Figure 4

TNPO3 K.D. cells block HIV-1 infection after nuclear import. (A) TNPO3 K.D. and shRNA control HeLa cells were challenged with HIV-1 or HIV-1-N74D. Formation of 2-LTR circles and productive infection was measured twenty-four and forty-eight hours post-infection, respectively. Formation of 2-LTR circles was measured by real-time PCR. Productive infection was determined by measuring the percentage of GFP-positive cells by flow cytometry 48 hours post-infection. (B) Similarly, TNPO3 K.D. and shRNA control HeLa cells were challenged with HIV-1 in the presence of the HIV-1 integrase inhibitor raltegravir, and the formation of 2-LTR circles and productive infection was determined as described above. (C) Formation of 2-LTR circles and productive infection was also measured by infecting TNPO3 K.D. and shRNA control HeLa cells with HIV-1 and HIV-1-D116N, which is a virus that contain a defective integrase. Similar results were obtained in three independent experiments and standard deviations are shown. Mock refers to control cells that were not infected. Viruses were normalized by quantifying the particle-associated reverse transcriptase activity on viral supernatants, as described in Methods.