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microRNA machinery is an integral component of drug-induced transcription inhibition in HIV-1 infection.

Carpio L, Klase Z, Coley W, Guendel I, Choi S, Van Duyne R, Narayanan A, Kehn-Hall K, Meijer L, Kashanchi F - J RNAi Gene Silencing (2010)

Bottom Line: Also, after transfection with HIV-1 clone (pNL4.3), CR8 and CR8#13 derivatives were shown to be more effective viral transcription inhibitors in cell lines that contained Dicer (T-cells) as compared to Dicer deficient lines (monocytes).In conclusion, our results indicate that viral microRNA, specifically the TAR microRNA produced from the HIV-1 LTR is responsible for maintaining latent infections by manipulating host cell mechanisms to limit transcription from the viral LTR promoter.With the microRNA machinery present, cdk inhibitors are able to significantly increase the amount of TAR microRNA, leading to downregulation of viral LTR transcription.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Disease, 10900 University Blvd MS 1H8, George Mason University, Manassas, VA 20110, USA.

ABSTRACT
RNA interference plays a significant role in manipulating cellular and viral mechanisms to maintain latency during HIV-1 infection. HIV-1 produces several microRNAs including one from the TAR element which alter the host's response to infection. Since cyclin/cdk complexes are important for viral transcription, these studies focus on the possible cdk inhibitors that inhibit viral transcription, without affecting normal cellular mechanisms. Roscovitine and Flavopiridol are well-studied cdk inhibitors that are effective at suppressing their target cdks at a low IC50. These cdk inhibitors and possibly future generations of drugs are affected by microRNA mechanisms. From our studies, we developed a third generation derivative called CR8#13. In cells that lack Dicer there was a higher level of basal viral LTR-reporter transcription. When drugs, specifically Flavopiridol and CR8#13 were added, the transcriptional inhibition of the LTR was less potent in cells that lacked Dicer. Also, after transfection with HIV-1 clone (pNL4.3), CR8 and CR8#13 derivatives were shown to be more effective viral transcription inhibitors in cell lines that contained Dicer (T-cells) as compared to Dicer deficient lines (monocytes). We next asked whether the addition of CR8 or CR8#13 could possibly increase levels of TAR microRNA in HIV-1 LTR containing cells. We demonstrate that the 3'TAR microRNA is produced in higher amounts after drug treatment, resulting in microRNA recruitment to the LTR. MicroRNA recruitment results in chromatin alteration, changes in Pol II phosphorylation and viral transcription inhibition. In conclusion, our results indicate that viral microRNA, specifically the TAR microRNA produced from the HIV-1 LTR is responsible for maintaining latent infections by manipulating host cell mechanisms to limit transcription from the viral LTR promoter. With the microRNA machinery present, cdk inhibitors are able to significantly increase the amount of TAR microRNA, leading to downregulation of viral LTR transcription.

No MeSH data available.


