FIG 6.

CRNKL1 knockdown increases expression of Gag after acute HIV infection. (A) Knockdown of CRNKL1 expression by siRNAs. CRNKL1-targeting siRNAs (Si.) and nontargeting control siRNAs (Ctr.) were cotransfected with myc-tagged CRNKL1 expression plasmids to test their efficacy and specificity. Exogenous CRNKL1 expression and endogenous α-tubulin expression in transfected 293T cells were monitored by anti-myc and anti-α-tubulin Western blot analyses, respectively. (B) Effect of CRNKL1 knockdown on Gag-BFP expression in transiently transfected cells. Targeting or nontargeting siRNAs were cotransfected with the HIV-dual-GT plasmid into 293T cells. Gag-BFP reporter expression was monitored by FACS analysis. (C) Effect of CRNKL1 knockdown on Gag-BFP expression in stably transduced cells. 293T cells stably transduced with HIV-dual-GT were transfected with the siRNAs and analyzed by flow cytometry. Shown are representative results from one experiment (left panel) and the percentage of BFP⁺ DsRed⁺ cells plotted from three independent experiments (right panel). (D) Effect of CRNKL1 knockdown on Gag expression in cells acutely infected with HIV-1. 293T cells were transfected twice within 4 days with siRNAs and then infected with VSV-G-pseudotyped HIV-1 env mutants with (HIV.r⁻e⁻) or without (HIV.R⁺e⁻) inactivating mutations of Rev. A reporter virus expressing wild-type Gag (HIV.r⁻e⁻DsRed) and a lentiviral vector expressing GFP from an internal promoter (pWPXL) were also included. Gag, GFP, and α-tubulin expression was analyzed by Western blotting using antibodies targeting the proteins indicated. The dotted line indicates excision of blank lanes from the Western blot image. (E) Effect of CRNKL1 knockdown on Gag expression in cells acutely infected with HIV-1 by flow cytometry. 293T cells were infected with CRNKL1 targeting or nontargeting CRISPR lentivirus and VSV-G-pseudotyped HIV.r⁻e⁻. Gag expression was analyzed on the single-cell level by intracellular staining for Gag and flow cytometry.