Biology Reference
In-Depth Information
(Sodora et al., 1999). GHOST cells proved to be instrumental in a high-
throughput screening assay of neutralizing activity of sera from the patients
who are long-term nonprogressors and were receiving highly active antiretro-
viral therapy (HAART ) (Dreyer et al., 1999).
Murine cell line NIH 3T3, which stably expresses a reporter of HIV-1 in-
fection, the LTR-GFP cassette, was used to de®ne molecular determinants for
HIV-1 infection (Mariani et al., 2000). Infection and replication were assessed
by ¯ow cytometry. Stable expression of human CD4, chemokine receptor
CCR5, and cyclin T1 allowed an M-tropic virus to enter e½ciently, form pro-
virus, and be expressed. However, the virus failed to replicate, presumably
because of a major block at virion assembly. A murine model for HIV-1
replication would allow investigation of features of AIDS such as the immune
response to the virus, the dynamics of virus replication, and the mechanism of
T-cell depletion. The suggested LTR-GFP reporter system can assist in de-
termining all factors that are needed for the development of such a model.
LABELING OF VIRAL PROTEINS WITH GFP
Intracellular protein tra½cking is an important component of function that can
regulate protein activity and determine involvement in certain processes. GFP
has been used widely as a unique tool to monitor dynamic processes in living
cells (reviewed by Whitaker, 2000) and it was used extensively for studying the
function of HIV proteins. HIV-1 encodes 15 distinct proteins (Frankel and
Young, 1998). Substantial progress has been made toward understanding the
function of each protein, the Gag and Env structural proteins MA (matrix), CA
(capsid), NC (nucleocapsid), p6, gp120 (surface envelope), and gp41 (trans-
membrane envelope); the Pol enzymes PR ( protease), RT (reverse transcrip-
tase), and IN (integrase); the gene regulatory proteins Tat and Rev; and the
accessory proteins Nef, Vif, Vpr, and Vpu.
Rev
The Rev protein was the ®rst HIV-1 protein to be fused to GFP (Stauber et al.,
1995). Rev activates export of unspliced viral RNA from the nucleus ( Pollard
and Malim, 1998). It shuttles between the nucleus and the cytoplasm and har-
bors both a nuclear localization signal and a nuclear export signal. Therefore,
HIV-1 Rev represents an excellent system for studying the transport across the
nuclear envelope. Fusion to the C-terminus of GFP did not a¨ect the biological
activity of Rev (Stauber et al., 1995). A GFP-fusion protein was used to study
the mechanisms of Rev inactivation by mutant Rev proteins displaying a trans-
dominant phenotype. Upon actinomycin D treatment, Rev-GFP was trans-
ported to the cytoplasm, whereas transdominant Rev( TDRev), although par-
tially dissociated from the nucleolus, was retained in the nucleus. Coexpression
of Rev-GFP and TDRev in the same cell demonstrated that TDRev inhibited