Related in: MedlinePlus

HIV microRNAs specifically induce formation of repressive chromatin markers on the HIV-1 LTR. A. Model of TZM-bl cells suppression and activation. Trichostatin-A (TSA), a widely used HDAC inhibitor were used to activate the integrated HIV-1 LTR-Luc transcription in TZM-bl and abolish the repressive heterochromatic state. Seven days post treatment of TSA, the TZM-bl were transfected with the TAR microRNA. B. ChIP assays were performed with the TSA treated TZM-bls. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. MicroRNA machinery (Ago2), histone methyltransferases (Suv39H1), chromatin remodeling markers (SETDB1, SETMAR), and transcription repressors (PIAS?) were downregulated after TSA treatment on the integrated HIV-LTR. C. ChIP assays were performed on several markers of chromatin repression (HDAC1) and microRNA machinery (Ago2) in TZMb1 cells. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. Lane 1 shows basal levels of repressive markers on the HIV-1 LTR. Lane 2 shows that seven days of TSA treatment removes the markers of repressive chromatin. Lane 3 shows that the TAR-D mutant does not initiate a recruitment of repressive enzymes. Lane 4 demonstrates that addition of the WT-TAR molecule is sufficient to recruit Ago2 and HDAC1 back to the HIV-1 LTR region. D. TZM-bl cells were treated with Flavopiridol (50nM), CR8 (100nM) and CR8#13 (100nM) after 7-day TSA treatment. RNA was extracted 48hrs-post drug treatment. 500ng of RNA from the microRNA-enriched fraction was used to generate cDNA using the Quantimir kit (SBI) in order to poly-adenylate small RNA species. RT reactions were performed followed by PCR in which a universal reverse primer was provided by the manufacturer. Specific microRNA forward primers are identical in sequence to the microRNA of interest. PCR products corresponding to the amplified microRNAs were separated in a 3.5% (w/v) agarose gel. The PCR products are at around 67bp as compared with the Fermentas 1kb DNA Plus Ladder. Increased levels of 3'TAR microRNA were produced post CR8#13 treatment. E. ChIP assays were performed on several markers of chromatin repression (HDAC1, Suv39H1) and microRNA machinery (Ago2) in TZMb1 cells. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. Lane 1 indicates basal levels of repressive markers on the HIV-1 LTR. Lane 2 indicates that seven days of TSA treatment removes the markers of repressive chromatin and Lane 3 shows results that the CR8#13 treatment is sufficient to recruit HDAC1, Ago2 and Suv39H1 back to the HIV-1 LTR region. F. Luciferase assays were performed on the cells used in Figure 6E. Luciferase activity increased with TSA treatment and then decreased post-CR8#13 treatment.
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Figure 6: HIV microRNAs specifically induce formation of repressive chromatin markers on the HIV-1 LTR. A. Model of TZM-bl cells suppression and activation. Trichostatin-A (TSA), a widely used HDAC inhibitor were used to activate the integrated HIV-1 LTR-Luc transcription in TZM-bl and abolish the repressive heterochromatic state. Seven days post treatment of TSA, the TZM-bl were transfected with the TAR microRNA. B. ChIP assays were performed with the TSA treated TZM-bls. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. MicroRNA machinery (Ago2), histone methyltransferases (Suv39H1), chromatin remodeling markers (SETDB1, SETMAR), and transcription repressors (PIAS?) were downregulated after TSA treatment on the integrated HIV-LTR. C. ChIP assays were performed on several markers of chromatin repression (HDAC1) and microRNA machinery (Ago2) in TZMb1 cells. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. Lane 1 shows basal levels of repressive markers on the HIV-1 LTR. Lane 2 shows that seven days of TSA treatment removes the markers of repressive chromatin. Lane 3 shows that the TAR-D mutant does not initiate a recruitment of repressive enzymes. Lane 4 demonstrates that addition of the WT-TAR molecule is sufficient to recruit Ago2 and HDAC1 back to the HIV-1 LTR region. D. TZM-bl cells were treated with Flavopiridol (50nM), CR8 (100nM) and CR8#13 (100nM) after 7-day TSA treatment. RNA was extracted 48hrs-post drug treatment. 500ng of RNA from the microRNA-enriched fraction was used to generate cDNA using the Quantimir kit (SBI) in order to poly-adenylate small RNA species. RT reactions were performed followed by PCR in which a universal reverse primer was provided by the manufacturer. Specific microRNA forward primers are identical in sequence to the microRNA of interest. PCR products corresponding to the amplified microRNAs were separated in a 3.5% (w/v) agarose gel. The PCR products are at around 67bp as compared with the Fermentas 1kb DNA Plus Ladder. Increased levels of 3'TAR microRNA were produced post CR8#13 treatment. E. ChIP assays were performed on several markers of chromatin repression (HDAC1, Suv39H1) and microRNA machinery (Ago2) in TZMb1 cells. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. Lane 1 indicates basal levels of repressive markers on the HIV-1 LTR. Lane 2 indicates that seven days of TSA treatment removes the markers of repressive chromatin and Lane 3 shows results that the CR8#13 treatment is sufficient to recruit HDAC1, Ago2 and Suv39H1 back to the HIV-1 LTR region. F. Luciferase assays were performed on the cells used in Figure 6E. Luciferase activity increased with TSA treatment and then decreased post-CR8#13 treatment.

Mentions: Recent studies have suggested that TAR derived microRNA may have effects on chromatin structure (Klase et al, 2009). To test the ability of TAR derived microRNA to direct chromatin remodeling at the viral LTR, chromatin immunoprecipitation (ChIP) assays were performed to examine the recruitment of factors to the HIV-1 LTR. One such factor, HDAC-1, is a histone deacetylase shown to be involved in silencing of HIV-1 promoter (Easow et al, 2007). TZM-bl cells carrying integrated HIV-1 LTR were utilized for these experiments (Figure 6A). In these cells LTR is already silenced. Previous work in the field has shown that chronic treatment of HeLa cells with the HDAC inhibitor TSA abolishes the heterochromatic state (Cobb et al, 2005). We therefore treated the cells for seven days with a sub-lethal dose of the HDAC inhibitor TSA and assayed for factor occupancy on the promoter. Chromatin changes were verified by performing ChIP assays before and after TSA treatment using antibodies specific for inhibitory factors including the components of the RNAi machinery (Figure 6B). The rationale here was to see if silenced state (absence of TSA) would have differing factor occupancy as compared to active state (presence of TSA). The results in panel B established that the RNAi protein Argonaute and the histone modifiers Suv39H1 and SETDB1 are present at the latent LTR and are removed once they are treated with TSA.


microRNA machinery is an integral component of drug-induced transcription inhibition in HIV-1 infection.

