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. 2007 Sep 24:4:68.
doi: 10.1186/1742-4690-4-68.

Both TRIM5alpha and TRIMCyp have only weak antiviral activity in canine D17 cells

Julie Bérubé  1 , Amélie Bouchard, Lionel Berthoux
Affiliations

Both TRIM5alpha and TRIMCyp have only weak antiviral activity in canine D17 cells

Julie Bérubé et al. Retrovirology. .

Abstract

Background: TRIM5alpha, which is expressed in most primates and the related TRIMCyp, which has been found in one of the New World monkey species, are antiviral proteins of the TRIM5 family that are able to intercept incoming retroviruses early after their entry into cells. The mechanism of action has been partially elucidated for TRIM5alpha, which seems to promote premature decapsidation of the restricted retroviruses. In addition, through its N-terminal RING domain, TRIM5alpha may sensitize retroviruses to proteasome-mediated degradation. TRIM5alpha-mediated restriction requires a physical interaction with the capsid protein of targeted retroviruses. It is unclear whether other cellular proteins are involved in the inhibition mediated by TRIM5alpha and TRIMCyp. A previous report suggested that the inhibition of HIV-1 by the rhesus macaque orthologue of TRIM5alpha was inefficient in the D17a canine cell line, suggesting that the cellular environment was important for the restriction mechanism. Here we investigated further the behavior of TRIM5alpha and TRIMCyp in the D17 cells.

Results: We found that the various TRIM5alpha orthologues studied (human, rhesus macaque, African green monkey) as well as TRIMCyp had poor antiviral activity in the D17 cells, despite seemingly normal expression levels and subcellular distribution. Restriction of both HIV-1 and the distantly related N-tropic murine leukemia virus (N-MLV) was low in D17 cells. Both TRIM5alpharh and TRIMCyp promoted early HIV-1 decapsidation in murine cells, but weak levels of restriction in D17 cells correlated with the absence of accelerated decapsidation in these cells and also correlated with normal levels of cDNA synthesis. Fv1, a murine restriction factor structurally unrelated to TRIM5alpha, was fully functional in D17 cells, showing that the loss of activity was specific to TRIM5alpha/TRIMCyp.

Conclusion: We show that D17 cells provide a poor environment for the inhibition of retroviral replication by proteins of the TRIM5 family. Because both TRIM5alpha and TRIMCyp are poorly active in these cells, despite having quite different viral target recognition domains, we conclude that a step either upstream or downstream of target recognition is impaired. We speculate that an unknown factor required for TRIM5alpha and TRIMCyp activity is missing or inadequately expressed in D17 cells.

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Figures

Figure 1
Figure 1
Expression and subcellular distribution. A, FLAG-tagged TRIM5α (rhesus, human, or African green monkey orthologues) and TRIMCyp were stably expressed in MDTF and in D17 cells, and expression was assessed by western blotting with antibodies directed against FLAG (top) or actin (bottom). The percentage of transduced cells was roughly similar for all cell lines created, as judged by the percentage of puromycin-resistant cells (not shown). The presence of the FLAG tag in TRIM5αhu was confirmed by sequencing of the plasmid DNA. B, MDTF or D17 cells expressing TRIM5αrh or TRIMCyp were fixed and stained using an antibody against FLAG and counterstained with Hoechst33342 to reveal DNA.
Figure 2
Figure 2
Restriction of N-MLV. A, MDTF or D17 cells expressing TRIM5αhu, TRIM5αAGM, or control cells were infected with multiple dilutions of N-MLV and B-MLV vectors expressing GFP. The percentage of infected cells was determined by flow cytometry. B, MDTF or D17 cells expressing various orthologues of TRIM5α, or expressing TRIMCyp, were infected with GFP-expressing N-MLV or B-MLV vectors. The virus dose used in each cell type was first adjusted so that 5–10% of control cells would be infected and the same volume of virus preparation was then used to infect the various TRIM5-expressing cell lines from that cell type. The percentage of infected cells was determined 2 days later by flow cytometry, and results are shown as % of the values obtained for the control cells. The experiment was carried out in triplicates, and standard deviations are shown.
Figure 3
Figure 3
Restriction of HIV-1. A, MDTF or D17 cells expressing TRIM5αrh, TRIMCyp, or control cells were infected with multiple dilutions of HIV-1GFP, an HIV-1 vector expressing GFP. The percentage of infected cells was determined by flow cytometry. B, MDTF, HeLa or D17 cells expressing TRIM5αrh or TRIMCyp, and control cells were infected with HIV-1GFP. The virus dose used in each cell type was first adjusted so that 5–10% of control cells would be infected and the same volume of virus preparation was then used to infect the various TRIM5-expressing cell lines from that cell type. The percentage of infected cells was determined 2 days later by flow cytometry, and results are shown as % of the values obtained for the control cells. The experiment was carried out in triplicates, and standard deviations are shown.
Figure 4
Figure 4
Enhancement of HIV-1 infection in cells expressing TRIMCyp by cyclosporine. A, MDTF or D17 cells, expressing TRIMCyp or not (control cells), were infected with HIV-1GFP. The virus dose was adjusted so that 1% to 3% of cells would be infected in the absence of cyclosporine for each cell line, and the infections were done in the presence of various cyclosporine concentrations. The percentage of infected cells was determined 2 days later by flow cytometry. B, as above, except that CsA concentration was constant (6 μM) and cells were infected with multiple doses of HIV-1GFP.
Figure 5
Figure 5
Fate-of-capsid assay. MDTF or D17 cells expressing TRIM5αrh or TRIMCyp and control cells were infected with HIV-1 VLPs for 4 hours, then cells were allowed to grow for 2 more hours in a virus-free medium. Following the infection, cells were submitted to hypotonic lysis and the protein suspension was sedimented through a 50% sucrose gradient. HIV-1 CA was detected by western blotting of whole lysates, post-sedimentation pellets and supernatants (materials that did not enter the sucrose cushion). The mature CA (24 kDa) band was quantitated for the blot showing the pellet fractions and quantitation data are shown expressed as relative values.
Figure 6
Figure 6
Fate-of-capsid assay. MDTF and D17 cells expressing TRIMCyp, and control cells were infected with HIV-1 VLPs as in Fig. 5. The cells expressing TRIMCyp were infected in the presence or not of cyclosporine (5 μM). CA was detected in post-sedimentation pellets and supernatants. Pellet CA was quantitated as in Fig. 5.
Figure 7
Figure 7
Retroviral cDNA synthesis. MDTF or D17 cells expressing the indicated TRIM5 orthologues were infected for 12 hours with HIV-1GFP at a MOI yielding about 20% infected cells for the control cells. In addition, infection of cells expressing TRIMCyp was carried out in the presence or absence of 5 μM cyclosporine, and infection of control cells was done in the presence or absence of the reverse transcriptase inhibitor nevirapine (80 μM). Top panel, total cellular DNAs were extracted and an aliquot of each DNA sample was subjected to a 30-cycle PCR amplification using ODNs annealing to GFP sequences. PCR products were separated on an agarose gel and revealed with ethidium bromide. Bottom panel, as above but HIV-1GFP-specific DNAs were quantitated by real-time PCR, using dilutions of a plasmid containing the GFP sequence as a standard.
Figure 8
Figure 8
Restriction by Fv1. MDTF or D17 cells, expressing Fv1b or not (control cells), were infected with multiple dilutions of N-MLV and B-MLV vectors expressing GFP. The percentage of infected cells was determined by flow cytometry 2 days later.
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