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. 2019 Aug 20;10(4):e01344-19. doi: 10.1128/mBio.01344-19

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Copyright © 2019 Wang and Su.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

PMC Copyright notice

FIG 4 — The Vpr-TET2 axis enhances HIV-Yu-2 Env processing and virion infectivity in MDMs. (A) MDMs were infected with Yu-2 or Yu-2 ΔVpr viruses (MOI = 0.1). Levels of p24 in the supernatant were assessed at 2 and 4 dpi. Cells were treated with 2 μM nevirapine (NVP) at 2 dpi where indicated, to inhibit the second round of HIV-Yu-2 infection. (B) Western blots of cell lysates from MDMs infected with Yu-2 or Yu-2 ΔVpr for 4 days with NVP added at 2 dpi. Env proteins gp160 and gp120 were detected with anti-gp120 antibody, gp41 was detected with anti-gp41 antibody, and p24 was detected with anti-p24 antibody. (C) Vpr-enhanced gp120/p24 and gp120/gp160 ratios in MDMs are summarized with MDMs from with two different donors. (D) Viral supernatants were concentrated from MDMs infected with Yu-2 or Yu-2 ΔVpr viruses at 2 dpi by ultracentrifugation (over 20% sucrose). Western blotting was performed by using anti-gp120, -p24, and -gp41 antibodies. (E) Vpr-enhanced gp120/p24 ratios are summarized from two different donors. (F) Supernatants with equal amounts of p24 from MDMs infected with Yu-2 or Yu-2 ΔVpr viruses were used to infect TZM-bl indicator cells. Luciferase activity was measured at 48 hpi. Data (luciferase/ng p24) from three wells were used from each donor MDM, and 3 technical repeats were performed with supernatant from each well. MDMs derived from 2 donors were used to repeat the experiments. (G) MDMs were transduced with control shRNA, shTET2, and shIFITM3. Viral supernatants were concentrated from the aforementioned MDMs infected with Yu-2 or Yu-2 ΔVpr viruses for 4 days, with NVP added at 2 dpi, by ultracentrifugation (over 20% sucrose). Five nanograms of p24 for each virion was added per lane for analysis of gp120, p24, and gp41 in virions. (H) Supernatants from MDMs infected with Yu-2 or Yu-2 ΔVpr viruses were used to infect TZM-bl indicator cells. The relative infectivity is defined by HIV-induced luciferase activity per ng of p24. Supernatant from control MDMs infected with HIV-YU-2 is defined as 100% (n = 2 donors).