Carpio L, Klase Z, Coley W, Guendel I, Choi S, Van Duyne R, Narayanan A, Kehn-Hall K, Meijer L, Kashanchi F - J RNAi Gene Silencing (2010)

HIV microRNAs specifically induce formation of repressive chromatin markers on the HIV-1 LTR. A. Model of TZM-bl cells suppression and activation. Trichostatin-A (TSA), a widely used HDAC inhibitor were used to activate the integrated HIV-1 LTR-Luc transcription in TZM-bl and abolish the repressive heterochromatic state. Seven days post treatment of TSA, the TZM-bl were transfected with the TAR microRNA. B. ChIP assays were performed with the TSA treated TZM-bls. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. MicroRNA machinery (Ago2), histone methyltransferases (Suv39H1), chromatin remodeling markers (SETDB1, SETMAR), and transcription repressors (PIAS?) were downregulated after TSA treatment on the integrated HIV-LTR. C. ChIP assays were performed on several markers of chromatin repression (HDAC1) and microRNA machinery (Ago2) in TZMb1 cells. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. Lane 1 shows basal levels of repressive markers on the HIV-1 LTR. Lane 2 shows that seven days of TSA treatment removes the markers of repressive chromatin. Lane 3 shows that the TAR-D mutant does not initiate a recruitment of repressive enzymes. Lane 4 demonstrates that addition of the WT-TAR molecule is sufficient to recruit Ago2 and HDAC1 back to the HIV-1 LTR region. D. TZM-bl cells were treated with Flavopiridol (50nM), CR8 (100nM) and CR8#13 (100nM) after 7-day TSA treatment. RNA was extracted 48hrs-post drug treatment. 500ng of RNA from the microRNA-enriched fraction was used to generate cDNA using the Quantimir kit (SBI) in order to poly-adenylate small RNA species. RT reactions were performed followed by PCR in which a universal reverse primer was provided by the manufacturer. Specific microRNA forward primers are identical in sequence to the microRNA of interest. PCR products corresponding to the amplified microRNAs were separated in a 3.5% (w/v) agarose gel. The PCR products are at around 67bp as compared with the Fermentas 1kb DNA Plus Ladder. Increased levels of 3'TAR microRNA were produced post CR8#13 treatment. E. ChIP assays were performed on several markers of chromatin repression (HDAC1, Suv39H1) and microRNA machinery (Ago2) in TZMb1 cells. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. Lane 1 indicates basal levels of repressive markers on the HIV-1 LTR. Lane 2 indicates that seven days of TSA treatment removes the markers of repressive chromatin and Lane 3 shows results that the CR8#13 treatment is sufficient to recruit HDAC1, Ago2 and Suv39H1 back to the HIV-1 LTR region. F. Luciferase assays were performed on the cells used in Figure 6E. Luciferase activity increased with TSA treatment and then decreased post-CR8#13 treatment.
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Figure 6: HIV microRNAs specifically induce formation of repressive chromatin markers on the HIV-1 LTR. A. Model of TZM-bl cells suppression and activation. Trichostatin-A (TSA), a widely used HDAC inhibitor were used to activate the integrated HIV-1 LTR-Luc transcription in TZM-bl and abolish the repressive heterochromatic state. Seven days post treatment of TSA, the TZM-bl were transfected with the TAR microRNA. B. ChIP assays were performed with the TSA treated TZM-bls. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. MicroRNA machinery (Ago2), histone methyltransferases (Suv39H1), chromatin remodeling markers (SETDB1, SETMAR), and transcription repressors (PIAS?) were downregulated after TSA treatment on the integrated HIV-LTR. C. ChIP assays were performed on several markers of chromatin repression (HDAC1) and microRNA machinery (Ago2) in TZMb1 cells. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. Lane 1 shows basal levels of repressive markers on the HIV-1 LTR. Lane 2 shows that seven days of TSA treatment removes the markers of repressive chromatin. Lane 3 shows that the TAR-D mutant does not initiate a recruitment of repressive enzymes. Lane 4 demonstrates that addition of the WT-TAR molecule is sufficient to recruit Ago2 and HDAC1 back to the HIV-1 LTR region. D. TZM-bl cells were treated with Flavopiridol (50nM), CR8 (100nM) and CR8#13 (100nM) after 7-day TSA treatment. RNA was extracted 48hrs-post drug treatment. 500ng of RNA from the microRNA-enriched fraction was used to generate cDNA using the Quantimir kit (SBI) in order to poly-adenylate small RNA species. RT reactions were performed followed by PCR in which a universal reverse primer was provided by the manufacturer. Specific microRNA forward primers are identical in sequence to the microRNA of interest. PCR products corresponding to the amplified microRNAs were separated in a 3.5% (w/v) agarose gel. The PCR products are at around 67bp as compared with the Fermentas 1kb DNA Plus Ladder. Increased levels of 3'TAR microRNA were produced post CR8#13 treatment. E. ChIP assays were performed on several markers of chromatin repression (HDAC1, Suv39H1) and microRNA machinery (Ago2) in TZMb1 cells. Primers specific for the HIV-1 LTR were used to amplify DNA that was precipitated with each antibody. Lane 1 indicates basal levels of repressive markers on the HIV-1 LTR. Lane 2 indicates that seven days of TSA treatment removes the markers of repressive chromatin and Lane 3 shows results that the CR8#13 treatment is sufficient to recruit HDAC1, Ago2 and Suv39H1 back to the HIV-1 LTR region. F. Luciferase assays were performed on the cells used in Figure 6E. Luciferase activity increased with TSA treatment and then decreased post-CR8#13 treatment.
Mentions: Recent studies have suggested that TAR derived microRNA may have effects on chromatin structure (Klase et al, 2009). To test the ability of TAR derived microRNA to direct chromatin remodeling at the viral LTR, chromatin immunoprecipitation (ChIP) assays were performed to examine the recruitment of factors to the HIV-1 LTR. One such factor, HDAC-1, is a histone deacetylase shown to be involved in silencing of HIV-1 promoter (Easow et al, 2007). TZM-bl cells carrying integrated HIV-1 LTR were utilized for these experiments (Figure 6A). In these cells LTR is already silenced. Previous work in the field has shown that chronic treatment of HeLa cells with the HDAC inhibitor TSA abolishes the heterochromatic state (Cobb et al, 2005). We therefore treated the cells for seven days with a sub-lethal dose of the HDAC inhibitor TSA and assayed for factor occupancy on the promoter. Chromatin changes were verified by performing ChIP assays before and after TSA treatment using antibodies specific for inhibitory factors including the components of the RNAi machinery (Figure 6B). The rationale here was to see if silenced state (absence of TSA) would have differing factor occupancy as compared to active state (presence of TSA). The results in panel B established that the RNAi protein Argonaute and the histone modifiers Suv39H1 and SETDB1 are present at the latent LTR and are removed once they are treated with TSA.

