FIG. 4.

Nullbasic inhibits reverse transcription of lentiviral vector in cells. (a and b) HEK293T cell cultures were infected in duplicate with equal amounts of VLPs conveying either Nullbasic-EGFP or EGFP. After attachment of virus to cells at 4°C, the infections were initiated and incubated at 37°C for 3 hr. Parallel cultures transduced side by side were processed for either viral cDNA or RNA. Where indicated, cells were pretreated with 200 μM nevirapine (NVP) or VLPs were heat-inactivated. The experiments were performed three times and a representative experiment is shown. (a) The level of cytoplasmic VPL cDNA was determined by qPCR using primers specific for the EGFP region. DNA copy number was normalized to mitochondrial DNA levels measured in each sample. A representative result from three experiments with similar results is shown. (b) The level of viral RNA in transduced cells was determined. DNase I-treated RNA extracted from cells treated with NVP was used in qRT-PCRs using primers specific for the EGFP region. A synthetic kanamycin cassette RNA was included in RNA isolations to control for recovery and RT-PCR efficiency. A no-reverse transcriptase reaction was always included to control for DNA contamination. (c) VLP supernatant was measured in triplicate for CAp24 by ELISA and for RT activity, using a commercial assay. The VLP stocks used contained similar levels of CAp24, approximately 250–300 ng/ml. RT assays were performed with an equal volume of each VLP stock and the values shown represent the ratio of RT to CAp24. The experiments were performed twice with similar results and a representative experiment is shown. The Student t test (two-tailed distribution) was used to assess statistical significance between data sets.