ER-export and ARFRP1/AP-1–dependent delivery of SARS-CoV-2 Envelope to lysosomes controls late stages of viral replication

  1. Guy J. Pearson
  2. Harriet V. Mears
  3. Malgorzata Broncel
  4. Ambrosius P. Snijders
  5. David L. V. Bauer
  6. Jeremy G. Carlton

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Abstract

The β-coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the global COVID-19 pandemic. Coronaviral Envelope (E) proteins are pentameric viroporins that play essential roles in assembly, release, and pathogenesis. We developed a nondisruptive tagging strategy for SARS-CoV-2 E and find that, at steady state, it localizes to the Golgi and to lysosomes. We identify sequences in E, conserved across Coronaviridae , responsible for endoplasmic reticulum–to–Golgi export, and relate this activity to interaction with COP-II via SEC24. Using proximity biotinylation, we identify an ADP ribosylation factor 1/adaptor protein–1 (ARFRP1/AP-1)–dependent pathway allowing Golgi-to-lysosome trafficking of E. We identify sequences in E that bind AP-1, are conserved across β-coronaviruses, and allow E to be trafficked from Golgi to lysosomes. We show that E acts to deacidify lysosomes and, by developing a trans-complementation assay for SARS-CoV-2 structural proteins, that lysosomal delivery of E and its viroporin activity is necessary for efficient viral replication and release.

