Skip to main content
. 2021 Mar 16;12(2):e00219-21. doi: 10.1128/mBio.00219-21

FIG 2.

FIG 2

MARCH8-mediated targeting of viral envelope glycoproteins exhibits differential dependence on the CT. (A) HEK293T cells in a 12-well plate were cotransfected with the Env-defective HIV-1 molecular clone pNL4-3/KFS (1 μg) and vectors expressing WT or mutant VSV-G (100 ng) and hemagglutinin (HA)-tagged WT or mutant MARCH8 (50 ng). TZM-bl cells were infected with VSV-G pseudotyped HIV-1, and luciferase activity was measured 2 days postinfection and normalized for viral p24. The infectivity of particles pseudotyped with WT VSV-G in the absence of MARCH8 is set at 100%. Abbreviations: Vect, vector, M8, MARCH8; M8CS, MARCH8-CS; MARCH8-W114A (M8W). (B) HEK293T cells were cotransfected with WT or CT-deleted (CTdel144) pNL4-3 HIV-1 molecular clones (1 μg), and vectors expressing HA-tagged MARCH8 (WT or mutants) (100 ng). Two days posttransfection, virus supernatants were collected, analyzed for p24 content by western blotting, and infections were carried out in TZM-bl cells and normalized to p24 as described above for panel A. Infectivity of WT HIV-1 in the absence of MARCH8 was set at 100%. (C) HEK293T cells were cotransfected with Env-defective (pNL4-3/KFS) HIV-1 molecular clone (1 μg) and vectors expressing EboV-GP (WT or CT mutants) (500 ng) and HA-tagged MARCH8 (WT or mutants) (250 ng), and infections were carried out in TZM-bl cells. The infectivity of particles pseudotyped with WT EboV-GP in the absence of MARCH8 was set at 100%. (D) HEK293T cells in a 6-well plate were cotransfected with the Env-defective, luciferase-expressing pNL4-3 derivative pNL4-3.Luc.R-E- (3 μg) and vectors expressing SARS-CoV-2-S full-length (FL) and mutants (FL-K-to-R, Truncated, and Truncated-K-to-R) (300 ng) and HA-tagged MARCH8 (WT or CS mutant) (200 ng). Two days posttransfection, virus supernatant was collected, and infectivity was measured in HEK293T cells stably expressing hACE2 with RT-normalized virus. The infectivity of virus particles pseudotyped with full-length S protein (S-FL) in the absence of MARCH8 was set at 100%. Data shown are means plus standard deviations (SD) (error bars) from three to five independent experiments. Values that are significantly different by two-tailed unpaired t test are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Values that are not significantly different (NS) are also indicated.