Cloning and detection of HIV-1-encoded microRNA.

MicroRNAs (miRNAs) are 21-to 25-nucleotides (nt) long and interact with messenger RNAs to trigger either translational repression or RNA cleavage through RNA interference (RNAi). We have shown that HIV-1 nef double-stranded RNA from AIDS patients who are long-term nonprogressors, inhibits HIV-1 transcription; and that nef-derived miRNA, miR-N367, is produced in human T-cells persistently infected with HIV-1. The miR-N367 can block HIV-1 Nef expression and long terminal repeat (LTR) transcription, suggesting that miR-N367 might suppress both Nef function and HIV-1 transcription through the RNAi pathway. Protocols are presented here for cloning HIV-1-encoded miRNA and confirming miRNA expression by Northern blot hybridization.