Biology Reference
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FL, and the resulting conjugate was 97.6% self-quenched due to ¯uorescence
resonance energy transfer between neighboring Bodipy molecules. Fluorescence
microscopy and ¯ow cytometry experiments with cells incubated with Bodipy-
VSV revealed intracellular relaxation of ¯uorescence self-quenching resulted
from viral disassembly and viral protein degradation.
The large size and the high protein content of the adenovirus and VSV par-
ticles facilitate speci®c conjugation with ¯uorescent dyes and detection, but may
not be applicable to other viral systems. The approach did not prove to be
e½cient in the case of HIV-1 and was complicated by extensive labeling of
contaminating microvesicles that are always present, even in the purest viral
preparations (Bess et al., 1997; Gluschankof et al., 1997). Incorporation of
¯uorescent components during self-assembly of viral particles allows for the
most speci®c labeling of the virions. Virions of potato virus X ( PVX) were the
®rst ones to be successfully labeled with the GFP (Cruz et al., 1996). GFP was
introduced as a fusion with the coat protein within the viral genome. However,
assembly of ¯uorescent virions required the presence of free coat protein in
addition to the fusion protein subunits. Fluorescent virions could be imaged in
infected plant tissue using confocal laser scanning microscopy. It is noteworthy
that the resulting virions had an altered morphology, being twice as large as
wild-type virions. Thus, GFP attachment in¯uences PVX coat protein oligo-
merization and virion assembly.
For su½cient intensity of ¯uorescence, the protein that is chosen for labeling
of viral particles during self-assembly should be incorporated in virions in
multiple copies. However, modi®cation of major structural viral proteins with a
label of a comparable size is likely to interfere with correct assembly of viral
core or coat. In case of HIV-1, accessory proteins seem to be good candidates
for labeling of whole virions. Among them, HIV-1 viral protein R ( Vpr) ap-
pears to be the optimal choice because it is incorporated into virions in rela-
tively large quantities. Vpr is a 96-amino acid protein that is involved in the
nuclear import of viral DNA and the growth arrest of host cells ( Bukrinsky and
Adzhubei, 1999). Recent studies have demonstrated that Vpr is present in viral
particles in a molar ratio of approximately 1:7 compared with capsid (Muller et
al., 2000). However, the extent of incorporation of HIV-1 Vpr into the virus
particles is ¯exible and can be modulated by expression level in cells (Lai et al.,
2000). Vpr was suggested as a carrier for targeting foreign proteins into primate
lentiviruses ( Park and Sodroski, 1996). It was successfully used for incorpora-
tion of chloramphenicol acetyltransferase (CAT ) enzyme into SIV (Park and
Sodroski, 1996) and HIV-1 ( Yao et al., 1999). An anti-integrase single-chain
variable fragment moiety delivered into viral particles by fusing it to Vpr was
used for inactivation of HIV-1 virions (BouHamdan et al., 2000; Okui et al.,
2000).
Vpr fused to the C-terminus of GFP incorporated e½ciently in trans during
HIV-1 assembly and produced highly ¯uorescent virions (Stauber et al., 1999).
GFP-labeled virions were prepared by cotransfection of 293 cells with HIV-1
molecular clones and a plasmid expressing highly ¯uorescent variant of GFP
