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. 2006 Oct 2:3:66.
doi: 10.1186/1742-4690-3-66.

Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4+ T cells

Tzu-Ling Sung  1 , Andrew P Rice
Affiliations

Effects of prostratin on Cyclin T1/P-TEFb function and the gene expression profile in primary resting CD4+ T cells

Tzu-Ling Sung et al. Retrovirology. .

Abstract

Background: The latent reservoir of human immunodeficiency virus type 1 (HIV-1) in resting CD4+ T cells is a major obstacle to the clearance of infection by highly active antiretroviral therapy (HAART). Recent studies have focused on searches for adjuvant therapies to activate this reservoir under conditions of HAART. Prostratin, a non tumor-promoting phorbol ester, is a candidate for such a strategy. Prostratin has been shown to reactivate latent HIV-1 and Tat-mediated transactivation may play an important role in this process. We examined resting CD4+ T cells from healthy donors to determine if prostratin induces Cyclin T1/P-TEFb, a cellular kinase composed of Cyclin T1 and Cyclin-dependent kinase-9 (CDK9) that mediates Tat function. We also examined effects of prostratin on Cyclin T2a, an alternative regulatory subunit for CDK9, and 7SK snRNA and the HEXIM1 protein, two factors that associate with P-TEFb and repress its kinase activity.

Results: Prostratin up-regulated Cyclin T1 protein expression, modestly induced CDK9 protein expression, and did not affect Cyclin T2a protein expression. Although the kinase activity of CDK9 in vitro was up-regulated by prostratin, we observed a large increase in the association of 7SK snRNA and the HEXIM1 protein with CDK9. Using HIV-1 reporter viruses with and without a functional Tat protein, we found that prostratin stimulation of HIV-1 gene expression appears to require a functional Tat protein. Microarray analyses were performed and several genes related to HIV biology, including APOBEC3B, DEFA1, and S100 calcium-binding protein genes, were found to be regulated by prostratin.

Conclusion: Prostratin induces Cyclin T1 expression and P-TEFb function and this is likely to be involved in prostratin reactivation of latent HIV-1 proviruses. The large increase in association of 7SK and HEXIM1 with P-TEFb following prostratin treatment may reflect a requirement in CD4+ T cells for a precise balance between active and catalytically inactive P-TEFb. Additionally, genes regulated by prostratin were identified that have the potential to regulate HIV-1 replication both positively and negatively.

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Figures

Figure 1
Figure 1
Prostratin induces expression of CD69 without promoting cell cycle progression in resting CD4+ T cells. Resting CD4+ T cells were analyzed immediately after isolation (Untreated), or were cultured for 48 hours in the presence of DMSO as a control or prostratin (250 ng/ml) before analysis. (A) Cells were assayed for expression of CD25 and CD69 by flow cytometry. (B) Cells were stained with propidium iodide to evaluate DNA content by flow cytometry.
Figure 2
Figure 2
Effects of prostratin on the expression levels of Cyclin T1, Cyclin T2a, and CDK9. Cell extracts were prepared from resting CD4+ T cells from different donors cultured in DMSO or prostratin for 48 hours, and immunoblots were performed to examine the levels of Cyclin T1, CDK9, β-actin (A) and Cyclin T2a (B). The immunoblots were quantified as described in Material and Methods using β-actin for normalization; the value for fold-induction is given below the panels.
Figure 3
Figure 3
Levels of 7SK snRNA and HEXIM1 and association with P-TEFb. (A) Total RNA was isolated from DMSO control and prostratin-treated resting CD4+ T cells. Northern blots were performed to measure 7SK snRNA and U1 snRNA levels; amounts of 7SK and U1 snRNA were quantified using a Phosphoimager and are shown below each panel. Levels of 7SK snRNA were normalized to U1 snRNA. (B) Immunoprecipitations were performed using α-CDK9 antibodies with extracts from control and prostratin-treated cells. CDK9 levels present in a portion of immunoprecipitates were examined by immunoblots (IP-Immunoblot). RNA was extracted from the remaining protein of immunoprecipitates and the levels of 7SK snRNA were determined by Northern blots (IP-Northern blot). Amounts of 7SK snRNA were quantified by a PhosphoImager and normalized the amounts of CDK9 present in immunoprecipitates. (C) Cell extracts were prepared from resting CD4+ T cells cultured in DMSO or prostratin for 48 hours to examine the levels of HEXIM1 and β-actin. Protein levels were quantified by the Densitometer and are shown below each panel. Levels of HEXIM1 were normalized to β-actin levels. (D) Immunoprecipitations were performed using antiserum against CDK9 with extracts adjusted to precipitate equivalent amount of CDK9. Immunoprecipitation products were subjected to immunoblot analysis to evaluate the levels of HEXIM1 associated with CDK9. Levels of HEXIM1 were normalized to CDK9 levels in immunoprecipitates.
Figure 4
Figure 4
Effects of prostratin on CDK9 kinase activity. The amount of cell extracts from DMSO and prostratin-treated cells used in immunoprecipitations were adjusted to precipitate equivalent amount of CDK9 using antiserum against CDK9. Immunoprecipitates were subjected to CTD kinase assays to examine relative kinase activities. Products of kinase assay were examined on SDS-polyacrylamide gels, and the CTD substrate hyperphosphorylated form (CTDo) and hypophosphorylated form (CTDa) are shown at the top. PHA-treated cells were used as a positive control. A portion of immunoprecipitates were analyzed in immunoblots shown at the bottom to confirm that equivalent amounts of CDK9 were precipitated; Cyclin T1 levels present in immunoblots were also evaluated by immunoblots.
Figure 5
Figure 5
Cyclin T1/P-TEFb is likely to be important for prostratin stimulation of HIV reporter virus expression. Resting CD4+ T cells were infected with wild type HIV NL4-3-Luc-Tat+ luciferase reporter virus (A) or NL4-3-Luc-Tat- (B), a mutant reporter virus with a non-functional Tat. After overnight incubation, cells were washed and cultured with DMSO or prostratin. Flavopiridol, a selective P-TEFb inhibitor, was added as indicated simultaneously with prostratin. Cells were harvest 48 hours after prostratin/flavopiridol treatment and reporter plasmid expression was examined by luciferase assays. Dashed lines indicate the background signal in the luciferase assay (~0.025) as determined from the signal in uninfected cell extract.
Figure 6
Figure 6
An aliquot of RNA from the three donors for microarray analysis were reverse transcribed for quantitative real-time PCR assays for Cyclin T1, CDK9, and control α-tubulin mRNAs. Two sets of primers were designed to amplify different regions of Cyclin T1 mRNA (see Methods). Fold-change was calculated as the change in transcript levels in prostratin-treated cells relative to DMSO-treated cells after normalization to α-Tubulin levels.
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