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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

Increased production of TAR microRNA due to drug treatment. A. Model of the effect of RNA polymerase II phosphorylation on transcription. RNA polymerase II CTD is hypo-phosphorylated at the initiation complex; Ser5 is only phosphorylated at the promoter clearance stage; and Ser2 is mostly phosphorylated at the elongation phase. HIV-1 genome is unique in that it contains both Ser2 and Ser5 phosphorylation at the elongation stage (Zhou et al, 2004). Phosphorylation of Ser2 and Ser5 could be seen by multiple cyclin/cdk complexes. B. Ten micrograms of total RNA from TNF treated CEM (lane 1) and TNF treated ACH2 cells (lanes 2-6) were hybridized to a radiolabeled TAR 5' probe and then treated with RNase A. Arrows indicate the probe protected by TAR at 27 nucleotides and the probe protected by a TAR miRNA at approximately 22nt. Cyc202 concentration at 500nM, CR8 at 100nM, CR8#13 at 50nM, and Flavopiridol at 50nM were used for these experiments. C. ACH2 cells were treated with Flavopiridol (50nM), CR8 (100nM) and CR8#13 (100nM). 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). RT reactions are performed followed by PCR in which a universal reverse primer is 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 resolved 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 amounts of 3' and 5' TAR microRNA were observed post drug treatment. D. Total RNA (1ug) from each samples was separated in a 1% (w/v) agarose gel. The location of both 18S and 28S are shown. E. RT assay was performed to detect viral levels in ACH2 cells after TNF and drug treatments. ACH2 cells were treated with Flavopiridol (50nM), Cyc202 (500nM), CR8 (100nM) and CR8#13(100nM). Supernatents were collected 48hrs later and used for RT assay. TNF treatment significantly increased RT levels in ACH2 cells and drug treatment was able to decrease RT levels.
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Figure 5: Increased production of TAR microRNA due to drug treatment. A. Model of the effect of RNA polymerase II phosphorylation on transcription. RNA polymerase II CTD is hypo-phosphorylated at the initiation complex; Ser5 is only phosphorylated at the promoter clearance stage; and Ser2 is mostly phosphorylated at the elongation phase. HIV-1 genome is unique in that it contains both Ser2 and Ser5 phosphorylation at the elongation stage (Zhou et al, 2004). Phosphorylation of Ser2 and Ser5 could be seen by multiple cyclin/cdk complexes. B. Ten micrograms of total RNA from TNF treated CEM (lane 1) and TNF treated ACH2 cells (lanes 2-6) were hybridized to a radiolabeled TAR 5' probe and then treated with RNase A. Arrows indicate the probe protected by TAR at 27 nucleotides and the probe protected by a TAR miRNA at approximately 22nt. Cyc202 concentration at 500nM, CR8 at 100nM, CR8#13 at 50nM, and Flavopiridol at 50nM were used for these experiments. C. ACH2 cells were treated with Flavopiridol (50nM), CR8 (100nM) and CR8#13 (100nM). 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). RT reactions are performed followed by PCR in which a universal reverse primer is 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 resolved 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 amounts of 3' and 5' TAR microRNA were observed post drug treatment. D. Total RNA (1ug) from each samples was separated in a 1% (w/v) agarose gel. The location of both 18S and 28S are shown. E. RT assay was performed to detect viral levels in ACH2 cells after TNF and drug treatments. ACH2 cells were treated with Flavopiridol (50nM), Cyc202 (500nM), CR8 (100nM) and CR8#13(100nM). Supernatents were collected 48hrs later and used for RT assay. TNF treatment significantly increased RT levels in ACH2 cells and drug treatment was able to decrease RT levels.

Mentions: Latently infected cells produce a high level of short, abortive RNA transcripts only 50-100nt in length, that contain the HIV TAR stemloop (Adams et al, 1994). It has been shown that TAR serves as a substrate for Dicer resulting in short 21-22 RNA molecules capable of silencing HIV-1 transcription (Klase et al, 2007). Since these TAR-containing short transcripts are the dominant HIV-1 RNA produced in appreciable quantities during latency, it is possible that these RNA molecules could suppress viral gene expression. Our current studies point to cdk inhibitors functionally interacting with TAR microRNA, which affects RNA polymerase II processing, and may increase the efficacy of the drugs' inhibition on the HIV-1 promoter. As seen in Figure 5A, a model for transcription argues that RNA polymerase II is phosphorylated at Ser5 at the initiation complex and Ser2 on the elongating complex. RNA polymerase II associated with HIV-1 promoter is, however, phosphorylated on both Ser2 and 5 in the presence of Tat (Zhou et al, 2004). In order to ascertain whether or not cells treated with CDK inhibitor produce additional TAR derived miRNA, we utilized RNase protection assays to detect small RNA fragments corresponding to TAR sequence (Klase et al, 2007). Briefly, a probe complementary to the entire length of the 5' portion of the TAR stem loop was designed, which would detect the generation of ~21nt RNAs from any position within that sequence. The results were considered positive if the 32nt probe was cleaved to ~21nt, indicating protection by a microRNA. The latently infected T-cell clone, ACH2, was treated with TNF (for viral induction) followed by treatment with Cyc202, CR8, and CR8#13. Thirty micrograms of total RNA from each condition was used for RPA analysis to detect the presence of the 5' TAR miRNA. Higher levels of TAR and miTAR RNA were observed in CR8#13 treated cells as compared to Flavopiridol treatment (Figure 5B, Lanes 5 and 6). This lead to further experimentation to determine levels of each of the microRNA produced from TAR region (3'TAR stem and 5'TAR stem) in presence of these drugs.


