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A novel HIV-1-encoded microRNA enhances its viral replication by targeting the TATA box region.

Zhang Y, Fan M, Geng G, Liu B, Huang Z, Luo H, Zhou J, Guo X, Cai W, Zhang H - Retrovirology (2014)

Bottom Line: It interacts with the TATA box in HIV-1 5' LTR and sequence-specifically activates the viral transcription.In addition, chemically-synthesized small RNAs targeting HIV-1 TATA box activate HIV-1 production from resting CD4+ T cells isolated from HIV-1-infected patients on suppressive highly active antiretroviral therapy (HAART).We have identified a novel HIV-1-encoded miRNA which specifically enhances viral production and provide a specific method to activate HIV-1 latency.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, China. zhangh92@mail.sysu.edu.cn.

ABSTRACT

Background: A lot of microRNAs (miRNAs) derived from viral genomes have been identified. Many of them play various important roles in virus replication and virus-host interaction. Cellular miRNAs have been shown to participate in the regulation of HIV-1 viral replication, while the role of viral-encoded miRNAs in this process is largely unknown.

Results: In this report, through a strategy combining computational prediction and deep sequencing, we identified a novel HIV-1-encoded miRNA, miR-H3. MiR-H3 locates in the mRNA region encoding the active center of reverse transcriptase (RT) and exhibits high sequence conservation among different subtypes of HIV-1 viruses. Overexpression of miR-H3 increases viral production and the mutations in miR-H3 sequence significantly impair the viral replication of wildtype HIV-1 viruses, suggesting that it is a replication-enhancing miRNA. MiR-H3 upregulates HIV-1 RNA transcription and protein expression. A serial deletion assay suggests that miR-H3 targets HIV-1 5' LTR and upregulates the promoter activity. It interacts with the TATA box in HIV-1 5' LTR and sequence-specifically activates the viral transcription. In addition, chemically-synthesized small RNAs targeting HIV-1 TATA box activate HIV-1 production from resting CD4+ T cells isolated from HIV-1-infected patients on suppressive highly active antiretroviral therapy (HAART).

Conclusions: We have identified a novel HIV-1-encoded miRNA which specifically enhances viral production and provide a specific method to activate HIV-1 latency.

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Related in: MedlinePlus

MiR-H3 targets the TATA box in HIV-1 5′ LTR via sequence specific manner. (A) Predicted binding site of miR-H3 in HIV-1 5′ LTR with the TATA box highlighted. The MFE of this interaction is -19.2 kcal/mol. (B) Mutations (labeled in red with underline) were introduced to the promoter, named HIV 5′ LTR mut. The effects of miR-H3 on promoter activity of wildtype or mutated promoter were determined with dual-luciferase assay. The TATA box motif was indicated with a box. (C) Top, the TATA box motif of CMV promoter was replaced by the binding site of miR-H3 on HIV-1 promoter (CMV-HIV_bs mt). The mutated nucleotides are indicated in red with underline. Bottom, the effects of miR-H3 on indicated promoter activities were determined with Dual-Luciferase assay as described above. The TATA box motif was indicated with a box. (D) ChIP assay of HEK239T cells co-transfected with pNL4-3-deltaE-EGFP and pre-miR-H3 or the empty vector with antibody against Pol II or TBP to examine the binding of these general transcription factors to the HIV-1 core promoter region. The normal IgG was used as a control. (E) The effects of miR-H3 on HIV-1 promoter activity without Tat expression. (F) Effect of miR-H3 on the activity of a TAR region deficient (deletion of 470 to 492 bp in HIV-1 5′ LTR) HIV-1 promoter. (G) The effect of miR-H3 on another retrovirus promoter. A RSV promoter-derived luciferase reporter construct was co-transfected with miR-H3 precursor or control vector, and the promoter activity was investigated by Dual-Luciferase assay. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances, n = 3. *p < 0.05, ***p < 0.001.
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Figure 6: MiR-H3 targets the TATA box in HIV-1 5′ LTR via sequence specific manner. (A) Predicted binding site of miR-H3 in HIV-1 5′ LTR with the TATA box highlighted. The MFE of this interaction is -19.2 kcal/mol. (B) Mutations (labeled in red with underline) were introduced to the promoter, named HIV 5′ LTR mut. The effects of miR-H3 on promoter activity of wildtype or mutated promoter were determined with dual-luciferase assay. The TATA box motif was indicated with a box. (C) Top, the TATA box motif of CMV promoter was replaced by the binding site of miR-H3 on HIV-1 promoter (CMV-HIV_bs mt). The mutated nucleotides are indicated in red with underline. Bottom, the effects of miR-H3 on indicated promoter activities were determined with Dual-Luciferase assay as described above. The TATA box motif was indicated with a box. (D) ChIP assay of HEK239T cells co-transfected with pNL4-3-deltaE-EGFP and pre-miR-H3 or the empty vector with antibody against Pol II or TBP to examine the binding of these general transcription factors to the HIV-1 core promoter region. The normal IgG was used as a control. (E) The effects of miR-H3 on HIV-1 promoter activity without Tat expression. (F) Effect of miR-H3 on the activity of a TAR region deficient (deletion of 470 to 492 bp in HIV-1 5′ LTR) HIV-1 promoter. (G) The effect of miR-H3 on another retrovirus promoter. A RSV promoter-derived luciferase reporter construct was co-transfected with miR-H3 precursor or control vector, and the promoter activity was investigated by Dual-Luciferase assay. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances, n = 3. *p < 0.05, ***p < 0.001.

