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. 1999 Feb;19(2):1210-7.
doi: 10.1128/MCB.19.2.1210.

The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals

R Truant  1 , B R Cullen
Affiliations

The arginine-rich domains present in human immunodeficiency virus type 1 Tat and Rev function as direct importin beta-dependent nuclear localization signals

R Truant et al. Mol Cell Biol. 1999 Feb.

Abstract

Protein nuclear import is generally mediated by basic nuclear localization signals (NLSs) that serve as _targets for the importin alpha (Imp alpha) NLS receptor. Imp alpha is in turn bound by importin beta (Imp beta), which _targets the resultant protein complex to the nucleus. Here, we report that the arginine-rich NLS sequences present in the human immunodeficiency virus type 1 regulatory proteins Tat and Rev fail to interact with Imp alpha and instead bind directly to Imp beta. Using in vitro nuclear import assays, we demonstrate that Imp alpha is entirely dispensable for Tat and Rev nuclear import. In contrast, Imp beta proved both sufficient and necessary, in that other beta-like import factors, such as transportin, were unable to support Tat or Rev nuclear import. Using in vitro competition assays, it was demonstrated that the _target sites on Imp beta for Imp alpha, Tat, and Rev binding either are identical or at least overlap. The interaction of Tat and Rev with Imp beta is also similar to Imp alpha binding in that it is inhibited by RanGTP but not RanGDP, a finding that may in part explain why the interaction of the Rev nuclear RNA export factor with _target RNA species is efficient in the cell nucleus yet is released in the cytoplasm. Together, these studies define a novel class of arginine-rich NLS sequences that are direct _targets for Imp beta and that therefore function independently of Imp alpha.

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Figures

FIG. 1
FIG. 1
Sequence of the Tat and Rev NLS. A potential alignment of the 9-amino-acid Tat NLS with the somewhat larger Rev NLS is shown. The Tat M1 mutant encodes glutamic acid residues in place of lysine 51 and arginine 55 while the Rev N40D mutation substitutes aspartic acid for asparagine 40, as indicated. The Rev M6 mutant has been previously described (27, 28) and bears an aspartic acid and a leucine residue in place of arginines 41 to 44.
FIG. 2
FIG. 2
In vitro nuclear import assays. Permeabilized HeLa cells were prepared as previously described (13, 42) and incubated in a buffer containing recombinant human Ran and p10/NTF2 as well as a source of energy. Import substrates consisted of wild-type (GST-Rev, GST-T NLS, GST-TatNLS) or mutant (GST-M6, GST-N40D, GST-TatM1) fusion proteins labeled with FLUOS. Recombinant purified import factors (Imp β, Imp α, Trn) were added as indicated. After incubation at 30°C for 15 min, the nuclei were fixed and then analyzed for nuclear import of the fluorescent substrate using a fluorescence microscope. Bar in panel M ≈ 20 μm.
FIG. 3
FIG. 3
Direct binding of Imp β by the Rev NLS. The indicated purified wild-type (Rev, TNLS) or mutant (Rev M6, Rev M40D, R5QR4) GST fusion proteins were coupled to agarose beads and used to construct micro-affinity columns that were then loaded with ∼2 μg of recombinant Imp β or Imp α. Proteins present in the column flow-through (FT) fraction were collected, and bound (B) proteins were eluted from the column by using 800 mM MgCl2. The FT and B fractions were then subjected to analysis by SDS-PAGE.
FIG. 4
FIG. 4
The interaction of the Tat NLS with Imp β is inhibited by Ran GTP. (A) Micro-affinity chromatography, using columns bearing the wild-type GST-TatNLS or mutant GST-TatM1 fusion proteins, was performed as described in the legend to Fig. 3. (B) Glutathione-Sepharose beads bearing the wild-type GST-TatNLS protein were loaded with recombinant Imp β and then incubated in buffer alone or with added RanGTP or RanGDP. After washing, the residual bound Imp β was released with SDS and analyzed by SDS-PAGE.
FIG. 5
FIG. 5
Competitive in vitro nuclear import assays. Nuclear import assays were performed using permeabilized HeLa cells in the presence of rabbit reticulocyte lysate as a source of native nuclear import factors. The FLUOS-labeled substrates analyzed in this assay are given at left. Competitor peptides were added at an ∼60-fold molar excess and are listed at the top of the figure. Nuclear uptake assays were performed for 20 min at 18°C and were then analyzed as described in the legend to Fig. 2. Bar in panel Y ≈ 20 μm.
FIG. 6
FIG. 6
Imp β inhibits RNA binding by HIV-1 Rev. The indicated electrophoretic mobility shift assay was performed essentially as previously described (28) using recombinant HIV-1 Rev protein and a radiolabeled HIV-1 RRE RNA probe. Recombinant Imp β or transportin was added to the RNA binding assays visualized in lanes C to F. In addition, RanGTP (lane D) or RanGDP (lanes E and F) was added as indicated.
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References

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