Biology Reference
In-Depth Information
region of the fusion protein to include CA and SP1 did not alter signi®cantly
the localization of the protein. When all or the N-terminal portion of nucleo-
capsid (NC) was included, ¯uorescence had punctate plasma membrane locali-
zation. The minimal region conferring plasma membrane localization (I domain)
was identi®ed as the N-terminal 14 amino acids of NC. Two arginine residues
were found to be critical for the function of the I-domain (Sandefur et al.,
2000). Thus, GFP labeling allowed straightforward identi®cation of a domain
involved in membrane targeting. An MA-GFP fusion protein was used to study
the role of the HIV-1 viral protein U (Vpu) in the release of virus particles.
Subcellular localization of Gag protein in the presence or absence of Vpu was
studied with the use of subcellular fractionation techniques and by confocal
microscopy with Gag-GFP fusion proteins (Deora et al., 2000). MA-GFP fusion
protein showed enhanced membrane binding in the presence of Vpu, thus sug-
gesting that Vpu action may be mediated by reinforcing Gag association with the
plasma membrane. Gag fusion to GFP was also used in an attempt to under-
stand the routing mechanism of the molecule from its site of synthesis in the
cytoplasm to the plasma membrane (Perrin-Tricaud et al., 1999). Double label-
ing with actin-speci®c probe showed no colocalization of Gag with actin, sug-
gesting that Gag may associate with other cytoskeletal components or that
oligomerization of Gag occurs before it gets to the membrane.
GFP was also successfully used in the studies of cellular receptors involved
in HIV-1 entry, mechanisms of their tra½cking, interactions with viral enve-
lopes, and impact of down-regulation on HIV-1 replication (Amara et al., 1997;
Orsini, 1999; Tarasova et al., 1998). However, this topic was extensively re-
viewed recently ( Kallal and Benovic, 2000; Pelchen-Matthews et al., 1999;
Tarasova et al., 2000) and thus will not be detailed here.
CONCLUSION
GFP has already become an indispensable instrument in retroviral research.
Many GFP-based tools have been developed and many of them are available
for investigators through the AIDS Research and Reference Reagent Program
established by the National Institute of Allergy and Infectious Diseases at the
National Institute of Health. Because of their simplicity, GFP-based assays are
becoming increasingly popular. It can be foreseen that application of GFP for
studies of HIV will grow rapidly in the future. Many ¯uorescence-based meth-
ods of analysis continue to develop. More sensitive and selective methods of
¯uorescence detection and novel microscopic techniques, such as ¯uorescence
correlation spectroscopy (Van Craenenbroeck and Engelborghs, 2000), ¯uo-
rescence polarization spectroscopy ( Fernandes, 1998), multiphoton laser scan-
ning microscopy (Buehler et al., 1999), time-resolved ¯uorescence spectroscopy
(Millar, 1996), and others stimulate even broader studies with application of
GFP-tagged proteins and viruses. Fluorescent proteins with a variety of spec-
tral properties (red, blue, or yellow proteins) with high quantum yields are
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