Biology Reference
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the transport of Rev-GFP from the nucleus to the cytoplasm. This inhibition
was speci®c for Rev, because the export of the functionally analogous Rex
protein of the human T-cell leukemia virus type I was not inhibited by TDRev.
These studies have suggested that Rev and TDRev form heteromultimers in the
nucleolus and that this interaction prevents Rev's export from the nucleus to
the cytoplasm. Rev mutants that are retained in the cytoplasm were also labeled
with GFP (Stauber et al., 1998b). Application of dual-color GFP tagging re-
vealed that coexpression of the nucleolar blue-tagged WT Rev protein together
with green-labeled cytoplasmic Rev mutants did not result in the retention of
WT Rev in the cytoplasm but, on the contrary, in colocalization of the mutants
to the nucleolus. The cytoplasmic mutants were not transdominant and thus are
not suitable for Rev inhibition. Rev-GFP fusion protein was used in studies of
the protein function in human astrocytes ( Ludwig et al., 1999). Astrocytes are
known to harbor the virus in a nonproductive manner, and structural HIV-1
proteins that depend on Rev in their expression were very rarely detected in this
type of cell. In all astrocytic cell cultures, Rev-GFP was found in both cyto-
plasmic and nuclear compartments, in contrast to the typical nuclear/nucleolar
localization in HIV-1-permissive control cells. The authors have suggested that
diminished Rev response in astrocytes is associated with cytoplasmic localiza-
tion of Rev.
To study nuclear export of Rev in isolation, investigators generated a GFP-
labeled, hormone-inducible Rev chimeric protein ( Love et al., 1998). The se-
quence encoding full-length Rev and the hormone-responsive element from the
rat glucocorticoid receptor (GR) was inserted into pGreenLantern from Life
Technologies ( Baltimore, MD). Rev/Gr/GFP behaved like wild-type Rev in
living cells. Steroid removal switches o¨ import, allowing direct visualization of
the Rev export pathway in living cells. Studies have demonstrated that a func-
tional nuclear export sequence ( NES), adenosine triphosplate (ATP), and frac-
tionated cytosol were su½cient for nuclear export in vitro. Nuclear pore-speci®c
lectins and leptomycin B were potent export inhibitors. Nuclear export was not
inhibited by antagonists of calcium metabolism that block nuclear import. The
distinct requirements for nuclear import and export suggest that these compet-
ing processes may be regulated independently.
Rev-GFP construct was used for the comparison of ten Rev-type nuclear
export sequences found in di¨erent proteins and for structure-activity studies of
NES ( Henderson and Eleftheriou, 2000). NES were found to vary profoundly
in activity and were inactivated by mutation of key hydrophobic residues. GFP
has proved to be instrumental in studying Rev tra½cking between nucleus and
cytoplasm and thus can be used for further detailed analysis of nuclear import
and export.
Tat
Fusion of HIV-1 transactivator protein to GFP was used to study intracellular
localization, tra½cking, and interactions of Tat in human cells (Stauber and
