MARCH8 inhibits viral infection by two different mechanisms

  1. Yanzhao Zhang
  2. Takuya Tada
  3. Seiya Ozono
  4. Satoshi Kishigami
  5. Hideaki Fujita
  6. Kenzo Tokunaga  Is a corresponding author
  1. Department of Pathology, National Institute of Infectious Diseases, Japan
  2. Faculty of Life and Environmental Sciences, University of Yamanashi, Japan
  3. Faculty of Pharmaceutical Sciences, Nagasaki International University, Japan
3 figures, 1 table and 3 additional files

Figures

MARCH8 targets and ubiquitinates cytoplasmic lysine residues of VSV-G but not of HIV-1 Env.

(A) Schematic structure of the lysine mutants of VSV-G (CT5K/R; upper) and HIV-1 Env (CT2K/R; lower). SP, signal peptide; EC, extracellular domain; TM, transmembrane domain; CT, cytoplasmic tail; SU, surface subunit. (B) Infectivity of viruses prepared from 293T cells cotransfected with Env-defective HIV-1 luciferase (luc) reporter proviral DNA and either the VSV-G wild-type (WT) or CT5K/R mutant plasmid together with either a control (Ctrl) (black) or HA-MARCH8 (gray) plasmid. Data are shown as a percentage of the viral infectivity in the absence of MARCH8 when WT VSV-G was used (mean + s.d. from three independent experiments). (C) Infectivity of viruses prepared as shown in B, except for using either the WT HIV-1 Env or its CT2K/R mutant plasmid (mean + s.d. from three independent experiments). (D) The VSV-G lysine mutant is resistant to MARCH8-mediated intracellular degradation. Shown are immunofluorescence-based analyses of the intracellular expression of either the WT or CT5K/R mutant VSV-G with or without MARCH8 in transfected HOS cells. Scale bars, 10 μm. Note that the cell-staining for VSV-G cannot be performed because VSV-G is tagged with the T7 epitope (T7e) at the C-terminus. (E) The lysine mutant of HIV-1 Env is still sensitive to MARCH8-induced downregulation from the cell surface. Immunofluorescence images show cell-surface expression of either the WT or CT2K/R mutant HIV-1 Env with or without MARCH8 in transfected HOS cells. Scale bars, 10 μm. (F) MARCH8 sequesters HIV-1 Env in the trans-Golgi network (TGN). Immunofluorescence images show intracellular localization of either the WT or CT2K/R mutant HIV-1 Env and the TGN marker TGN46, with or without MARCH8 in transfected HOS cells. Scale bars, 10 μm. (G) Lysine residues at the CT domain of VSV-G are ubiquitinated by MARCH8. The ubiquitination of the WT or CT5K/R mutant VSV-G tagged with T7e in cells expressing control or MARCH8 (WT or RING-CH mutant (CS)) was examined by immunoprecipitation (IP) of either ubiquitinated proteins with an anti-ubiquitin antibody (left panel) or of T7e-tagged VSV-G with an anti-T7e antibody (right panel), followed by immunoblotting with an antibody to either T7e (left panel) or ubiquitin (right panel), respectively.

The tyrosine motif of MARCH8 mediates downregulation of HIV-1 Env but not of VSV-G.

(A) Schematic structure of YxxΦ motif mutants of MARCH8 (222YxxL225 and 232YxxV235). (B) Western blot analysis was performed by using extracts from 293T cells transfected with HA-tagged MARCH8 expression plasmids. Antibodies specific for HA were used to detect MARCH8 proteins. (C, D) Infectivity of viruses prepared from 293T cells cotransfected with Env-defective HIV-1 luciferase (luc) reporter proviral DNA and either a control (Ctrl), HA- WT, HA-222AxxL225 or HA-232AxxV235 MARCH8 plasmid, together with either (C) the VSV-G expression plasmid or (D) the HIV-1 Env expression plasmid. Data are shown as a percentage of the viral infectivity in the absence of MARCH8 (mean + s.d. from three independent experiments). ns; ***p<0.0005 compared with the Ctrl using two-tailed unpaired t-tests. (E, F) BlaM-Vpr-based viral entry assay using VSV-G-pseudotyped viruses (E) or NL4-3 whole viruses (F) produced from cells expressing either control, WT MARCH8, or the 222AxxL225 mutant. Representative FACS dot plots are shown from four independent experiments. (G) VSV-G is downregulated by both WT and 222AxxL225 mutant MARCH8, (H) whereas the cell-surface expression of HIV-1 Env is not affected by the mutant MARCH8. (I) 222AxxL225 MARCH8 expression in producer cells is unable to decrease HIV-1 gp120 levels in viral supernatants. ELISA-based levels of Env gp120 in viral supernatants from 293T cells cotransfected with luc reporter proviral DNA and NL-Env plasmid, together with either MARCH8 WT or its 222AxxL225 mutant. Representative data from three independent experiments are shown as percent Env gp120/Gag p24 in the supernatants relative to that from control cells. (mean + s.d. from three independent experiments). ns; ***p<0.0005 compared with the Ctrl using two-tailed unpaired t-tests.

