Table 1.
Phenotypes of TRIM5α RING Y63 variants
| TRIM5αrh variant | Restriction potency against HIV-1a | HIV-1 reverse transcriptionb | HIV-1 2-LTR circle formationc | % TRIM5 self-ubiquitylation (± SD)d | Particulate HIV-1 cytoplasmic capsidse | Mean binding to HIV-1 CA-NC complexes (± SD)f | % HOSA (± SD)g |
|---|---|---|---|---|---|---|---|
| WT | + | − | − | 100.0 | − | 1 | 100.0 |
| Y63A | + | − | − | 50.0 (8.3)*** | − | 0.9 (0.2) NS | 18.6 (4.3) |
| Y63F | + | − | − | 80.9 (11.5)* | ND | 0.9 (0.1) NS | 79.3 (7.3) |
| Y63D | + | + | + | 31.0 (4.5)*** | + | 1.2 (0.1) NS | 8.7 (5.7) |
| Y63E | + | + | + | 40.6 (5.6)*** | + | 0.8 (0) NS | 19.4 (7.2) |
| Y63K | + | + | + | 30.6 (10.1)*** | + | 0.4 (0.2)*** | 18.6 (5.4) |
| Y63S | + | − | − | 50.6 (9.7)*** | − | 1.0 (0.1) NS | 15.7 (4.3) |
Restriction was measured by infecting cells expressing the indicated TRIM5αrh variants with HIV-1 expressing the GFP protein. After 48 h, the percentage of GFP-positive cells (infected cells) was determined by flow cytometry. +, Restriction comparable to that of the wild-type protein. Experiments were performed at least three times.
HIV-1 reverse transcription was measured by real-time PCR 7 h after infection, as described in Materials and Methods. −, Absence of reverse transcription. +, Occurrence of reverse transcription at levels similar to those observed in control cells transduced with the empty LPCX vector when infected with HIV-1 (or N-MLV, as shown in Table 2). Experiments were performed at least three times.
Formation of HIV-1 2-LTR circles was measured by real-time PCR 24 h after infection, as described in Materials and Methods. The presence (+) or absence (−) of HIV-1 2-LTR circles is indicated. Experiments were performed at least three times.
To investigate the E3 ubiquitin ligase activity of TRIM5αrh RING domain variants, the ability of these variants to undergo self-ubiquitylation was assayed. Semipurified Flag-tagged TRIM5α variants from transfected 293T cells by immunoprecipitation were incubated with E1, E2, myc-tagged ubiquitin, and ATP, for 1 h at 37°C as described in Materials and Methods. Samples were analyzed by Western blotting using antibodies against FLAG and myc for the detection of TRIM5α and ubiquitin, respectively. The amount of TRIM5α ubiquitylated protein was determined by subtracting the amount of nonubiquitylated TRIM5α protein remaining in the reaction mixture incubated with E1 and E2 from the TRIM5α protein in the control reaction mixture, which was not incubated with E1 and E2. The value of nonubiquitylated TRIM5α for the reactions was quantified by using fluorescent Western blotting. TRIM5α self-ubiquitylation was expressed as the percentage of total TRIM5α variant input protein. Experiments were performed at least three times and standard deviations (SD) are shown. Statistical differences when compared with wild-type TRIM5α are given as follows: ***P < 0.001; *P < 0.05 (two-way analysis of variance [ANOVA] followed by the Bonferroni posttest).
The amount of particulate capsids for HIV-1 during infection of cells expressing the indicated TRIM5α RING domain variants was measured as described in Materials and Methods. −, Absence of particulate capsids during infection, similar to what is observed in the presence of the wild-type TRIM5α. +, Presence of particulate capsids in an amount similar to that observed in HIV-1 (or N-MLV, as shown in Table 2) infection of control cells that were transduced with the empty vector LPCX. Experiments were performed at least twice. ND, not determined.
Binding to the HIV-1 capsid complexes was determined for each TRIM5αrh RING domain variant as described in Materials and Methods. Binding is expressed as the amount of the TRIM5αrh variant bound to HIV-1 capsid complexes divided by the amount of bound wild-type TRIM5αrh at a similar input level. Experiments were repeated at least three times; the means and standard deviations (SD) are shown. Note that, because the binding ratios are calculated at input levels at which some binding of the mutant TRIM5αrh protein to the HIV-1 capsid complexes can be detected, these ratios overestimate the relative CA-binding affinities of the mutant proteins. Statistical differences when compared with wild-type TRIM5α binding are given as follows: ***P < 0.001; NS, not significant with P > 0.05; two-way ANOVA followed by the Bonferroni posttest.
Each TRIM5αrh RING domain variant was assayed for HOSA as described in Materials and Methods. The percentage represents the fraction of the TRIM5αrh variant coprecipitated with itself, relative to the coprecipitation of wild-type TRIM5αrh with itself. Experiments were performed at least three times.