Gene expression profile of HIV-1 Tat expressing cells: a close interplay between proliferative and differentiation signals

Abstract

Background: Expression profiling holds great promise for rapid host genome functional analysis. It is plausible that host expression profiling in an infection could serve as a universal phenotype in virally infected cells. Here, we describe the effect of one of the most critical viral activators, Tat, in HIV-1 infected and Tat expressing cells. We utilized microarray analysis from uninfected, latently HIV-1 infected cells, as well as cells that express Tat, to decipher some of the cellular changes associated with this viral activator.

Results: Utilizing uninfected, HIV-1 latently infected cells, and Tat expressing cells, we observed that most of the cellular host genes in Tat expressing cells were down-regulated. The down-regulation in Tat expressing cells is most apparent on cellular receptors that have intrinsic receptor tyrosine kinase (RTK) activity and signal transduction members that mediate RTK function, including Ras-Raf-MEK pathway. Co-activators of transcription, such as p300/CBP and SRC-1, which mediate gene expression related to hormone receptor genes, were also found to be down-regulated. Down-regulation of receptors may allow latent HIV-1 infected cells to either hide from the immune system or avoid extracellular differentiation signals. Some of the genes that were up-regulated included co-receptors for HIV-1 entry, translation machinery, and cell cycle regulatory proteins.

Conclusions: We have demonstrated, through a microarray approach, that HIV-1 Tat is able to regulate many cellular genes that are involved in cell signaling, translation and ultimately control the host proliferative and differentiation signals.

Figure 1

Gene expression analysis of uninfected and HIV-1 infected cells. A) Both CEM (uninfected) and ACH₂ (latently HIV-1 infected) cells were grown to mid-log phase of growth and processed for RNA isolation. Total RNA was labeled with ³²P-ATP and hybridized to human cDNA filters (Clontech, 588 genes). Blots were hybridized overnight, washed the next day, and exposed to a PhosphorImager cassette. B) Same as in panel A, except all the 588 genes were plotted as fold change vs. gene index (individual genes). Examples of three genes such as prothymosin-α, C-myc, and p21/Waf1, is shown on the diagram. C) Northern blot analysis of prothymosin-α, C-myc, p21/Waf1 and ubiquitin using 10 μg of total RNA, separated on 0.8% formaldehyde gel, and probed with 40 mer anti-sense oligos against respective genes. Bottom of panel C, last insert shows RNA ethidium bromide stain from CEM and ACH₂ cells.

Figure 2

Gene expression analysis from Tat expressing cells. Both H9 and H9/Tat cells were grown to mid-log phase of growth, processed for RNA preparation, and labeled with Tyramide linked Cy-5 (H9) or Cy-3 (H9/TAT). Labeled RNAs were hybridized simultaneously to a glass slide containing 2400 known cDNA genes (NEN Inc.). All genes were plotted similar to Figure 1 and genes above & below 1 fold change were plotted (on the right hand side) to show activation and suppression of all genes.

Figure 3

Functional and physical confirmation of few genes from Tat expressing cells. A) Infection of mono- and T-tropic viruses into Tat expressing cells. Both HXB-2 and BaL strains of HIV-1 were infected into H9 and H9/TAT cells. Supernatants were collected every 3 days and further processed for p24 gag ELISA assays. B) Western blot analysis from H9 and H9/TAT expressing cells using co-activators (SRC-1), DNA damage (DNA-PK), activator (p300), and signal transduction (Ras, RAF, and MAPK) antibodies. TBP stands for TATA binding protein, which served as positive control in western blots. C) Western blot analysis from CEM (uninfected T-cell), ACH2 (infected T-cell), U937 (uninfected promonocytic), U1 (infected promonocytic), and PBMCs treated with purified Tat wild type or K41A mutant (100 ng/ml) proteins. Fifty microgram of whole cells lysates were processed for western blots with anti-DNA-PK, p300, RAF, and TBP antibodies.

Figure 4

Synthesis of IL-8 in Tat expressing cells. Hela cells (pCEP4, and eTat) were either unblocked (unt), or blocked with hydroxyurea (Hu) (2 mM) for 14 h, released, washed twice with phosphate-buffered saline (PBS) and subsequent addition of complete medium [103]. Supernatants were collected at 9 hrs after release for ELISA. All remaining suspension cells were treated with 1% serum for 48 hrs prior to addition of Hu. PHA-activated PBMCs were kept in culture for 2 days prior to addition of Tat protein. Approximately 5 × 10⁶ PBMCs were used for treatment with either wild type or K41A Tat mutant (100 ng/ml) proteins. After an initial incubation for one hr with Tat proteins, cells were washed and cultured in complete media for 24 hrs, prior to IL-8 ELISA.

Figure 5

Predictive model for control of gene expression and signal transduction by constitutive Tat expressing cells. Down-regulation of SWI/SNF components such as BAF 170 and 60 along with coactivators CBP/p300 and SRC-1 may down-regulate a subset of cellular genes that depend on chromatin remodeling and/or co-activator function for their gene expression. Such genes depend on the presence of ligand receptors that require either both SRC-1 and p300 or individual co-activator for their activity.

Figure 6

Proposed model for changes in signal transduction. A) Down-regulation of receptor tyrosine kinases (RTK) by Tat which modulates the phosphorylation and transcription of downstream effectors such as Ras, Raf, MEK, MAPK, and control transcription factor phosphorylation. B) Role of Tat in the increase of genes necessary for proliferation, such as Cdc2, Cdc37, and Prothymosin α, and down-regulation of differentiation genes, such as receptors, co-receptors, and signal transduction genes.