Schematic diagram of two different molecular mechanisms by which MARCH8 inhibits viral infection.

Left, MARCH8 (red) downregulates VSV-G (violet) in a ubiquitin-dependent manner. The RING-CH domain (pink) of MARCH8 recognizes VSV-G’s cytoplasmic lysine residues, which results in ubiquitin conjugation (shown as orange beads), leading to lysosomal degradation; Right, MARCH8 downregulates HIV-1 Env (green) in a YxxΦ motif-dependent manner. The tyrosine motif located in the C-terminal CT of MARCH8 likely interacts with the adaptor protein μ-subunits (navy) (if this is the case with μ2 or μ1, clathrin (brown) is involved in this step), resulting in the intracellular retention of HIV-1 Env in the TGN without degradation. It should be noted that the downregulation of these viral glycoproteins might not necessarily occur at the plasma membrane. The nucleus and other organelles are not shown.

Tables

Appendix 1—key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene(Homo sapiens)MARCH8NCBIBC025394
Recombinant DNA reagentpNL4-3Adachi et al., 1986
Recombinant DNA reagentpNL-E(-)Iwabu et al., 2009
Recombinant DNA reagentpNL-Luc2-E(-)Tada et al., 2015
Recombinant DNA reagentpC-GagPol-RRETada et al., 2015
Recombinant DNA reagentpC-NLenvTada et al., 2015
Recombinant DNA reagentpCa-RevIwabu et al., 2009
Recombinant DNA reagentpC-VSVgTada et al., 2015
Recombinant DNA reagentpVSVg-T7ETada et al., 2015
Recombinant DNA reagentpCa-EGFPIwabu et al., 2009
Recombinant DNA reagentpMM310Tobiume et al., 2003
Recombinant DNA reagentpC-MARCH8Tada et al., 2015
Recombinant DNA reagentpC-HA-MARCH8Tada et al., 2015
Recombinant DNA reagentpC-HA-MARCH8-CSTada et al., 2015
Recombinant DNA reagentpC-NLenv-CT2K/RThis paperSee Materials and methods
Recombinant DNA reagentpC-VSVg-CT5K/RThis paperSee Materials and methods
Recombinant DNA reagentpC-VSVg-CT5K/R-T7EThis paperSee Materials and methods
Recombinant DNA reagentpC-MARCH8-222AxxL225This paperSee Materials and methods
Recombinant DNA reagentpC-HA-MARCH8-222AxxL225This paperSee Materials and methods
Recombinant DNA reagentpC-MARCH8-232AxxV235This paperSee Materials and methods
Recombinant DNA reagentpC-HA-MARCH8-232AxxV235This paperSee Materials and methods
Sequence-basedreagentNL-env-MfeI-SThis papergacaattggagaagtgaattSense primer for pC-NLenv-CT2K/R
Sequence-basedreagentNLenv-CT2KR-AThis paperctattcCttagttcctgactccaatactgtaggagattccaccaatatCtgagggcOverlapping PCR's antisense primer for pC-NLenv-CT2K/R
Sequence-basedreagentNLenv-CT2/KR-SThis papergccctcaGatattggtggaatctcctacagtattggagtcaggaactaaGgaatagOverlapping PCR's sense primer for pC-NLenv-CT2K/R
Sequence-basedreagentNL-env-XhoI-AThis paperccgCTCGAGttatagcaaaatcctttccaagAntisense primer for pC-NLenv-CT2K/R
Sequence-basedreagentVSVg-BsiWI-SThis paperatCGTACGatgaagtgccttttgtacttSense primer for pC-VSVg-CT5K/R-T7E
Sequence-basedreagentVSVg-CT5K/R-XhoI-AThis paperATctcgaGcCttccaagtcggttcatctctatgtctgtataaatctgtcttCtcCtggtgtgcCttaatCtaatg Antisense primer for pC-VSVg-CT5K/R-T7E
Sequence-basedreagentMARCH8-KpnI-SThis paperggGGTACCatgagcatgccactgcatcagSense primer for pC-MARCH8-222AxxL225 and pC-MARCH8-232AxxV235 
Sequence-basedreagentMARCH8-XhoI-SThis paperccgCTCGAGagcatgccactgcatcagatSense primer for pC-HA-MARCH8-222AxxL225 and pC-HA-MARCH8-232AxxV235 
Sequence-basedreagentMARCH8-222AxxL225-SThis papergtgtaaagtgGCtgtgcaGttgtggaagagOverlapping PCR's sense primer for pC-MARCH8-222AxxL225 and pC-HA-MARCH8-222AxxL225
Sequence-basedreagentMARCH8-222AxxL225-AThis paperctcttccacaaCtgcacaGCcactttacacOverlapping PCR's antisense primer for pC-MARCH8-222AxxL225 and pC-HA-MARCH8-222AxxL225
