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of the proteins. All these approaches provide bene®cial tools with a variety of
applications and thus will be described in the present chapter.
LABELING OF HIV-1 VIRIONS IN CIS
Immunohistochemistry has been widely used for detection of HIV-1-infected
cells both in peripheral blood mononuclear cells (PBMCs) of patients with ac-
quired immunode®ciency syndrome (AIDS) and in cultured cells (reviewed by
McSharry, 2000). HIV-1 recombinants producing GFP allow for much more
rapid and easy detection of virus-infected cells by ¯uorescent microscopy,
¯uorescence-activated cell sorter ( FACS) analysis, and multi-well ¯uorescence
reading. Two groups have reported generation of replication-competent GFP-
tagged HIV-1 (Lee et al., 1997; Page et al., 1997). In both cases, the nef gene in
the molecular clone of HIV-1 was replaced with the gene encoding GFP. A
total of 293 cells were transfected with the plasmid-encoding GFP-tagged HIV-
1, and the labeled virions were harvested from the supernatant and used for
infecting H9, a human leukemia cell line (Page et al., 1997). Page and col-
leagues have found that virus labeled with wild-type GFP did not produce suf-
®cient ¯uorescence in infected cells, whereas S65A mutant generated 10- to 20-
fold greater ¯uorescence intensity, making it applicable for single-cell analysis
under standard conditions of ¯ow cytometry. Both groups have found that
insertion of the GFP gene did not a¨ect viral replication. Lee and co-workers
have analyzed the genomic stability of the recombinant GFP-tagged virus and
found no signi®cant reduction in the expression of viral and GFP genes, even
after 10 passages. GFP-tagged HIV-1 was suggested to be a useful tool in
studying the biology of HIV replication in vitro and in animal models and for
screening of anti-HIV-1 drugs. The HXB-2D clone of HIV-1 with the enhanced
GFP gene inserted in place of the nef reading frame was used to examine
the dependence of HIV-1 on its nuclear factor ( NF )-kB binding sites within
the long terminal repeats ( LTR) of the HIV-1 genome (Chen et al., 1997).
Comparison of wild-type HIV-1 with the virus mutated in the kB site of LTR
allowed for the demonstration that in T cells, the virus can use the transcription
factor NF-kB to enhance its growth but is also able to grow in its absence.
Application of a virus labeled with GFP in cis allowed easy analysis of viral
gene expression pattern in a single cell. GFP tagging has several advantages
over other methods of detection of viral infection in vitro. First, the infected
cells can be identi®ed without any additional processing steps. Second, GFP
allows for identi®cation of live infected cells without any damage to them.
Third, virus-infected cells can be easily separated by FACS analysis.
Similar to HIV-1, simian immunode®ciency virus (SIV ) was engineered to
express enhanced GFP in cis (Alexander et al., 1999). Unlike HIV-1, labeled
SIV can be used not only for in vitro, but also for in vivo studies. Interestingly,
replacement of the vpr sequence in SIV with EGFP resulted in a vector that
caused transfected cells to ¯uoresce but did not produce replication-competent
