To obtain an accurate IC50 for inhibition of HIV replication by flavopiridol in

The first is pNL43 deltaE KU-55933 臨床試験 EGFP vector, which contains the entire HIV 1 genome except for the env gene which was impaired and replaced with a gfp gene. The second is pCMV R8. 2 vector, which contains similar genes with pNL4 3 deltaE EGFP but lacks of 5 and 3 LTR regions. The third is psPAX2 vec tor, which only contains gag, pol, tat, rev genes and rre motifs. When co transfected these vec tors with miR H3 precursor or the empty vector, we found that miR H3 could only enhance the RNA expres sion of pNL4 3 deltaE EGFP, but not that of the other two vectors, suggesting its targeting site is located on 5 or 3 LTR region. To clarify which re gion is the target of miR H3, the LTR regions were cloned into a luciferase reporter plasmid, pMIR REPORT.

The 5 LTR sequence was inserted into the upstream of firefly luciferase gene to replace its CMV promoter, while the 3 LTR sequence was inserted to the 3 UTR region of the firefly luciferase gene with a MMLV promoter whose activity is similar to that of HIV 1 5 LTR. Ectopic expression of miR H3 substan tially buy Linifanib enhanced the luciferase activity of the construct containing HIV 1 5 LTR as the promoter, but not that of the construct containing HIV 1 3 LTR as the 3 UTR. These results implied that miR H3 targets the 5 LTR region of HIV 1 and most probably worked through enhancing the promoter transcriptional activity. MiR H3 targets HIV 1 TATA box sequence specifically With computational prediction, we surprisingly found a putative binding site of miR H3 which covers the core promoter in HIV 1 5 LTR region.

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. When mutations were LY3009104 1187594-09-7 introduced into the binding site in the TATA box region, the enhancement effect on promoter activity by miR H3 was impaired, suggesting that the direct binding between the core pro moter and miR H3 is required for its regulation. Fur thermore, 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. 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 investi gate 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. The result suggested miR H3 enhanced the association of both factors to the HIV 1 core promoter region. As Tat protein is a very important regula tory 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 pro moter activity. Alternatively, although the deletion of TAR significantly affected the promoter activity, the enhancement activity by miR H3 was not affected. Furthermore, miR H3 did not affect the promoter activities of another retrovirus, Rous sarcoma virus, arguing against a non specific transcription regulation on retroviruses.