Biology Reference
In-Depth Information
tested (the reverse transcriptase inhibitors zidovudine and nevirapine, the pro-
tease inhibitor saquinavir, the gag transport inhibitor cyclophilin, and the tat
inhibitor Ro24-7429) produced IC50 values that were equivalent to a standard
assay using p24 antigen reduction. The HIV-1 LTR can be activated by a
number of cellular proteins ( Pereira et al., 2000). However, upon stimulation
with the most potent cellular stimulants, such as tumor necrosis factor (TNF )-a
and 12-myristate 13-acetate, the ¯uorescence of CEM-GFP cells increased 2- to
3-fold compared with over 100-fold with HIV-1 infection (Gervaix et al., 1997).
Psoralen-UV light-inactivated virus, which is able to bind and enter the cells
but unable to replicate, produced no GFP in inoculated CEM-GFP cells. CEM-
GFP cells were used to demonstrate Nef-induced down-regulation of major his-
tocompatibility complex class 1 (MHC-1) (Akari et al., 2000). Immunostaining
of the cell-surface MHC-1 in infected cells followed by FACS analysis revealed
that ¯uorescence intensity for GFP inversely correlated with MHC-1 expression
for the wild-type NL-432 HIV-1 isolate. In contrast, infection with Nef-defective
mutants was not accompanied by MHC-1 down-regulation. The CEM-GFP re-
porter system seems to be a valuable tool that can be applied to high-throughput
assays for anti-HIV-1 drugs and rapid determination of viral infectivity titers.
However, CEM that can be infected with T-cell-tropic HIV-1 isolates lack CCR5
receptor that is mandatory for macrophage-tropic or non-scyncytium-inducing
( NSI ) isolates of HIV-1. Engineering the cells that express a variety of corecep-
tors used by HIV-1 for cell entry will make the assay applicable for broad
screening for anti-HIV-1 agents. The method is rapid, easy and inexpensive.
The results can be read by ¯ow cytometry, cyto¯uorometry, or ¯uorescent
microscopy.
Human osteosarcoma ( HOS) cells stably transfected with a reporter con-
struct consisting of the HIV-2 LTR upstream of humanized GFP was used to
generate a panel of cell lines (GHOST cells) expressing CD4 and one of eight
chemokine receptors that mediate HIV-1 entry into cells (Cecilia et al., 1998).
The system allowed for testing of a large number of primary HIV-1 isolates for
their coreceptor preference. Coreceptor usage was discerned for each virus by
measuring the number of ¯uorescent cells by ¯ow cytometry in preparations of
infected cells. The assay was also used to study the parameters that contribute
to the sensitivity of HIV-1 isolates to neutralization by human monoclonal and
polyclonal antibodies (Cecilia et al., 1998). GHOST cells are now available
through the National Institutes of Health AIDS Research and Reference Re-
agent Program ( URL available at http://www.aidsreagent.org) due to the gen-
erous donation of Dr. Littman, and they are becoming a popular tool in
studying inhibition of HIV-1 replication. They were used to investigate whether
the identity of the coreceptor used by primary HIV-1 isolates in¯uences the
sensitivity of these isolates to neutralization by monoclonal antibodies and
CD4-reagents. These studies showed that neutralizing antibodies are unlikely
to be the major selection pressure in the evolution of HIV-1 coreceptor usage
( Trkola et al., 1998). GHOST cells were used to characterize the phenotype of
SIV viruses that emerge in immunization with attenuated strains of the virus
