Biology Reference
In-Depth Information
Fluorescent HIV-1 virions were also generated with the help of Vpr fused
to GFP through the C-terminus of Vpr (Muthumani et al., 2000). Vpr-GFP
exhibited the same activity in arresting the cell cycle in G
2
phase as the wild-
type protein and replication kinetics showed no signi®cant di¨erences between
Vpr-GFP and native complemented pseudovirus. In this study, amphotropic
pseudotype viruses lacking vpr were used. Production of ¯uorescent virions was
accomplished by cotransfection of human rhabdosarcoma (RD) cells with three
constructs encoding envelope protein, viral genome lacking vpr and env, and
Vpr-GFP plasmid. The resulting particles were tested for the ability to infect
target cells and deliver detectable amounts of GFP. Twenty-four hours post-
infection, GFP-labeled virus was seen to have infected normal human PBMCs,
macrophages, and dendritic cells. The infected cells were ¯uorescent and could
be identi®ed by ¯uorescence microscopy and FACS analysis. GFP ¯uorescence
exhibited perinuclear localization in infected cells. The study strongly suggests
that Vpr-GFP fusion protein can be used to identify subsets of important cells
infected in a complex cell background.
GFP AS A TRANSCRIPTIONAL REPORTER FOR HIV-1 LTR
Transcriptional activation of reporter genes is a widely used approach in the
development of highly sensitive detection systems. LTR are promoters that are
the hallmarks of the retroviruses. DNA sequences within LTR interact with a
variety of cellular factors (reviewed in Pereira et al., 2000). Tat protein encoded
by the virus is a potent transactivator for HIV gene expression. It binds to the
TAR region within the LTR. A number of HIV-1 detection systems have been
developed that use a reporter gene (luciferase, b-galactosidase, alkaline phos-
phatase, CAT ) under the control of HIV-1 LTR. The cells of interest are ini-
tially transfected with the reporter plasmid containing HIV-1 LTR upstream
from the marker gene. Upon infection with HIV-1, the viral tat product in-
creases transcription from LTR promoter, leading to high level of expression of
the reporter protein. Unlike previously used reporter systems, GFP-based de-
tection does not require processing of the cells by ®xation, lysis, or incubation
with a substrate. HIV-1 infected cells can be detected with high sensitivity by
either ¯uorescence microscopy or FACS analysis. Originally, the system was
introduced by Dorsky and colleagues (Dorsky et al., 1996) as a tool for titering
HIV-1 and sorting HIV-1 infected cells. The authors have modi®ed the com-
mercial plasmid-expressing S65T mutant of GFP ( pS65T-C1) from Clontech by
substitution of the HCMV IE promoter with HIV-1 LTR. HeLa-CD4 cells
transfected with the plasmid became brightly ¯uorescent when infected by HIV-
1 or after cotransfection with tat-expressing vector. The background ¯uores-
cence of the cells carrying only marker plasmid was very weak.
Transcriptional activity of the LTR of four di¨erent HIV-1 primary isolates
was compared with the help of a transient expression system utilizing LTR-
GFP vectors ( Kar-Roy et al., 2000). The comparison conducted in HeLa cells
