Scientific Report 2007

Molecular and Experimental Medicine

Division of Rheumatology Research

Transcriptional Modulation of HIV Type 1 Expression and Latency

K.V. Morris

RNA-mediated transcriptional gene silencing occurs in human cells through the antisense strand of small interfering RNAs and the protein Argonaute 1. The use of RNA to regulate gene expression represents a shift in the current understanding of gene regulation. We wish to understand the mechanism of RNA-mediated regulation of gene expression in human cells and to use the mechanism therapeutically to treat infection with HIV type 1 (HIV-1). We have learned that small interfering RNAs targeted to promoter regions can direct silent-state epigenetic modifications and that DNA methyltransferase 3A and a low-copy RNA spanning the targeted promoter are involved. However, until recently, a biological example of RNA-mediated transcriptional regulation had not been reported. We hypothesized that an HIV-1–expressed microRNA (miR-N367) is used by the virus to modulate the establishment and maintenance of latency (Fig. 1).

Fig. 1. A putative pathway for the establishment and reactivation of HIV latency. A, The integrated provirus could produce miRNAs either within the viral genome (miR-N367) or from the unelongated transactivating response (TAR) element. B, The miRNAs could be processed by the enzyme Dicer and the TAR RNA binding protein (TRBP) and thus be capable of targeting the LTR in a transcriptional fashion. C, The targeting of the LTR by small interfering RNAs can lead to H3K9me+ and subsequent gene silencing (i.e., induction of a latent state). D, When the T-cell receptor (TCR) is stimulated by IL-2 or TNF-α, the nuclear factors NF-AT and NF-κB can become activated. NF-κB can activate BRG-1, a subunit of a complex that can function with other factors to remodel the chromatin at the viral LTR and produce a favorable environment for transcription. Moreover, NF-AT and NF-κB can activate the LTR and induce expression of the viral transactivator Tat, which when present in high concentrations results in feedback to enhance the elongation of viral transcription from the TAR element, as well as inhibit Dicer by competitive interactions with TRBP. E, The result would be a reversal of the silent-state H3K9 methylation (due to the inhibition of Dicer/TRBP by Tat); an induction of viral expression (due to NF-AT, NF-κB, and Tat), which leads to H3K4 and H3K36 methylation; and a transcriptional active chromatin state. This process ultimately results in the production of full-length viral products and the emergence of HIV-1 from latency. Reprinted from Weinberg, M.S., Morris, K.V. Are viral-encoded microRNAs mediating latent HIV-1 infection? DNA Cell Biol. 25:223, 2006.

To more clearly assess the role that miR-N367 might be playing in the establishment or maintenance of HIV-1 latency, we generated phosphorothioate oligonucleotides (ODNs) targeted to either the sense or the antisense strand of miR-N367 (Fig. 2A). The ODNs were transfected into ACH-2 cells, which contain a stable integrated HIV-1 that expresses low levels of miR-N367. Despite a low transfection efficiency, a noticeable and reproducible suppressive trend that would be predicted on the basis of the particular miR-N367 ODN used (Fig. 2A, sense vs antisense) was observed (Fig. 2B). To determine if the observed effects on viral RNA expression were the result of epigenetic modifications at the putative miR-N367 target site in the HIV-1 long terminal repeat (LTR) of ACH-2 cells, we repeated the experiments and assayed the viral RNA at 96 hours. The ODN-treated cultures showed a similar trend in viral RNA expression (Fig. 2C). When these cultures were assessed by using chromatin immunoprecipitation, a distinct silent-state epigenetic methyl-mark (H3K27^me3+) was observed at the miR-N367 target site in the HIV-1 LTR in cultures treated with the sense ODN and not in those treated with the antisense ODN (Fig. 2D).

Fig. 2. Phosphorothioate targeting of miR-N367. A, The miR-N367 was generated and processed by Dicer as depicted. In order to determine if miR-N367 is active in ACH-2 cells, antisense ODNs were generated to either mimic miR-N367 (miR-N367S-ODN) or target and degrade miR-N367 (miR-N367AS-ODN) in an RNAse H–dependent manner. B, The effects of miR-N367 sense and antisense ODNs relative to the control R61 as ODN were determined by transfecting the ODNs into ACH-2 cells in triplicate, and viral RNA was assessed 24 and 48 hours later. Triplicate transfections are shown with the standard errors of the means. C, In order to determine the longer term effects of ODN treatment, the respective ODNs were transfected into ACH-2 cells in quadruplicate, and viral RNA was assessed 96 hours later. D, The miR-N367 target site in the HIV-1b LTR of ACH-2 ODN-treated cultures was also assessed by using chromatin immunoprecipitation 96 hours after transfection to determine methylation of histone 3 and lysine 27 (H3K27me3+). Error bars (C and D) indicate the standard deviations.

These data suggest that an endogenous mechanism involving the miR-N367 sequence is used by HIV-1 to regulate viral expression and latency in an epigenetic manner in ACH-2 cells. The knowledge gained of the mechanism of HIV-1 latency may one day be advantageous in therapeutic strategies to treat HIV-1 infections.

Publications

Han, J., Kim, D., Morris, K.V. Promoter-associated RNA is required for RNA-directed transcriptional gene silencing in human cells. Proc. Natl. Acad. Sci. U. S. A. 104:12422, 2007.

Kim, D.H., Villeneuve, L.M., Morris, K.V., Rossi, J.J. Argonaute-1 directs siRNA-mediated transcriptional gene silencing in human cells. Nat. Struct. Mol. Biol. 13:793, 2006.