Fig 2: MARCH proteins target envelope glycoproteins from a diverse number of viral families. (A) Ebola virus (EBOV) mature envelope glycoprotein Gp2, (B) lymphocytic choriomeningitis virus (LCMV) mature envelope glycoprotein GP2, (C) Nipah virus (NiV) fusion glycoprotein (F) (left) and NiV receptor binding glycoprotein (G) (right), (D) influenza A virus (IAV) hemagglutinin (HA), (E) Chikungunya virus (CHIKV) E1 and E2 protein, and (F) Zika virus (ZIKV) E levels in the presence of human MARCH1 (hM1), MARCH2 (hM2), MARCH8 (hM8), and empty vector (E.V). 293T cells were cotransfected with either (A) EBOV glycoprotein (Gp), (B) LCMV Gp, (C) NiV F or NiV G, (D) IAV HA, (E) a plasmid expressing the CHIKV structural proteins, or (F) ZIKV E in combination with either hM1, 2, 8, or E.V. At 24 h posttransfection, cells were lysed and analyzed by Western blotting using anti-V5 (for EBOV GP2 and IAV HA detection), anti-FLAG (for LCMV GP2 detection), anti-AU1 (for NiV G detection), anti-HA (for NiV F detection), anti-flavivirus E antibody (4G2, for ZIKV E detection), anti-myc (CHIKV E1 detection), anti-E2 (CHIKV E2 detection), anti-MARCH1, anti-MARCH2, anti-MARCH8, and anti-ß-actin antibodies. Shown are the results of a single experiment (representative of three independent experiments).

Fig 3: Mouse March1 and mouse March8 physically interact with MLV p15E. (A and B) MutuDC1940 cells, which express endogenous levels of both mouse March1 (mM1) and mM8, were infected with MLV (10 MOI). Cells were harvested 72 h postinfection, and lysates were immunoprecipitated with (A and B) an isotype control, (A) anti-MARCH1, and (B) anti-MARCH8 and analyzed in Western blots (WB) with anti-p15E, anti-MARCH1, anti-MARCH8, and anti-ß-actin. (C and D) 293T cells were cotransfected with an MLV infectious clone and either (C) mouse March1 (mM1) or (D) mM8 and their TM mutants used in Fig. 4. Cells were harvested 48 h posttransfection, and lysates were immunoprecipitated with anti-myc (mM1 and mM8) and anti-p15E followed by Western blot analyses probing with anti-p15E, anti-myc (mM1 and mM8), and anti-ß-actin antibodies. Results are shown for n = 3 independent experiments. Representative immunoblotting results are shown for panels A through D.

Fig 4: Expression levels of mouse March1, 2, 3, and 8 in the presence of IFN-ß and MLV infection. (A to C) Fold expression changes of mouse March1, 2, 3, and 8 (mM1, 2, 3, and 8) relative to untreated cells and normalized to GAPDH in (A) bone marrow-derived macrophages (BMDMs), (B) bone marrow-derived dendritic cells (BMDCs), and (C) MutuDC1940 cells at 0, 4, 8, 16, and 24 h after treatment with 500 units/ml of mouse IFN-ß. Purity of BMDMs and BMDCs was determined by flow cytometry staining with anti-CD11b and anti-CD11c, respectively, as shown in Fig. S1D and E in the supplemental material. (D) Fold expression changes of mM1 relative to untreated cells and normalized to GAPDH after treatment of MutuDC1940 cells with different concentrations of IFN-ß (0.005 to 5,000 units/ml) for 4 h. (E and F) Fold expression changes of mM1, 2, 3, and 8 relative to uninfected cells and normalized to GAPDH in (E) mouse T lymphocyte cell line EL4 and (F) MutuDC1940 cells at 4, 8, 16, and 24 h postinfection with MLV (5 MOI). Data shown represent averages of results from n = 3 independent experiments. All results are presented as means ± standard error of the mean (SEM). Statistical analysis performed using unpaired t test. *, P = 0.05; **, P = 0.01; ***, P = 0.001; ****, P = 0.001.

Fig 5: MARCH-mediated degradation of the MLV envelope glycoproteins occurs in the lysosome. (A and B) 293T cells were cotransfected with an MLV infectious clone and either mouse March1 (mM1), mM8, or empty vector (E.V.). Cells were incubated with either (A) 100 µM chloroquine or (B) 16 µM MG132. Cells were harvested 24 h posttransfection, and lysates were analyzed by immunoblotting using anti-MLV p30 (detects p65Gag), anti-MLV p15E, anti-myc (for mM1 and 8 detection), and anti-ß-actin antibodies. Results are shown for n = 3 independent experiments. Representative immunoblotting results are shown for panels A and B.