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Figure 7 | Retrovirology

Figure 7

From: The ability of TNPO3-depleted cells to inhibit HIV-1 infection requires CPSF6

Figure 7

Cytosolic CPSF6 expressed in human HeLa cells restricts HIV-1 replication before or at the nuclear import step. (A) HeLa cells were stably transduced with the indicated CPSF6 variant. Expression of the different CPSF6 variants was analyzed by Western blotting using anti-FLAG antibodies. (B) The cellular distribution of the different CPSF6 variants was studied by immunofluorescence using anti-FLAG antibodies (red). The nuclear compartment was stained using DAPI (blue). Image quantification is shown in Additional file 4. (C) HeLa cells stably expressing the indicated CPSF6 variants were challenged with HIV-1 or HIV-1-N74D expressing GFP as a reporter for infection. Forty-eight hours post-infection the percentage of GFP-positive cells was determined by flow cytometry. As control, HeLa cells stably transduced with the empty vector pLPCX were challenged with increasing amounts of HIV-1 and HIV-1-N74D. (D) HeLa cells stably expressing CPSF6 and NES-CPSF6 were challenged with the indicted viruses. Infection was determined by measuring the percentage of GFP-positive cells by flow cytometry 48 hours post-infection (upper panel). In parallel, similarly infected cells were lysed at 7 or 24 hours post-infection and total DNA was extracted. The DNA samples collected at 7 hours post-infection were used to determine the levels of late reverse transcripts by real-time PCR (middle panel). DNA samples collected at 24 hours post-infection were used to quantify HIV-1 2-LTR circles by real-time PCR (lower panel). Mock refers to control cells that were not infected. (E) HeLa cells expressing the different CPSF6 variants were challenged with HIV-2, SIVmac, FIV, BIV or EIAV. As control, HeLa cells stably transduced with the empty vector pLPCX were challenged with HIV-2, SIVmac, FIV, BIV or EIAV. Infectivity by the different viruses was determined by measuring the percentage of GFP-positive 48 hours post-infection. Similar results were obtained in three independent experiments and a representative experiments is shown.

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