Identification of T cell-signaling pathways that stimulate latent HIV in primary cells - PubMed (original) (raw)
Identification of T cell-signaling pathways that stimulate latent HIV in primary cells
David G Brooks et al. Proc Natl Acad Sci U S A. 2003.
Abstract
Eradication of HIV infection depends on the elimination of a small, but stable population of latently infected T cells. After the discontinuation of therapy, activation of latent virus can rekindle infection. To purge this reservoir, it is necessary to define cellular signaling pathways that lead to activation of latent HIV. We used the SCID-hu (Thy/Liv) mouse model of HIV latency to analyze a broad array of T cell-signaling pathways and show in primary, quiescent cells that viral induction depends on the activation of two primary intracellular signaling pathways, protein kinase C or nuclear factor of activated T cells (NF-AT). In contrast, inhibition or activation of other important T cell stimulatory pathways (such as mitogen-activated protein kinase, calcium flux, or histone deacetylation) do not significantly induce virus expression. We found that the activation of NF-kappaB is critical to viral reactivation; however, all pathways that stimulate NF-kappaBdonot reactivate latent virus. Our studies further show that inhibition of NF-kappaB does not prevent activation of HIV by NF-AT, indicating that these pathways can function independently to activate the HIV LTR. Thus, we define several molecular pathways that trigger HIV reactivation from latency and provide evidence that latent HIV infection is maintained by the functional lack of particular transcription factors in quiescent cells.
Figures
Fig. 1.
Costimulation induces latent HIV expression. HIVNL-r-HSAS-infected CD4 SP, muCD24-negative thymocytes (day 0) were cultured without stimulation or costimulated with antibodies to CD3 and CD28 for 3 days (day 3) and assessed for human CD45 and HIV (muCD24) expression by flow cytometry. The percentage of muCD24-positive cells is indicated in each panel. The concentration of viral p24 gag in the supernatant is shown next to each panel.
Fig. 2.
Signal transduction pathways involved in reactivating latent virus after costimulation. (A) CD4SP, muCD24-negative thymocytes from HIVNL-r-HSAS-infected Thy/Liv implants were either cultured without stimulation, costimulated, or costimulated in the presence of the indicated inhibitor for 2 days, and HIV reactivation (muCD24 expression) was quantitated by flow cytometry. Cells from many experiments costimulated (CS) in the presence of the indicated inhibitor are shown. Primary data can be viewed in Fig. 5, which is published as supporting information on the PNAS web site,
. The graph displays the fold increase (as assessed by flow cytometry) in HIV reporter expression for each condition compared with the unstimulated control, which is set at 1. The protein or pathway inhibited by each compound is provided within the parentheses. Each point represents results from a single experiment, and the horizontal lines indicate mean values. Use of each inhibitor was associated with minimal toxicity (90–100% viability compared with cells costimulated without any inhibitors) except for _N-_butyrate (50% viability compared with costimulated cells). *, P = <0.05 compared with unstimulated cells by Dunnett's two-tailed multiple comparisons test. (B) Effect on cell cycling by each inhibitor after costimulation. Uninfected CD4SP thymocytes from Thy/Liv implants were cultured as indicated for 2 days and stained for RNA and DNA content. The lower left quadrants of each panel indicate cells in the G0 or G1a phases of the cell cycle whereas the lower and upper right quadrants represent cells in the G1b and S through M phases, respectively. The percentage of cells in each quadrant is given. Events in the upper left quadrants are likely bald nuclei or doublets formed during the staining procedure.
Fig. 3.
Reactivation of latent HIV by different signal transduction pathways. (A) CD4SP,muCD24-negative thymocytes were cultured in the presence of the indicated agent for 2 days and analyzed as in Fig. 2_A_. The protein or pathway inhibited by each compound is stated in parentheses. Primary data can be viewed in Fig. 6, which is published as supporting information on the PNAS web site. Use of each agent was associated with minimal toxicity (90–100% viability compared with cells costimulated without any inhibitors) except for TsA and PHA (50% viability compared with costimulated cells). (B) Effect of each agent on cell cycle progression. Uninfected CD4SP thymocytes from Thy/Liv implants were cultured as in Fig. 2_B_.
Fig. 4.
NF-κB is required for HIV reactivation whereas de novo protein synthesis is not. (A) RNA was isolated from HIVNL-r-HSAS-infected CD4SP,muCD24-negative thymocytes after7hof culture without stimulation (stippled bars), after costimulation (filled bars), or after costimulation in the presence of cycloheximide (striped bars) or gliotoxin (diamond bars). For each condition, real-time RT-PCR was performed to quantify the levels of LTR/gag RNA (normalized to the number of β2m transcripts). The graph shows the fold increase in LTR/gag transcripts for each condition compared with the unstimulated control, which is set at 1. Each group of bars represents a separate experiment. (B) CD4SP,muCD24-negative thymocytes were incubated without stimulation (stippled bars), with the PKC activator prostratin (filled bars), or with prostratin plus cycloheximide (striped bars) or prostratin plus gliotoxin (diamond bars). RNA was isolated after 7 h of culture and quantitated as in A. (C) CD4SP,muCD24-negative thymocytes were incubated without stimulation (stippled bars), with PHA (filled bars), with PHA plus cycloheximide (striped bars), or with PHA plus gliotoxin (diamond bars). RNA was isolated and quantitated after 7 h of culture.
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