TABLE 4.
Number of arginine insertions required for functional restoration of Rev–MS-C protein deleted for the RNA binding and NLS domain of Rev
| Arginine substitutions in Rev–MS-C | RNA bindinga
|
Subcellular locationb |
trans-Activationc (SD)
|
||
|---|---|---|---|---|---|
| RRE | MS2 | RRE | RREZ-MS2 | ||
| Δ35/50Rev-i-1R/MS-C | ND | ND | Cyt>>>Nuc | 0.8 (0.5) | 4.8 (1.8) |
| Δ35/50Rev-i-3R/MS-C | ND | ND | Cyt>>>Nuc | 1.8 (0.8) | 10.8 (3.2) |
| Δ35/50Rev-i-4R/MS-C | ND | ND | Cyt>>>Nuc | 3.1 (1.2) | 18.2 (4.8) |
| Δ35/50Rev-i-5R/MS-C | ++ | +++++ | Cyt>>>Nuc | 0.4 (0.3) | 27 (3.8) |
| Δ35/50Rev-i-7R/MS-C | ND | ND | Cyt>>Nuc | 0.8 (0.4) | 45 (8.9) |
| Δ35/50Rev-i-8R/MS-C | ++++ | +++++ | Cyt≈Nuc | 17 (4.5) | 68 (9.9) |
| Δ35/50Rev-i-9R/MS-C | +++++ | +++++ | Nuc>>>Cyt | 97 (14.5) | 122 (16.7) |
| Δ35/50Rev-i-10R/MS-C | ND | ND | Nuc>>>Cyt | 133 (17.5) | 111 (18.3) |
| Δ35/50Rev-i-11R/MS-C | +++++ | +++++ | Nuc>>>Cyt | 89 (13.8) | 156 (8.7) |
| Δ35/50Rev-i-12R/MS-C | ND | ND | Nuc>>>Cyt | 107 (8.8) | 108 (12.1) |
RNA binding was evaluated by EMSA at fixed levels of the respective RNAs but with various levels of the individual proteins, expressed in E. coli as MBP fusion proteins. The binding potentials of mutants are expressed as follows: ++++, 75%; +++, 50%; ++, 25%; +, 1%; and +/−, <0.5% of wt Rev or Rev–MS-C. ND, not determined.
Subcellular localization data are from indirect immunofluorescence microscopy analyses. Nuc, nuclear; Cyt, cytoplasmic.
Mean GAG expression values from five experiments, normalized for transfection efficiency, are tabulated with the respective standard deviations. The positive values are in boldface. wt Rev-induced GAG expression from RRE RNA is assigned a value of 100.