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. 2021 Sep 20;12:5543. doi: 10.1038/s41467-021-25683-4

Fig. 1. HIV infection downregulates m6Am modification of cellular mRNA by Vpr-induced degradation of the m6Am methyltransferase PCIF1.

Fig. 1

a m6Am modification of cellular mRNA is decreased by HIV infection. m6Am levels were quantified in MT4 cells infected with HIVLAI (MOI = 0.4, 3 days) by LC–MS/MS. n = 3 biological independent experiments. Two-sided t-test. Mean ± SD, ***p = 0.0008. b PCIF1 mRNA levels are not changed by HIV infection. PCIF1 mRNA levels were quantified in MT4 cells infected with HIVLAI (MOI = 0.4) or HIVNL4-3 (MOI = 2). n = 3 biological independent experiments. Two-sided t-test. Mean ± SD. c HIV infection decreases PCIF1 in MT4 cells. Immunoblotting of PCIF1 and p24 in MT4 cells infected with HIVLAI or HIVNL4-3 for 3 days. d HIV infection decreases PCIF1 in primary CD4+ T cells. Immunoblotting of PCIF1 and p24 in activated primary CD4+ T cells infected with HIVLAI (MOI = 1). e PCIF1 is downregulated by HIV through proteasome degradation. Immunoblotting of PCIF1, p62, and p24 in MT4 cells infected with HIVLAI (MOI = 0.4, 3 days). Cells were incubated with DMSO or MG132 (0.25 µM) at 1 day before lysis. f PCIF1 is degraded by HIV viral protein Vpr. Immunoblotting of PCIF1 and the viral proteins in HeLa cells transfected with indicated expression vectors (E.V: empty vector). g Vpr deleted HIV does not degrade PCIF1 and ETS1 protein expression. Immunoblotting of indicated proteins in MT4 cells infected with HIVLAI or HIVLAI Vpr deleted virus (HIVLAI-∆Vpr) (MOI = 0.4, 3 days). h Vpr interacts with PCIF1. Flag immunoprecipitation was performed in HeLa cells co-transfected with plasmids expressing PCIF1 and Flag-tagged Vpr for 2 days. PCIF1 and Vpr expression and enrichment were detected by western blotting. i Vpr interacts with PCIF1 in T cells. MT4 cells expressing Flag-PCIF1 was infected with HIV (MOI = 1) for 3 days followed by Flag immunoprecipitation. Flag-PCIF1 and Vpr expression and enrichment were detected by western blotting. j The E3 complex binding site of Vpr is necessary to decrease PCIF1. Immunoblotting of PCIF1 and Vpr in HeLa cells transfected with plasmids expressing empty vector (E.V.), Vpr, or Vpr-Q65R mutant vector for 2 days. k Vpr induces PCIF1 ubiquitination. HA or FLAG immunoprecipitation was performed in HeLa cells co-transfected with plasmids expressing Flag-PCIF1, HA-Ub, either Vpr or Vpr-Q65R mutant for 2 days. HA-tagged ubiquitin, Flag-tagged PCIF1, and Vpr expression were detected by western blotting. Similar results were obtained from three independent experiments, and GAPDH expression was shown as a loading control (c–j).