FIG. 3.

Nullbasic inhibits reverse transcription of lentiviral VLPs. (a) Using partially purified VLPs, ERT assays were performed with equivalent amounts of RT activity determined in a homopolymeric primer:template RT assay. Briefly, DNase I-treated VLP supernatant was subjected to centrifugation at 100,000×g for 1 hr. The washed viral pellets were resuspended in PBS and used in ERT reactions as described in Materials and Methods (Warrilow et al., 2008). All ERT experiments included a no-dNTP control reaction. The viral DNA was obtained and subjected to qPCRs in triplicate, using primers described in the online supplement, to measure HIV-1 negative-strand strong stop DNA. The mean value and the standard deviations are indicated. (b) Lentiviral VLPs made with pLOX-CW-EGFP were made in HEK293T cells by cotransfection with a Nullbasic expression vector, pCDNA3.1-NB-FLAG, or an empty vector. VLP supernatant was collected 48 hr posttransfection and ERT assays were performed and analyzed by qPCR as described previously. (c) Western blot analysis of protein lysates prepared from the transfected HEK293T using an anti-Tat (top) or anti-tubulin (bottom) antibody.