Sequence-basedreagentMARCH8-232AxxV235-SThis papergagactcaaggccGCtaatagagtgatcOverlapping PCR's sense primer for pC-MARCH8-232AxxV235
Sequence-basedreagentMARCH8-232AxxV235-AThis papergatcactctattaGCggccttgagtctcOverlapping PCR's antiense primer for pC-MARCH8-232AxxV235
Sequence-basedreagentMARCH8-XhoI-AThis paperccgCTCGAGtcagacgtgaatgatttctgAntisense primer for pC-MARCH8-222AxxL225 and pC-MARCH8-232AxxV235 
Sequence-basedreagentMARCH8-NotI-AThis paperattGCGGCCGCtcagacgtgaatgatttctgAntisense primer for pC-HA-MARCH8-222AxxL225 and pC-HA-MARCH8-232AxxV235 
Cell line (H. sapiens)293TATCCCRL-3216
Cell line (H. sapiens)MT4JCRB1216 RRID:CVCL_2632
Cell line (H. sapiens)HeLaATCCCVCL_0030
Cell line (H. sapiens)MAGIC5Mochizuki et al., 1999
Cell line (H. sapiens)HOSATCCCRL-1543
Commercial assayor kitPCR Mycoplasma Detection SetTakaraTKR-6601Mycoplasma detection
Chemicalcompound,drugFuGENE6PromegaE2691Transfection reagent
Commercial assayor kitHIV-1 p24 ELISA KitXpressBio XBR-1000HIV-1 p24 antigen capture ELISA
Commercial assayor kitHIV-1 gp120 ELISA Kit Advanced BioScience Laboratories5429HIV-1 gp120 ELISA
Commercial assayor kitOne-Glo Luciferase Assay ReagentPromegaE6110Luciferase assay
Chemicalcompound,drugProtein A-SepharoseGE Healthcare17-0780-01Immunoprecipitation
Chemicalcompound,drugComplete protease inhibitor cocktailRoche11697498001Protease inhibitor
Chemicalcompound,drugn-octyl-β-D-glucosideDojindoO001Nonionic surfactant
Chemicalcompound,drugSaponinSigma-Aldrich47036Nonionic surfactant
AntibodyAnti-HASigma-AldrichH9658 RRID:AB_260092WB (1:10,000 )Mouse monoclonal
AntibodyAnti-HASigma-AldrichH3663 RRID:AB_262051IF (1:200)Mouse monoclonal
AntibodyAnti-HASigma-AldrichH6908 RRID:AB_260070IF (1:200)Rabbit polyclonal
Antibodyanti-HAGenScriptA00168-40IF (1:200)Goat polyclonal
AntibodyAnti-β-actinSigma-AldrichA5316 RRID:AB_476743WB (1:5,000) Mouse monoclonal
AntibodyAnti-T7 epitope tagMBLPM022 RRID:AB_592788IP (4 μg); WB (1:1,000)Rabbit polyclonal
AntibodyAnti-T7 epitope tagNovagen69522-4 RRID:AB_11211744IF (1:200)Mouse monoclonal
AntibodyAnti-ubiquitinCayman14220IP (4 μg); WB (1:500)Mouse monoclonal (Clone FK2)
AntibodyAnti-gp120AbcamAb21179 RRID:AB_732949FACS (1:150); IF (1:200)Goat polyclonal
AntibodyAnti-gp120Matsushita et al., 1988IF (1:100)Mouse monoclonal (0.5β)kindly provided by S. Matsushita
AntibodyAnti-TGN46AbcamAb50595 RRID:AB_2203289IF (1:200)Rabbit polyclonal 
AntibodyAnti-VSV-G Sigma-AldrichV5507 RRID:AB_261877FACS (1:150)Mouse monoclonal
AntibodyGoat anti-mouse IgG conjugated with R-phycoerythrin Molecular ProbesP-852 RRID:AB_143191FACS (1:500)
AntibodyAlexa 488 donkey anti-mouse IgGMolecular ProbesA-21202 RRID:AB_141607IF (1:400)
AntibodyAlexa 488 donkey anti-goat IgGMolecular ProbesA-11055 RRID:AB_2534102IF (1:400)
AntibodyAlexa 568 donkey anti-rabbit IgGMolecular ProbesA-10042 RRID:AB_2534017 IF (1:400)
AntibodyAlexa 647 donkey anti-goat IgGMolecular ProbesA-21447 RRID:AB_141844FACS (1:500); IF (1:400)
AntibodyAlexa 647 donkey anti-mouse IgG Molecular ProbesA-31571 RRID:AB_162542IF (1:400)
AntibodyAlexa 647 donkey anti-rabbit IgG Molecular ProbesA-31573 RRID:AB_2536183IF (1:400)
Commercial assayor kitECL Western blotting detectionsystemGE HealthcareRPN2109Chemiluminescence
Commercial assayor kit EzWestLumi plus ATTOWSE-7120Chemiluminescence
Chemicalcompound,drugHBSSThermo Fisher14025076Wash buffer
Chemicalcompound,drugCCF2-AM dyeInvitrogenK1023Fluorescent substrate for BlaM
Chemicalcompound,drugPluronic F-127InvitrogenP2443Nonionic surfactant for CCF2-AM dye
Chemicalcompound,drugSaponinSigma-Aldrich47036Non-ionic surfactant for immunofluorescence
Software,algorithmBD FACS Diva SoftwareBD Bioscience
Software,algorithmGraphPadPrism 8.04GraphPad

