March 2006
Volume 5
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March 2006
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Contents
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Finding New Approaches to Attacking the Latent Reservoir of HIV-Infected Cells
In an expression-profiling study for cellular genes differentially expressed during HIV replication, we found that host-cell gene expression changed in an ordered, temporally dependent pattern, and some of the differentially expressed genes had known relationships to important aspects of the viral replication cycle. However, we also unexpectedly found that the expression patterns of HIV latently infected cell lines differed from those of their uninfected parental cells. The cellular gene expression differences in the latently infected cell lines likely arose during their production and cloning. The production of cloned cell lines typically yields derivative cell lines that vary in expression patterns from the parental lines. These variations may be informative, because during production of the latently infected cells, there is likely selection for the differential expression of sets of cellular genes that help maintain the virus in latency (Figure 1, part A). We further hypothesized that targeting the products of the differentially expressed genes would activate HIV replication in the latently infected cell lines and potentially in the latently infected cells of HIV patients. The genes that were differentially expressed in the latently infected cell lines included genes that were already known to be involved in HIV replication and the control of HIV latency, supporting the hypothesis. Figure 1. A) Model to account for the differences in gene expression in latently infected cell lines compared with their uninfected parental cell lines. During the production of an HIV latently infected cell line, HIV is added to cells, which are then maintained in culture for a long period of time. During this maintenance in culture, there is selection for cells that can maintain HIV in latency (latency-favoring cellular environment [LFCE] cells), because the large majority of cells that do not maintain HIV in latency die (replication-favoring cellular environment [RFCE] cells). The remaining, living cells are then subjected to limiting dilution cloning to produce HIV latently infected cell lines. These cell lines are further characterized to show that they contain an HIV provirus that can be induced into active viral replication, and the cells’ gene expression profiles are determined and compared with the gene expression profiles of the uninfected parental cells. Such studies have revealed significant differences in the expression profiles of the latently infected cells compared with the uninfected parental cells. Studies to determine whether different HIV latently infected cell lines, produced using the same host cell and cloned virus, have similar or different patterns of cellular gene expression, are in progress. B) Cellular genes differentially expressed in an HIV latently infected cell line and the effect on HIV latency of targeting products of the differentially expressed genes. The figure shows results for two agents, clastolactacystin-β-lactone, a proteasome inhibitor, and resveratrol, an upstream activator of transcription factor Egr1. In the top left of each panel is a color map of the expression profile of the gene(s) in the latently infected cells compared with the uninfected parental cell line before (No) and after (in hours) activation into lytic replication. (Green indicates relative expression lower than the uninfected control; red indicates relative expression higher than the uninfected control.) The proteasome genes were overexpressed, and Egr1 was underexpressed in the latently infected cells compared with their uninfected parental cells before induction. The bar graphs show the fold change in HIV p24 antigen, a marker for viral replication, following treatment with the indicated agent. Tumor necrosis factor-α (TNF-α) serves as a positive control agent. All experiments except the “no AZT” data were produced from cells that were also treated with AZT to ensure that HIV p24 antigen production resulted only from reactivated latent infection and not subsequent rounds of viral replication. Clastolactacystin-β-lactone and resveratrol activated HIV replication in the latently infected cells. These agents, therefore, represent two new classes of agents that can activate HIV replication in at least some latently infected cell lines. To test the hypothesis that targeting cellular genes differentially expressed in HIV latently infected cells would activate HIV replication, we paid special attention to genes whose products could be targeted by available small molecule inhibitors. Figure 1, part B shows the results obtained using two different inhibitors targeting host cell genes that were differentially expressed in the latently infected cell lines. We found that genes encoding several proteasome components were upregulated in latently infected cells and showed that treating the cells with a proteasome inhibitor, clastolactacystin-β-lactone (CLC), activated HIV replication. We found that transcription factor Egr1 was downregulated in the latently infected cells and showed that targeting Egr1 with an upstream activator, resveratrol, activated HIV replication. HIV latently infected cell lines, therefore, appear to constitute model systems that enable the discovery of host cell genes involved in the maintenance of latency; studying these host cell genes can identify new agents that eject HIV from latency. These new agents may also be able to eject HIV from latency in the latently infected cells of HIV patients, which could lead to new ways to attack the latent reservoir of HIV infected cells in vivo. Additional Reading
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persistence of latent reservoirs of HIV-infected cells is a critical issue in
HIV disease. Highly active antiretroviral therapy (HAART) can control but not
eliminate HIV infection, due to the long-lived reservoirs. One potential approach
to addressing this problem involves treatment with agents that activate HIV
from latency, while blocking new rounds of infection with HAART. However, no
clinically useful, highly effective agents capable of depleting HIV reservoirs
have been developed. Most known agents are highly toxic and/or incompletely
effective. To construct improved reservoir depletion strategies, we must better
understand the mechanisms that control HIV latency. Recent findings suggest
that model systems may enable the systematic identification of cellular genes
involved in latency maintenance and that this information can inform the search
for new agents that target the products of those differentially expressed genes
and activate HIV replication in latently infected cells. 
