FIG 3.

Riplet enhances degradation of unspliced, partially spliced, and multiply spliced viral mRNAs. (A) 293TrexhZAP cells were left unmanipulated (−) or transfected with either an empty vector control (EV) or DNAs overexpressing the indicated Riplet constructs. Cells were infected with HIV-1 reporter virus (pNL4.3env-luc⁺), and 6 h later, doxycycline or DMSO was added. At 24 h postinfection, total cytoplasmic RNAs were extracted, and the abundance of three viral mRNA species was analyzed by qRT-PCR, using PCR primer pairs targeting gag, vif, nef (in this vector, nef-luc), and GAPDH as standard. Fold inhibition of the viral mRNAs was calculated as the ratio of mRNA levels in DMSO-treated cells to those in doxycycline-treated cells after normalization to mRNA levels of the housekeeping gene GAPDH. An additional sample containing no template (NT) for PCR amplification was included to control for DNA contamination.