Bottom Line: Also, after transfection with HIV-1 clone (pNL4.3), CR8 and CR8#13 derivatives were shown to be more effective viral transcription inhibitors in cell lines that contained Dicer (T-cells) as compared to Dicer deficient lines (monocytes).In conclusion, our results indicate that viral microRNA, specifically the TAR microRNA produced from the HIV-1 LTR is responsible for maintaining latent infections by manipulating host cell mechanisms to limit transcription from the viral LTR promoter.With the microRNA machinery present, cdk inhibitors are able to significantly increase the amount of TAR microRNA, leading to downregulation of viral LTR transcription.

View Article: PubMed Central - PubMed

Affiliation: National Center for Biodefense and Infectious Disease, 10900 University Blvd MS 1H8, George Mason University, Manassas, VA 20110, USA.

ABSTRACT
RNA interference plays a significant role in manipulating cellular and viral mechanisms to maintain latency during HIV-1 infection. HIV-1 produces several microRNAs including one from the TAR element which alter the host's response to infection. Since cyclin/cdk complexes are important for viral transcription, these studies focus on the possible cdk inhibitors that inhibit viral transcription, without affecting normal cellular mechanisms. Roscovitine and Flavopiridol are well-studied cdk inhibitors that are effective at suppressing their target cdks at a low IC50. These cdk inhibitors and possibly future generations of drugs are affected by microRNA mechanisms. From our studies, we developed a third generation derivative called CR8#13. In cells that lack Dicer there was a higher level of basal viral LTR-reporter transcription. When drugs, specifically Flavopiridol and CR8#13 were added, the transcriptional inhibition of the LTR was less potent in cells that lacked Dicer. Also, after transfection with HIV-1 clone (pNL4.3), CR8 and CR8#13 derivatives were shown to be more effective viral transcription inhibitors in cell lines that contained Dicer (T-cells) as compared to Dicer deficient lines (monocytes). We next asked whether the addition of CR8 or CR8#13 could possibly increase levels of TAR microRNA in HIV-1 LTR containing cells. We demonstrate that the 3'TAR microRNA is produced in higher amounts after drug treatment, resulting in microRNA recruitment to the LTR. MicroRNA recruitment results in chromatin alteration, changes in Pol II phosphorylation and viral transcription inhibition. In conclusion, our results indicate that viral microRNA, specifically the TAR microRNA produced from the HIV-1 LTR is responsible for maintaining latent infections by manipulating host cell mechanisms to limit transcription from the viral LTR promoter. With the microRNA machinery present, cdk inhibitors are able to significantly increase the amount of TAR microRNA, leading to downregulation of viral LTR transcription.

No MeSH data available.


Related in: MedlinePlus