427, 848-853]. Here, we show that the human and African green monkey (AGM) TRIM5alpha genes encode Ref1 and Lv1 antiretroviral activities, respectively. Expression of TRIM5alpha in permissive cat cells renders them resistant to restriction-sensitive murine leukemia virus but not closely related insensitive virus. Disruption of TRIM5alpha expression in human and AGM cells with small interfering RNA rescues infectivity of restricted virus without affecting unrestricted virus. We also demonstrate that the activity of the murine restriction factor Fv1 depends on TRIM5alpha expression when Fv1 is expressed in human cells. Furthermore, a drug that modifies the behavior of the related promyelocytic leukemia protein PML specifically rescues infection by viruses restricted by human TRIM5alpha. Alignment of the TRIM5alpha proteins from rhesus macaque and AGM indicates an 18-aa insertion. We speculate that this insertion may contribute to the broader specificity of the AGM TRIM5alpha restriction as compared with the human and rhesus macaque proteins.

Fig. 1.

Expression of human or AGM TRIM5α in cat cells enables them to restrict MLV-N but not MLV-B. Cat CRFK cells were transduced by a retroviral vector encoding TRIM5α from human (b and e) or AGM (c and f), and single-cell clones were isolated. Untransduced CRFK cells and TRIM5α-positive clones were infected with equivalent doses of MLV-N (a–c) or MLV-B (d–f) encoding GFP. The multiplicity of infection was 0.1. Forty-eight hours later, the cells were analyzed for green fluorescence by FACS. Side scatter is shown on the y axis, and GFP or MLV infectivity is shown on the x axis. Percentages given indicate the proportion of GFP-positive cells (MLV infected). Data shown are representative of experiments performed on 12 independent TRIM5α-positive CRFK clones. eGFP, enhanced GPP.

Fig. 5.

Arsenic trioxide specifically rescues viral infectivity from restriction by human but not AGM TRIM5α. Human TE671 cells were infected with titrations of restricted EIAV (a), restricted MLV-N (b), and unrestricted MLV-NB (c). Infections were performed in the presence • or absence ▴ of 4 μM arsenic trioxide (AT). Viral doses are measured in nanograms of reverse transcriptase (EIAV) or SC1 infectious units (MLV). Untransduced (d and e), human TRIM5α-expressing (f and g), and AGM TRIM5α-expressing (h and i) murine MDTF cells were infected with MLV-N GFP at a multiplicity of infection of 0.4 in the presence and absence of 8 μM AT. Forty-eight hours later, infected cells were enumerated by FACS. In d–i, percentages given indicate the proportion of GFP-positive cells (MLV-infected). Data shown are representative of three independent experiments.

Fig. 2.

Disruption of TRIM5α expression by siRNA in human or AGM cells increases permissivity for restricted virus but not unrestricted virus. Human HeLa cells (a) or AGM COS7 cells (b) were transfected with siRNA to TRIM5α (filled bars) or left untransfected as control (open bars) and infectious titer of restricted and nonrestricted viruses were determined by infection followed by assay of GFP by FACS 48 h later. Infections were performed such that between 0.5% and 10% target cells were infected to ensure linearity. Errors are SEM. Results are representative of two independent experiments.

Fig. 3.

Disruption of TRIM5α expression by siRNA in human cells increases MLV-N but not MLV-B viral DNA synthesis. Human HeLa cells were transfected with siRNA to TRIM5α (filled bars) or left untransfected as control (open bars) and infected with equivalent doses of MLV-N and MLV-B encoding GFP. Six hours after infection, total DNA was purified and subjected to TaqMan quantitative PCR as described in Materials and Methods. DNA copy number per 100 ng of total DNA is plotted. Errors are SEM. Results are representative of two independent experiments.

Fig. 4.

Disruption of TRIM5α expression by siRNA in human cells expressing Fv1 N rescues infectivity of both TRIM5α and Fv1 restricted infection but not unrestricted infection. Human TEN cells expressing Fv1 N were transfected with siRNA to TRIM5α (filled bars) or left untransfected as control (open bars), and infectious titers of restricted and nonrestricted virus were determined by infection followed by assay of GFP by FACS 48 h later. Infections were performed such that between 0.5% and 10% target cells were infected to ensure linearity. Errors are SEM. Results are representative of two independent experiments.

Fig. 6.

Comparison of protein sequence of TRIM5α from rhesus macaque (Macacca mulatta) and AGM (Cercopithicus sp.) deduced from the cDNA sequence.