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  1. Version published to 10.1126/sciadv.adl5012
  2. Version published to 10.1101/2021.06.30.450614v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.06.30.450614: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    RandomizationE phenotype quantification was performed on 50 cells per condition, with sample identification randomised and blinded during scoring.
    BlindingE phenotype quantification was performed on 50 cells per condition, with sample identification randomised and blinded during scoring.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: Cell Culture: STR-profiled, mycoplasma-free vials of 293T and VeroE6 cells were obtained from the Crick Cell Services Science Technology Platform.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies and fluorescent labels: An antibody against GAPDH (MAB374) was from Millipore; an antibody against SARS-CoV-2 Spike (GTX632604) was from GeneTex; an antibody against SARS-CoV-2 Nucleocapsid (BS-41408R) was from Bioss; an antibody against SARS-CoV Membrane (101-401-A55) was from (Rockland); an antibody against ERGIC53 (E1031) was from Sigma-Aldrich; an antibody against GM130 (610822) was from BD Biosceinces; an antibody against TGN46 (ab50595) was from Abcam; an antibody against EEA1 (610457) was from BD Biosciences; an antibody against O-GlcNAc (CTD110.6) was from Sigma; an antibody against HA.11 (16B12) was from Biolegend; an antibody against HaloTag (G9211) was from Promega; an antibody against GFP (7.1/13.1) was from Roche; an antibody against RER1 (HPA051400) was from Sigma-Aldrich; an antibody against PALS1 (17710-1-AP) was from Proteintech; an antibody against GORAPS2 (10598-1-AP) was from Proteintech; an HRP-conjugated antibody against Streptavidin (S911) was from Invitrogen; Alexa conjugated secondary antibodies were from Invitrogen and HRP-conjugated secondary antibodies were from Millipore.
    GAPDH
    suggested: (Millipore Cat# MAB374, RRID:AB_2107445)
    GTX632604
    suggested: (GeneTex Cat# GTX632604, RRID:AB_2864418)
    ERGIC53
    suggested: (Sigma-Aldrich Cat# E1031, RRID:AB_532237)
    GM130
    suggested: (BD Biosciences Cat# 610822, RRID:AB_398141)
    TGN46
    suggested: (Abcam Cat# ab50595, RRID:AB_2203289)
    EEA1
    suggested: (BD Biosciences Cat# 610457, RRID:AB_397830)
    O-GlcNAc
    suggested: None
    HA.11
    suggested: None
    antibody against HaloTag (G9211
    suggested: None
    GFP
    suggested: None
    RER1
    suggested: (Atlas Antibodies Cat# HPA051400, RRID:AB_2681469)
    HPA051400
    suggested: (Atlas Antibodies Cat# HPA051400, RRID:AB_2681469)
    PALS1
    suggested: (Proteintech Cat# 17710-1-AP, RRID:AB_2282012)
    GORAPS2
    suggested: None
    antibody against Streptavidin (S911
    suggested: None
    Cells were then prepared for fixed cell imaging, with the presence of HA tagged Dynamin2-K44A detected by detection by an HA antibody.
    HA
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Fixed cell imaging: VeroE6 cells were plated at 40,000 per well on 13mm No. 1.5 coverslips and transfected as described the following day.
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    TurboID Proximity Biotinylation, Pull Down, and Mass Spectrometry: HEK293T cells were grown for at least 5 days in biotin-free growth media to remove all sources of biotin.
    HEK293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    For analysis of post-translational modification on E, 600,000 293T cells in a 35 mm dish were transfected with 2 mg pCR3.1 E-HTSite3, or derivatives.
    293T
    suggested: None
    Recombinant DNA
    SentencesResources
    Insertion of HaloTag in the E coding sequence was performed using HiFi DNA Assembly, with HaloTag amplified by PCR from pHTN-HaloTag CMV-neo (Promega), with a Gly-Gly-Gly-Ser linker placed either side of the HaloTag at site 3 and 4, a single linker placed between E N/C-terminus and HaloTag at tag sites 1 or 5, and a Gly-Gly-Gly-Ser-HaloTag-Gly-Gly-Gly-Ser-Glu-Glu inserted at site 2.
    pHTN-HaloTag
    suggested: None
    Hemagglutinin (HA)-TurboID tagging at sites 3 and 4 was performed by HiFi DNA Assembly, with HA-TurboID amplified by PCR from 3xHA-TurboID-NLS_pCDNA3 (Addgene #107171) and inserted with a Gly-Gly-Gly-Ser linker either side of the HA-TurboID sequence.
    3xHA-TurboID-NLS_pCDNA3
    suggested: RRID:Addgene_107171)
    Emerald-TurboID was used for a cytosolic control in proximity biotinylation experiments and was generated by using HiFi DNA Assembly to assemble Emerald-TurboID in a pLXIN vector.
    pLXIN
    suggested: RRID:Addgene_132935)
    mEmerald was amplified from mEmerald-Sec61b-C1 (Addgene #90992) and TurboID amplified from 3xHA-TurboID-NLS_pCDNA3, with AgeI and EcoRI restriction sites for insertion into pEGFP-C1. pHLARE plasmids were a kind gift from Prof. Diana Barber (University of California, San Francisco).
    mEmerald-Sec61b-C1
    suggested: RRID:Addgene_90992)
    pEGFP-C1
    suggested: None
    pHLARE
    suggested: None
    Immunoprecipitation Assay: 25 million 293T cells in a 150 mm dish were transfected with 40 mg pCR3.1 E-HTSite3 or pCR3.1 using Polyethyleneimine.
    pCR3.1
    suggested: RRID:Addgene_106457)
    For analysis of post-translational modification on E, 600,000 293T cells in a 35 mm dish were transfected with 2 mg pCR3.1 E-HTSite3, or derivatives.
    pCR3.1 E-HTSite3
    suggested: None
    Virus Like Particle Production Assay: 7.5 million 293T cells in a 100 mm dish were transfected with a mixture comprising 5 mg pCR3.1 SARS-CoV-2 S (codon optimised), 3 mg pCR3.1 SARS-CoV-2 M, 3 mg pCR3.1 SARS-CoV-2 E (or derivatives) and 1 mg of pCR3.1 SARS-CoV-2 N (codon optimised).
    pCR3.1 SARS-CoV-2 N
    suggested: None
    Software and Algorithms
    SentencesResources
    A sequence corresponding to the native sequence of Membrane was synthesised by GeneWIZ.
    GeneWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Antibodies and fluorescent labels: An antibody against GAPDH (MAB374) was from Millipore; an antibody against SARS-CoV-2 Spike (GTX632604) was from GeneTex; an antibody against SARS-CoV-2 Nucleocapsid (BS-41408R) was from Bioss; an antibody against SARS-CoV Membrane (101-401-A55) was from (Rockland); an antibody against ERGIC53 (E1031) was from Sigma-Aldrich; an antibody against GM130 (610822) was from BD Biosceinces; an antibody against TGN46 (ab50595) was from Abcam; an antibody against EEA1 (610457) was from BD Biosciences; an antibody against O-GlcNAc (CTD110.6) was from Sigma; an antibody against HA.11 (16B12) was from Biolegend; an antibody against HaloTag (G9211) was from Promega; an antibody against GFP (7.1/13.1) was from Roche; an antibody against RER1 (HPA051400) was from Sigma-Aldrich; an antibody against PALS1 (17710-1-AP) was from Proteintech; an antibody against GORAPS2 (10598-1-AP) was from Proteintech; an HRP-conjugated antibody against Streptavidin (S911) was from Invitrogen; Alexa conjugated secondary antibodies were from Invitrogen and HRP-conjugated secondary antibodies were from Millipore.
    GeneTex
    suggested: (GeneTex, RRID:SCR_000069)
    Bioss
    suggested: (Bioss Inc, RRID:SCR_013539)
    BD Biosceinces
    suggested: None
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Sigma
    suggested: (SigmaPlot, RRID:SCR_003210)
    Biolegend
    suggested: (BioLegend, RRID:SCR_001134)
    Promega
    suggested: (Promega, RRID:SCR_006724)
    Proteintech
    suggested: (Proteintech Group, RRID:SCR_008986)
    Acquired images were processed using Zeiss’ “Auto” 2D Airyscan processing, and image brightness levels and image crops were adjusted and performed using the FIJI distribution of ImageJ.
    Zeiss’
    suggested: None
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Raw files were then exported into FLIMfit software35 for analysis.
    FLIMfit
    suggested: (FLIMfit, RRID:SCR_016298)
    Intensity based absolute quantification (iBAQ) in MaxQuant was performed using a built-in quantification algorithm36 enabling the ‘Match between runs’ option (time window 0.7 minutes) within replicates.
    MaxQuant
    suggested: (MaxQuant, RRID:SCR_014485)
    MaxQuant output files were processed with Perseus, v1.4.0.238.
    Perseus
    suggested: (Perseus, RRID:SCR_015753)
    Statistical analysis: 2-tailed Student’s T-tests, or ordinary 1-way ANOVA with the indicated post-hoc tests were used to assess significance between test samples and controls and were performed using GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33 and 30. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2021.06.30.450614v1 on bioRxiv