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. 2018 Sep 28;8:14551. doi: 10.1038/s41598-018-32785-5

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© The Author(s) 2018

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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Knockdown of IFITMs rescues HIV-1 output and viral protein production in primary human T cells. Activated human CD4⁺ T cells were transduced with lentivirus expressing the indicated shRNAs against IFITMs or scrambled sequence (sc) for 48 hours. (A) Level of IFITM expression was analyzed by immunoblotting and (B) densitometry; data was normalized to GAPDH and scrambled control. Cells were then infected with HIV-1 89.6 with equivalent p24 concentrations. Cells were pre-treated with AMD3100 for 2 hours prior to infection and throughout. Viral proteins in infected cells was analyzed by immunoblotting. Immediately after infection (3 h post-infection), cells were treated with trypsin and washed with PBS before intracellular staining of p24 and flow cytometry to measure virus uptake. Intracellular levels of p24 and median fluorescence intensity are shown in (D) representative histograms and (E) summary bar chart. (F) Level of virus production was measured by p24 ELISA 72 hours post-infection and normalized. Levels of viral proteins in infected cells from one of the blood donors were shown by (G) immunoblotting and (H) densitometry. Data show mean + S.E.M. of 2 blood donors. Differences were assessed with Student’s t-test and *indicates p < 0.05.