Figure 6

Cytosolic CPSF6 expressed in Cf2Th cells restricts HIV-1 replication before or at the nuclear import step. (A) Cf2Th cells stably expressing the indicated CPSF6 constructs were challenged with HIV-1 or HIV-1-N74D expressing GFP as a reporter. Forty-eight hours post-infection the percentage of GFP-positive cells was determined by flow cytometry. As control, Cf2Th cells stably transduced with the empty vector pLPCX were challenged with HIV-1 and HIV-1-N74D. (B) Cf2Th 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, cells from similar infections 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). Separately, DNA samples collected at 24 hours post-infection were used to quantify HIV-1 2-LTR circles by real-time PCR in DNA (lower panel). Mock refers to control cells that were not infected. (C) Cf2Th cells stably expressing the different CPSF6 variants were challenged with HIV-2, SIVmac, FIV, BIV, EIAV or N-MLV. As control, Cf2Th cells stably transduced with the empty vector pLPCX were challenged with HIV-2, SIVmac, FIV, BIV, EIAV or N-MLV. Infectivity by the different viruses was determined by measuring the percentage of GFP-positive 48 hours post-infection. (D) Cf2Th cells stably expressing the indicated CPSF6 variant or TRIM5α_rh were challenged using similar amounts of HIV-1, and performed the fate of the capsid assay to separate pelletable from soluble cytosolic capsids 16 hours post-infection as described in Methods. Input, soluble and pellet fractions were analyzed by Western blotting using antibodies against HIV-1 CA p24. Similar results were obtained in three independent experiments and a representative experiments is shown.