Fig. 2: PCIF1 restricts HIV infection by inhibiting its transcription.
From: HIV reprograms host m6Am RNA methylome by viral Vpr protein-mediated degradation of PCIF1
a–c PCIF1 inhibits HIV. a Illustration of the 4 sgRNAs. b, c p24 ELISA was performed in MT4 cells transduced with indicated lentivirus vectors, and then infected with HIVLAI (b MOI = 0.01, c MOI = 0.2) for 3 days. sgRNA resistant wild-type (PCIF1) or inactive mutant (APPA) was overexpressed in either control or PCIF1 KO cells. PCIF1 protein expressions were detected by western blotting. b *p = 0.02, ****p = 0.0000047, ***p = 0.0009, **p = 0.0013. c *p = 0.0036, ****p = 4.13E-06, ****p = 9.65701E-06, ns, not significant. d, e PCIF1 does not affect entry or release of HIV. The infection of HIV was detected using luciferase assays performed in PCIF1 knockout 293FT cells infected with HIV pseudovirus (HIVpp-luc, MOI = 0.2, 2 days). ****p = 0.000064. e p24 ELISA was performed in the supernatant of control or PCIF1 knockout 293FT cells transfected with HIVLAI infectious clone (2 days). **p = 0.0061. f, g PCIF1 does not affect pre-integration and integration of HIV. Control and PCIF1 KO Jurkat cells were infected with single-cycle HIVpp-luc. The formation of late reverse transcriptase cDNA (late-RT) at 10 h (f) as well as integrated proviral DNA at 48 h (g) were assessed by qPCR. ns, not significant. h, i PCIF1 inhibits transcription but not translation of HIV. h Control or PCIF1 knockout Jurkat cells were infected with HIV pseudovirus (HIVpp-GFP, MOI = 0.2). HIV transcription was quantified by gp120 RT-qPCR. **p = 0.0012, ***p = 0.00073. i HIV protein expression was detected by quantifying GFP reporter levels using flow cytometry (3 days). ****p = 1.4053E-08. j, k PCIF1 inhibits HIV replication in CD4+ primary T cell. Activated Primary CD4+ T cells from Donor 1 and 2 were transduced with PCIF1 shRNA or control shRNA (shNC). After re-activation, cells were infected with HIVLAI for 3 days, p24 levels were measured by ELISA. PCIF1 mRNA levels were detected by RT-qPCR. j ***p = 0.002, **p = 0.029. k ***p = 0.0002, *p = 0.04. All data are represented as mean ± SD and analyzed by a two-sided t-test in b–k. n = 3 (c–k), or 4 (b) independent experiments. Similar immunoblotting results were obtained from three independent experiments in b and c. GAPDH expression was shown as a loading control.