Additional files

Source data 1

Source Data File for Figures 1B, C, 2C, D and I.

https://cdn.elifesciences.org/articles/57763/elife-57763-data1-v2.xlsx
Source data 2

Source data for Figure 1G and 2B.

Original uncropped images of IP-western blot (ubiquitination assays) in Figure 1G. The PVDF membranes were incubated with an anti-T7-epitope tag antibody, or with an anti-ubiquitin antibody. Images shown in Figure 1G were cropped from the boxed areas, and the brightness/contrast was adjusted equally across the entire image using Photoshop CS6 Figure 2B source data. Original uncropped images of western blot in Figure 2B. The PVDF membrane was incubated with an anti-HA antibody, then stripped and reprobed with an anti-β-actin antibody for a loading control. Images shown in Figure 2B were cropped from the boxed areas, and the brightness/contrast was adjusted equally across the entire image using Photoshop CS6.

https://cdn.elifesciences.org/articles/57763/elife-57763-data2-v2.pdf
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https://cdn.elifesciences.org/articles/57763/elife-57763-transrepform-v2.docx

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  1. Yanzhao Zhang
  2. Takuya Tada
  3. Seiya Ozono
  4. Satoshi Kishigami
  5. Hideaki Fujita
  6. Kenzo Tokunaga
(2020)
MARCH8 inhibits viral infection by two different mechanisms
eLife 9:e57763.
https://doi.org/10.7554/eLife.57763