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. 2009 Aug 5;83(20):10737–10751. doi: 10.1128/JVI.01307-09

TABLE 2.

Phenotypes of TRIM5α B-box 2 mutants

TRIM5αrh variant Restrictiona
Mean reverse transcription (%) ± SDb,h Dimerizationc,h Higher-order association (%)d,h
Mean binding to HIV-1 CA-NC complexes ± SDe,h Localizationf Half-life (min)g
HIV-1 N-MLV Association with wild type Self-association
Wild type ++++ ++ 0 Yes 100 100 1 ± 0 Wild-type 48
E120D ++++ + 0 ND 100 ND 1.26 ± 0.05 L reticular 80
L104E ++++ + 0 Yes ND ND 1.09 ± 0.29 L reticular >1,000
K93A ++++ + 0 Yes ND ND 1.23 ± 0.04 Wild-type 60
Q92A ++++ + 0 Yes ND ND 1.14 ± 0.12 Wild-type 65
F131E ++++ + ND ND ND ND 0.84 ± 0.06 L reticular 60
Q123E ++++ - ND ND ND 73 1.00 ± 0.30 Wild-type 70
R121K ++++ - 0 ND 7 81 1.34 ± 0.07 Wild-type 42
R99E ++++ - 0 Yes ND 85 0.95 ± 0.23 L reticular 240
K103E ++++ - 0 ND ND ND 0.90 ± 0.08 L reticular >1,000
L105E +++ - ND ND ND ND 1.04 ± 0.16 Reticular >1,000
E120R/R121E +++ - 56.5 ± 10 Yes ND 15 0.76 ± 0.15 Reticular >1,000
E120D/R121K +++ - 25 ± 11 ND 3 ND 1.12 ± 0.24 Reticular 400
E120K/R121K +++ - ND ND 0 70 0.71 ± 0.12 L reticular 110
L132E +++ - 63.5 ± 7 ND 0 0 0.84 ± 0.13 L reticular 120
E102R +++ - ND ND ND ND 0.97 ± 0.23 M reticular 400
E102R/R121E +++ - 81.5 ± 26 Yes 0 28 0.86 ± 0.04 M reticular 600
E120R ++ - ND ND 6 ND 0.45 ± 0.12 M reticular ND
E120R/R121K ++ - ND ND 0 80* 0.45 ± 0.06 L reticular 240
E120K ++ - 5 ± 5 ND 0 14 0.84 ± 0.10 L reticular 300
E102R/E120R/R121E ++ - ND ND 0 ND ND L reticular 400
E120D/R121D ++ - ND Yes 0 ND 0.85 ± 0.06 L reticular 360
E120R/R121D ++ - 19 ± 11 ND 0 ND 0.72 ± 0.10 Reticular 400
E120K/R121E + - ND Yes 0 5 0.73 ± 0.07 L reticular >1,000
E120K/R121D + - ND Yes 0 0 0.42 ± 0.00 Reticular >1,000
E120D/R121E + - ND ND 0 0 0.55 ± 0.06 L reticular >1,000
R121S + - 49 ± 8 Yes ND ND 0.48 ± 0.13 Reticular 340
R121Q + - 55 ± 5 Yes 0 ND 0.54 ± 0.07 Reticular 300
L118E + - 100 Yes 0 10 0.4 ± 0.03 Reticular 240
W117E + - 60 Yes 6 0* 0.43 ± 0.08 Reticular 300
R121E - - 100 Yes 0 0* 0.43 ± 0.05 Reticular >1,000
R121D - - 100 Yes 0 0* 0.55 ± 0.06 Reticular 173
a

Restriction was measured by infecting cells expressing the indicated TRIM5αrh variants with HIV-1 and N-MLV expressing the GFP protein. After 48 h, the percentage of GFP-positive cells (infected cells) was determined by flow cytometry. For HIV-1: ++++, 100% restriction; +++, ∼75% restriction; ++, ∼50% restriction; +, ∼25% restriction; and −, <25% or no restriction. For N-MLV: ++, ∼50% restriction; +, ∼25% restriction; and −, <25% restriction. Experiments were performed at least three times, and typical results are shown.

b

HIV-1 reverse transcription was measured by real-time PCR 6 h after infection as described in Materials and Methods. The number represents the percentage of late reverse transcripts observed in cells expressing the indicated TRIM5αrh variant relative to the level of late reverse transcripts in control HIV-1-infected cells transduced with the empty LPCX vector. Experiments were performed at least two times.

c

Dimerization of TRIM5αrh variants was measured by the use of the cross-linker EGS as described in Materials and Methods.

d

Each TRIM5αrh variant was assayed for higher-order association with wild-type TRIM5αrh (association with wild type) or with itself (self-association), as described in Materials and Methods. In the case of association with wild-type TRIM5αrh, the percentage represents the fraction of the TRIM5αrh variant coprecipitated with wild-type TRIM5αrh relative to the amount of wild-type TRIM5αrh coprecipitated with itself. For self-association, the percentage represents the fraction of the TRIM5αrh variant coprecipitated with itself relative to the coprecipitation of wild-type TRIM5αrh with itself. *, A high background was observed for the TRIM5αrh variant in the control sample without a TRIM5α target protein. The reported values may be less reliable as a result of this background.

e

Binding to the HIV-1 capsid complexes was determined for each TRIM5αrh 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. 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 capsid-binding affinities of the mutant proteins.

f

The localization of each TRIM5αrh variant was determined as described in Materials and Methods. For each TRIM5αrh variant, cytoplasmic bodies were counted for five independent cells and compared to the number in cells expressing wild-type TRIM5αrh. “L” means fewer cytoplasmic bodies than wild-type TRIM5αrh; “M” means more cytoplasmic bodies than the wild type, and “reticular” indicates a diffuse, reticular pattern of staining throughout the cytoplasm.

g

The half-life of each TRIM5αrh variant was estimated by pulse-chase experiments. Every experiment was repeated at least two times.

h

ND, not determined.