Figure 4

hsa-mir-29a and b inhibited Nef expression and HIV-1 replication. (A) Nef expression was inhibited by hsa-miR-29a and 29b. HEK293T cells were co-transfected with miRNA clones or control vector along with pCDNA-HA-Nef using calcium phosphate precipitation. After 36 hours of transfection, cells were lysed and expression of Nef was analyzed by immunoblotting using HA antibody. Immunoreactive actin bands were used as loading control. (B and C) hsa-miR-29a and hsa-miR-29b miRNA clones inhibited virus production in HEK293T (B) and Jurkat cells (C). Cells were co-transfected with miRNA clones or control vector along with HIV-1 molecular clone pNL4.3 (Materials and Methods). Cells were lysed post-transfection and expression of Nef was analyzed by immunoblotting using Nef antibody (upper panels); culture supernatant was used for p24 antigen ELISA (lower panels). Asterisks in (B) represent significant p-value of 0.016 for inhibition mediated by 29a compared to control. The difference observed with 29b is not significant. Asterisks in (C) represent significant p-value of 0.014 and 0.016 for inhibition by 29a and 29b respectively, as compared to control vector.