Biology Reference
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Pavlakis, 1998). Fusion of the C-terminus of Tat to GFP had no e¨ect on the
transactivating activity of the protein. When expressed at relatively low levels,
Tat-GFP was found in the nucleus, whereas overexpression resulted in nucleo-
lar accumulation. A Tat-GFP hybrid containing the Rev nuclear export signal
localized mostly in the cytoplasm. Nevertheless, it was able to transactivate
HIV-1 LTR, suggesting that even transient presence of Tat in the nucleus is
su½cient for activity.
Vpr
Vpr fusions with GFP were used for labeling of HIV-1 virions in trans
(Muthumani et al., 2000; Stauber et al., 1999) When expressed in cells, both
Vpr-GFP and GFP-Vpr had nuclear localization that is characteristic for
nonlabeled protein. Fusion to GFP was applied for identi®cation of the Vpr
domains that are involved in nuclear localization ( Kamata and Aida, 2000).
The ®rst predicted N-terminal a-helical domain (aH1) and the second helical
domain located close to the C-terminus (aH2) were fused individually with
GFP. Both constructs were localized predominantly in the nucleus. The nuclear
localization pattern was eliminated by mutations in the helixes, suggesting that
the two domains are involved in nuclear import of Vpr by certain cellular
factors.
Integrase
Karyophilic properties of HIV-1 integrase were studied by construction of an
N-terminal fusion protein of the enzyme and GFP (GFP-IN ) ( Pluymers et
al., 1999). Integrase is responsible for the integration of the retroviral cDNA
into the cellular genome, and integration is an essential part of the retroviral
replication cycle. However, the nuclear transport of integrase was not directly
visualized before the construction of the fusion protein with GFP. Transient
expression of GFP-IN in di¨erent mammalian cells allowed investigators to
obtain solid evidence for the nuclear localization of the enzyme. Mutations
in the conserved KRK motif (residues 186 to 189) that was suggested to be a
putative nuclear localization signal did not a¨ect the karyophilic property of
HIV-1 integrase, as was shown by using GFP fusion protein (Tsurutani et al.,
2000). Further mutational studies with the integrase-GFP construct may pro-
vide a better understanding of the nuclear transport mechanisms of integrase
and of the whole preintegration complex.
Nef
Chimeric proteins consisting of HIV-1 Nef fused to a strongly ¯uorescing mu-
tant of GFP were used to correlate Nef function with intracellular localization
in human CD4
Jurkat T cells (Greenberg et al., 1997). The Nef-GFP fusion
protein colocalizes with components of the clathrin coat, including clathrin and
