FIG. 5.

Infectivity of a lentiviral-based vector is inhibited by overexpressing NUP153C. (A) The level of EGFP transgene expression from an HIV-1-based vector is reduced in the presence of NUP153C. Nontransfected 293T cells (panel a) and cells transfected with an empty plasmid (panel b) or with a plasmid encoding NUP153C (panel c) were challenged with a VSV-G-pseudotyped HIV-1-based vector encoding EGFP at 8 h posttransfection. Twenty-four hours postinfection, the cells were collected and analyzed by flow cytometry for EGFP expression. Histograms are representative of results under the indicated experimental conditions. The number of EGFP-positive cells under each condition is expressed as the mean ± SEM for at least three experiments. (B) Graphical representation of EGFP expression normalized to the nontransfected control. An asterisk indicates a significant difference from the empty vector control (P = 0.0002). (C) NUP153C expression inhibits the nuclear import of viral cDNA. DNA was extracted from the same cell populations that were analyzed by flow cytometry in panel A and was subjected to PCR using primers to amplify the 1-LTR circle form of viral DNA (top), total viral DNA (middle), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (bottom) from genomic DNA. Lane 1 shows nontransduced 293T cells. Lane 2 shows 293T cells transduced with the VSV-G-pseudotyped HIV-1-based vector encoding EGFP. Lane 3 shows 293T cells transfected with an empty vector 8 h before transduction with the HIV-1 vector. Lane 4 shows 293T cells transfected with NUP153C 8 h prior to transduction with the HIV-1 vector. (D) NUP153C expression inhibits viral cDNA integration. Alu-PCR was used to quantify the copy number of provirus integrated in cells transfected with either an empty vector (green bar; n = 3) or the NUP153C-expressing vector (red bar; n = 9). The values are expressed as the copy number of provirus/ng genomic DNA. An asterisk indicates a significant difference from the empty vector control (P = 0.0002).