Mentions: With computational prediction, we surprisingly found a putative binding site of miR-H3 which covers the core promoter (the TATA box) in HIV-1 5′ LTR region (Figure 6A). The TATA box motif in HIV-1 5′ LTR starts two nucleotides further upstream and turns to the sequence CATATAA in all subtypes except for subtype E [36]. When mutations were introduced into the binding site in the TATA box region, the enhancement effect on promoter activity by miR-H3 was impaired (Figure 6B), suggesting that the direct binding between the core promoter and miR-H3 is required for its regulation. Furthermore, we mutated the TATA box region of CMV promoter to the same sequence as that of HIV-1 5′ LTR, and found that the transcription of this mutant could also be enhanced by miR-H3 (Figure 6C). These results suggest that the binding site in HIV-1 5′ LTR interacts with miR-H3 sequence-specifically and is required for the promoter activation induced by miR-H3. To investigate whether miR-H3 increases the binding of general transcription factors to the HIV-1 core promoter, we carried out ChIP assay with antibody against the RNA Polymerase II or the TATA box binding protein (TBP). The result suggested miR-H3 enhanced the association of both factors to the HIV-1 core promoter region (Figure 6D). As Tat protein is a very important regulatory factor for HIV-1 transcription, we investigated whether the interaction between Tat protein and TAR motif affected the HIV-1 promoter activation induced by miR-H3. Our data indicated that, in the absence of Tat, miR-H3 still upregulated HIV-1 promoter activity (Figure 6E). Alternatively, although the deletion of TAR significantly affected the promoter activity, the enhancement activity by miR-H3 was not affected (Figure 6F). Furthermore, miR-H3 did not affect the promoter activities of another retrovirus, Rous sarcoma virus (RSV), arguing against a non-specific transcription regulation on retroviruses (Figure 6G).


A novel HIV-1-encoded microRNA enhances its viral replication by targeting the TATA box region.

Zhang Y, Fan M, Geng G, Liu B, Huang Z, Luo H, Zhou J, Guo X, Cai W, Zhang H - Retrovirology (2014)

