Compound cytotoxicity was also greater in these primary cells decreasing the li

The resting primary CD4 T lymphocytes [You must be registered and logged in to see this link.] were then iso lated from PBMCs with CD4 T Cell Isolation Kit II. Human primary PBMCs and CD4 T cells were grown in the RPMI 1640 conditioned media supplemented with 10% fetal bovine serum, 50 U ml penicillin and 50 ug ml streptomycin. The infectious HIV 1 clone and Env defective HIV 1 clone were obtained through the AIDS Research and Reference Reagent Program, NIAID, US NIH. The precursor of miR H3 and hsa miR 150 were amplified by PCR and directionally cloned into the downstream of the EGFP gene in the pEGFP C1 vector. The pNL4 3 miR H3MT plasmid was constructed by introducing mutations in the region for miR H3 3p mature miRNA without changing amino acid code. Similar mutations were also introduced into non infectious HIV 1 clone, pNL4 3 deltaE EGFP.

HIV pro Luc plasmid was constructed by replacing the promoter of Luciferase gene in the pMIR REPORT [You must be registered and logged in to see this link.] Luciferase vector with HIV 1 5 LTR sequence. MMLV Luc HIV 3LTR plasmid was constructed by replacing the pro moter of Luciferase gene in the pMIR REPORT vector with the MMLV promoter and inserting the HIV 1 3 LTR downstream the luciferase gene. Several mutations were in troduced into miR H3 binding site in the 5 LTR sequence. HIV pro Luc mtTAR plasmid was constructed with delet ing 5 half region of TAR motif to abolish its functional secondary structure. RSV pro Luc plasmid was constructed by replacing the promoter of Luciferase gene in the pMIR REPORT vector with the RSV promoter. All the constructs were verified by sequencing. The siRNAs against Drosha and Exportin 5 genes were purchased from Dharmacon.

Anti B actin antibody was purchased from CST. Anti human CD3 and anti human CD28 antibodies were from BD. The rabbit anti P24 antibody was prepared by our lab. [You must be registered and logged in to see this link.] Infection and transfection Infectious HIV 1 clone pNL4 3 was transfected into 60% confluent HEK293T cells using Lipofecta mine 2000 according to the manufacturers protocol. Viral supernatant was collected 2 days after transfection and viral production was determined by P24 ELISA kit. Five ng P24 f infectious HIV 1 viruses were used to infect 2X 106 activated human CD4 T lymphocytes for 3 hrs at 37 C. The cells were then washed three times with cold PBS and add fresh conditioned medium containing IL 2. Supernatants of cell culture were collected in 2 3 days interval and subject to P24 ELISA detection.

Transfection of HEK293T and TZM bl cells was performed with Lipofectamine 2000 according to the manufacturers protocol. Transfection of primary CD4 cells with small RNA was performed with RNAiMAX according to the manufacturers protocol. Quantitative real time RT PCR analysis Total RNA from HEK293T or CD4 T cells was isolated with Trizol reagent and then subjected to cDNA synthesis using PrimeScript RT reagent Kit. All primers were annealed at 37 C and RT was processed at 42 C. Quantitative PCR was performed with SYBR Premix ExTaq II Kit by following the manufacturers instructions. The expressions of HIV 1 total RNAs were determined with the primer pair HIVTotRNA 5 F R. The HIV 1 RT activity assay was per formed by following the method described in Vermeire et al..