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)

Increased production of TAR microRNA due to drug treatment. A. Model of the effect of RNA polymerase II phosphorylation on transcription. RNA polymerase II CTD is hypo-phosphorylated at the initiation complex; Ser5 is only phosphorylated at the promoter clearance stage; and Ser2 is mostly phosphorylated at the elongation phase. HIV-1 genome is unique in that it contains both Ser2 and Ser5 phosphorylation at the elongation stage (Zhou et al, 2004). Phosphorylation of Ser2 and Ser5 could be seen by multiple cyclin/cdk complexes. B. Ten micrograms of total RNA from TNF treated CEM (lane 1) and TNF treated ACH2 cells (lanes 2-6) were hybridized to a radiolabeled TAR 5' probe and then treated with RNase A. Arrows indicate the probe protected by TAR at 27 nucleotides and the probe protected by a TAR miRNA at approximately 22nt. Cyc202 concentration at 500nM, CR8 at 100nM, CR8#13 at 50nM, and Flavopiridol at 50nM were used for these experiments. C. ACH2 cells were treated with Flavopiridol (50nM), CR8 (100nM) and CR8#13 (100nM). 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). RT reactions are performed followed by PCR in which a universal reverse primer is 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 resolved 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 amounts of 3' and 5' TAR microRNA were observed post drug treatment. D. Total RNA (1ug) from each samples was separated in a 1% (w/v) agarose gel. The location of both 18S and 28S are shown. E. RT assay was performed to detect viral levels in ACH2 cells after TNF and drug treatments. ACH2 cells were treated with Flavopiridol (50nM), Cyc202 (500nM), CR8 (100nM) and CR8#13(100nM). Supernatents were collected 48hrs later and used for RT assay. TNF treatment significantly increased RT levels in ACH2 cells and drug treatment was able to decrease RT levels.
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Figure 5: Increased production of TAR microRNA due to drug treatment. A. Model of the effect of RNA polymerase II phosphorylation on transcription. RNA polymerase II CTD is hypo-phosphorylated at the initiation complex; Ser5 is only phosphorylated at the promoter clearance stage; and Ser2 is mostly phosphorylated at the elongation phase. HIV-1 genome is unique in that it contains both Ser2 and Ser5 phosphorylation at the elongation stage (Zhou et al, 2004). Phosphorylation of Ser2 and Ser5 could be seen by multiple cyclin/cdk complexes. B. Ten micrograms of total RNA from TNF treated CEM (lane 1) and TNF treated ACH2 cells (lanes 2-6) were hybridized to a radiolabeled TAR 5' probe and then treated with RNase A. Arrows indicate the probe protected by TAR at 27 nucleotides and the probe protected by a TAR miRNA at approximately 22nt. Cyc202 concentration at 500nM, CR8 at 100nM, CR8#13 at 50nM, and Flavopiridol at 50nM were used for these experiments. C. ACH2 cells were treated with Flavopiridol (50nM), CR8 (100nM) and CR8#13 (100nM). 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). RT reactions are performed followed by PCR in which a universal reverse primer is 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 resolved 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 amounts of 3' and 5' TAR microRNA were observed post drug treatment. D. Total RNA (1ug) from each samples was separated in a 1% (w/v) agarose gel. The location of both 18S and 28S are shown. E. RT assay was performed to detect viral levels in ACH2 cells after TNF and drug treatments. ACH2 cells were treated with Flavopiridol (50nM), Cyc202 (500nM), CR8 (100nM) and CR8#13(100nM). Supernatents were collected 48hrs later and used for RT assay. TNF treatment significantly increased RT levels in ACH2 cells and drug treatment was able to decrease RT levels.
Mentions: Latently infected cells produce a high level of short, abortive RNA transcripts only 50-100nt in length, that contain the HIV TAR stemloop (Adams et al, 1994). It has been shown that TAR serves as a substrate for Dicer resulting in short 21-22 RNA molecules capable of silencing HIV-1 transcription (Klase et al, 2007). Since these TAR-containing short transcripts are the dominant HIV-1 RNA produced in appreciable quantities during latency, it is possible that these RNA molecules could suppress viral gene expression. Our current studies point to cdk inhibitors functionally interacting with TAR microRNA, which affects RNA polymerase II processing, and may increase the efficacy of the drugs' inhibition on the HIV-1 promoter. As seen in Figure 5A, a model for transcription argues that RNA polymerase II is phosphorylated at Ser5 at the initiation complex and Ser2 on the elongating complex. RNA polymerase II associated with HIV-1 promoter is, however, phosphorylated on both Ser2 and 5 in the presence of Tat (Zhou et al, 2004). In order to ascertain whether or not cells treated with CDK inhibitor produce additional TAR derived miRNA, we utilized RNase protection assays to detect small RNA fragments corresponding to TAR sequence (Klase et al, 2007). Briefly, a probe complementary to the entire length of the 5' portion of the TAR stem loop was designed, which would detect the generation of ~21nt RNAs from any position within that sequence. The results were considered positive if the 32nt probe was cleaved to ~21nt, indicating protection by a microRNA. The latently infected T-cell clone, ACH2, was treated with TNF (for viral induction) followed by treatment with Cyc202, CR8, and CR8#13. Thirty micrograms of total RNA from each condition was used for RPA analysis to detect the presence of the 5' TAR miRNA. Higher levels of TAR and miTAR RNA were observed in CR8#13 treated cells as compared to Flavopiridol treatment (Figure 5B, Lanes 5 and 6). This lead to further experimentation to determine levels of each of the microRNA produced from TAR region (3'TAR stem and 5'TAR stem) in presence of these drugs.

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