MiR-H3 targets the TATA box in HIV-1 5′ LTR via sequence specific manner. (A) Predicted binding site of miR-H3 in HIV-1 5′ LTR with the TATA box highlighted. The MFE of this interaction is -19.2 kcal/mol. (B) Mutations (labeled in red with underline) were introduced to the promoter, named HIV 5′ LTR mut. The effects of miR-H3 on promoter activity of wildtype or mutated promoter were determined with dual-luciferase assay. The TATA box motif was indicated with a box. (C) Top, the TATA box motif of CMV promoter was replaced by the binding site of miR-H3 on HIV-1 promoter (CMV-HIV_bs mt). The mutated nucleotides are indicated in red with underline. Bottom, the effects of miR-H3 on indicated promoter activities were determined with Dual-Luciferase assay as described above. The TATA box motif was indicated with a box. (D) ChIP assay of HEK239T cells co-transfected with pNL4-3-deltaE-EGFP and pre-miR-H3 or the empty vector with antibody against Pol II or TBP to examine the binding of these general transcription factors to the HIV-1 core promoter region. The normal IgG was used as a control. (E) The effects of miR-H3 on HIV-1 promoter activity without Tat expression. (F) Effect of miR-H3 on the activity of a TAR region deficient (deletion of 470 to 492 bp in HIV-1 5′ LTR) HIV-1 promoter. (G) The effect of miR-H3 on another retrovirus promoter. A RSV promoter-derived luciferase reporter construct was co-transfected with miR-H3 precursor or control vector, and the promoter activity was investigated by Dual-Luciferase assay. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances, n = 3. *p < 0.05, ***p < 0.001.
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Figure 6: MiR-H3 targets the TATA box in HIV-1 5′ LTR via sequence specific manner. (A) Predicted binding site of miR-H3 in HIV-1 5′ LTR with the TATA box highlighted. The MFE of this interaction is -19.2 kcal/mol. (B) Mutations (labeled in red with underline) were introduced to the promoter, named HIV 5′ LTR mut. The effects of miR-H3 on promoter activity of wildtype or mutated promoter were determined with dual-luciferase assay. The TATA box motif was indicated with a box. (C) Top, the TATA box motif of CMV promoter was replaced by the binding site of miR-H3 on HIV-1 promoter (CMV-HIV_bs mt). The mutated nucleotides are indicated in red with underline. Bottom, the effects of miR-H3 on indicated promoter activities were determined with Dual-Luciferase assay as described above. The TATA box motif was indicated with a box. (D) ChIP assay of HEK239T cells co-transfected with pNL4-3-deltaE-EGFP and pre-miR-H3 or the empty vector with antibody against Pol II or TBP to examine the binding of these general transcription factors to the HIV-1 core promoter region. The normal IgG was used as a control. (E) The effects of miR-H3 on HIV-1 promoter activity without Tat expression. (F) Effect of miR-H3 on the activity of a TAR region deficient (deletion of 470 to 492 bp in HIV-1 5′ LTR) HIV-1 promoter. (G) The effect of miR-H3 on another retrovirus promoter. A RSV promoter-derived luciferase reporter construct was co-transfected with miR-H3 precursor or control vector, and the promoter activity was investigated by Dual-Luciferase assay. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances, n = 3. *p < 0.05, ***p < 0.001.
Mentions: With computational prediction, we surprisingly found a putative binding site of miR-H3 which covers the core promoter (the TATA box) in HIV-1 5′ LTR region (Figure 6A). The TATA box motif in HIV-1 5′ LTR starts two nucleotides further upstream and turns to the sequence CATATAA in all subtypes except for subtype E [36]. When mutations were introduced into the binding site in the TATA box region, the enhancement effect on promoter activity by miR-H3 was impaired (Figure 6B), suggesting that the direct binding between the core promoter and miR-H3 is required for its regulation. Furthermore, we mutated the TATA box region of CMV promoter to the same sequence as that of HIV-1 5′ LTR, and found that the transcription of this mutant could also be enhanced by miR-H3 (Figure 6C). These results suggest that the binding site in HIV-1 5′ LTR interacts with miR-H3 sequence-specifically and is required for the promoter activation induced by miR-H3. To investigate whether miR-H3 increases the binding of general transcription factors to the HIV-1 core promoter, we carried out ChIP assay with antibody against the RNA Polymerase II or the TATA box binding protein (TBP). The result suggested miR-H3 enhanced the association of both factors to the HIV-1 core promoter region (Figure 6D). As Tat protein is a very important regulatory factor for HIV-1 transcription, we investigated whether the interaction between Tat protein and TAR motif affected the HIV-1 promoter activation induced by miR-H3. Our data indicated that, in the absence of Tat, miR-H3 still upregulated HIV-1 promoter activity (Figure 6E). Alternatively, although the deletion of TAR significantly affected the promoter activity, the enhancement activity by miR-H3 was not affected (Figure 6F). Furthermore, miR-H3 did not affect the promoter activities of another retrovirus, Rous sarcoma virus (RSV), arguing against a non-specific transcription regulation on retroviruses (Figure 6G).

Bottom Line: It interacts with the TATA box in HIV-1 5' LTR and sequence-specifically activates the viral transcription.In addition, chemically-synthesized small RNAs targeting HIV-1 TATA box activate HIV-1 production from resting CD4+ T cells isolated from HIV-1-infected patients on suppressive highly active antiretroviral therapy (HAART).We have identified a novel HIV-1-encoded miRNA which specifically enhances viral production and provide a specific method to activate HIV-1 latency.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong 510080, China. zhangh92@mail.sysu.edu.cn.

ABSTRACT

Background: A lot of microRNAs (miRNAs) derived from viral genomes have been identified. Many of them play various important roles in virus replication and virus-host interaction. Cellular miRNAs have been shown to participate in the regulation of HIV-1 viral replication, while the role of viral-encoded miRNAs in this process is largely unknown.

Results: In this report, through a strategy combining computational prediction and deep sequencing, we identified a novel HIV-1-encoded miRNA, miR-H3. MiR-H3 locates in the mRNA region encoding the active center of reverse transcriptase (RT) and exhibits high sequence conservation among different subtypes of HIV-1 viruses. Overexpression of miR-H3 increases viral production and the mutations in miR-H3 sequence significantly impair the viral replication of wildtype HIV-1 viruses, suggesting that it is a replication-enhancing miRNA. MiR-H3 upregulates HIV-1 RNA transcription and protein expression. A serial deletion assay suggests that miR-H3 targets HIV-1 5' LTR and upregulates the promoter activity. It interacts with the TATA box in HIV-1 5' LTR and sequence-specifically activates the viral transcription. In addition, chemically-synthesized small RNAs targeting HIV-1 TATA box activate HIV-1 production from resting CD4+ T cells isolated from HIV-1-infected patients on suppressive highly active antiretroviral therapy (HAART).

Conclusions: We have identified a novel HIV-1-encoded miRNA which specifically enhances viral production and provide a specific method to activate HIV-1 latency.

Show MeSH
Related in: MedlinePlus