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

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a, b Knockout of ETS1 promotes HIV infection. MT4 were edited with ETS1 sgRNAs for 7 days, and then control or edited cells were infected with HIVpp-luc for 2 days. ETS1 protein levels were detected using western blotting (g). HIV replication was detected using luciferase assays (h). **p = 0.0012, ***p = 0.00015, ***p = 0.00094. c–e Interfering ETS1 expression increases HIV replication. Levels of single-cycle HIV infection were determined using luciferase assays in control shRNA or two ETS1 knockdown shRNA transduced MT4 cells infected with HIVpp-luc at an MOI of 0.2 for 2 days (c). ETS1 (d) and gp120 (e) mRNA expression levels were quantified in control shRNA or ETS1 KD MT4 cells infected with HIV_LAI at an MOI of 0.01 for 3 days. c **p = 0017, **p = 0021. d **p = 001, **p = 002. e ****p = 2.99781E−06, ****p = 1.20386E−06. f ETS1 is recruited to the HIV promoter. HIV-infected MT4 cells were prepared for ETS1-CHIP analysis. RT-qPCR of the HIV promoter regions or c-myc or GAPDH region coimmunoprecipitated with ETS was performed. *p = 0.049, *p = 0.020, **p = 0.007, *p = 0.0309, ns not significant. g ETS1 inhibits HIV promoter activity. A plasmid containing HIV 5′ LTR and a luciferase reporter (HIV-LTR-luc) was used to quantify HIV promoter activity. The reporter plasmid was electroplated into control or ETS1 KD MT4 cells and then protein expression was quantified using luciferase assay after 2 days. ***p = 0.00017, **p = 0.007. h Model illustrating Vpr–PCIF1-ETS1 regulation axis in HIV replication. During HIV replication, PCIF1 restricts HIV transcription and is targeted by viral protein Vpr for proteasome-mediated degradation, which leads to an impaired m⁶Am modification in cellular mRNAs. PCIF1 inhibits HIV infection by stabilizing a transcription factor ETS1 (ETS Proto-Oncogene 1, transcription factor) mRNA that binds HIV promoter to regulate viral transcription. All data are represented as mean ± SD and analyzed by a two-sided t-test in b–g. n = 3 (c, d, g), or 4 (b, e, f) independent experiments. Similar results were obtained from three independent experiments in a.