TABLE 1.
Half-lives, retrovirus-restricting activities, and cytoplasmic staining of TRIM variants
| TRIM variant | Half-life (min)a
|
Retrovirus- restricting abilityb | Cytoplasmic stainingc | |
|---|---|---|---|---|
| CHX | CHX + MG115 | |||
| Wild-type TRIM5αrh | 48 | 59 | ++++ | CB |
| TRIM5αrh K113R/R121K | 23 | 29 | +++ | CB |
| TRIM5αrh R121E | 1,541 | >10,000 | − | D |
| TRIM5αrh K113A/R121A | 764 | 1,464 | + | CB |
| TRIM5αrh R121A | 675 | >10,000 | +++ | CB |
| TRIM5αrh R121D | 173 | 714 | − | D |
| TRIM5αrh C97A/H100A | 336 | 470 | − | CB |
| TRIM5αrh Δ(1-93) | 150 | 1,783 | ++ | D |
| TRIM5αrh Δ(97-129) | 629 | >10,000 | − | D |
| TRIM5αrh Δ(1-132) | 203 | 3,072 | − | D |
| TRIM6-5 (CC-B30.2) | 4,933 | >10,000 | ++++ | ND |
| TRIM34-5 (CC-B30.2) | >10,000 | >10,000 | ++++ | ND |
| Wild-type TRIM5αhu | 40 | 42 | ++++ | CB |
| TRIM5αhu R119K | 29 | 31 | ++++ | CB |
| TRIM5αhu R119A | 231 | 439 | + | CB |
| TRIM5αhu R119D | 361 | 602 | − | D |
| TRIM5αhu R119E | 749 | 1,518 | − | D |
| TRIM5αhu C95A | 253 | 768 | − | CB |
| Wild-type TRIMCyp | 56 | 63 | ++++ | CB |
| TRIMCyp R120K | 58 | 61 | ++++ | CB |
| TRIMCyp R120D | 381 | 577 | ++++ | D |
| TRIMCyp R120E | 804 | 1,350 | ++++ | D |
| TRIMCyp R120A | 320 | 680 | ++++ | CB |
The half-life of the indicated TRIM protein stably expressed in Cf2Th cells was determined in the presence of cycloheximide (CHX) or cycloheximide and MG115 (CHX + MG115), as described in Materials and Methods. The half-lives were estimated from semilog plots of TRIM level versus time, in some cases extrapolating to make the estimate. The values shown are typical of those obtained in at least two experiments. The half-lives of the TRIM6-5(CC-B30.2) and TRIM34-5(L2-B30.2) proteins were determined in HeLa cells stably expressing these proteins; the half-life of the wild-type TRIM5αrh protein in these cells was 52 min in the presence of CHX and 115 min in the presence of CHX + MG115.
The retrovirus-restricting ability of each variant was estimated, in comparison to the value (++++) observed for the corresponding wild-type protein, using the data shown in Fig. 2 to 6 and 12. The minus sign indicates no detectable restricting ability for the appropriate susceptible virus, ++++ indicates activity comparable to that of the wild-type protein, and + to +++ indicate intermediate levels of potency.
The intracellular localization of the TRIM variant was determined. The intracellular localization of the TRIM5αrh C97A/H100A, Δ(1-93) and Δ(1-132) variants was described in reference 11. The TRIM5αrhΔ(1-93) protein is also found in the nuclei of expressing cells, where it is associated with nuclear bodies. CB, cytoplasmic bodies; D, diffuse cytoplasmic staining; ND, not determined.