ARF6 is a host factor for SARS-CoV-2 infection in vitro

C. Mirabelli; J. Bragazzi Cunha; J. W. Wotring; E. J. Sherman; J. El Saghir; J. Harder; M. Kretzler; J. Z. Sexton; B. T. Emmer; C. E. Wobus

doi:10.1099/jgv.0.001868

Volume 104, Issue 6

Research Article

Open Access

ARF6 is a host factor for SARS-CoV-2 infection in vitro

C. Mirabelli^1,†, J. Bragazzi Cunha², J. W. Wotring³, E. J. Sherman^2,‡, J. El Saghir⁴, J. Harder⁴, M. Kretzler⁴, J. Z. Sexton³, B. T. Emmer² and C. E. Wobus¹
View Affiliations Hide Affiliations

Affiliations: ¹ Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA ² Department of Internal Medicine, Division of Hospital Medicine, University of Michigan, Ann Arbor, Michigan, USA ³ Department of Internal Medicine, Division of Gastroenterology and Hepatology, University of Michigan, Ann Arbor, Michigan, USA ⁴ Department of Internal Medicine, Division of Nephrology, University of Michigan, Ann Arbor, Michigan, USA ^† Present address: Institut für Virologie und Zellbiologie, University of Lübeck, Lübeck, Germany ^‡ Present address: Territory Manager at Takara Bio, Inc., MI, MN, IN, KY, USA
*Correspondence: C. Mirabelli, [email protected] *Correspondence: C. E. Wobus, [email protected]
Published: 21 June 2023 https://doi.org/10.1099/jgv.0.001868

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged beta-coronavirus that enter cells via two routes, direct fusion at the plasma membrane or endocytosis followed by fusion with the late endosome/lysosome. While the viral receptor, ACE2, multiple entry factors and the mechanism of fusion of the virus at the plasma membrane have been investigated extensively, viral entry via the endocytic pathway is less understood. By using a human hepatocarcinoma cell line, Huh-7, which is resistant to the antiviral action of the TMPRSS2 inhibitor camostat, we discovered that SARS-CoV-2 entry is not dependent on dynamin but on cholesterol. ADP-ribosylation factor 6 (ARF6) has been described as a host factor for SARS-CoV-2 replication and is involved in the entry and infection of several pathogenic viruses. Using CRISPR/Cas9 genetic deletion, a modest reduction in SARS-CoV-2 uptake and infection in Huh-7 was observed. In addition, pharmacological inhibition of ARF6 with the small molecule NAV-2729 showed a dose-dependent reduction of viral infection. Importantly, NAV-2729 also reduced SARS-CoV-2 viral loads in more physiological models of infection: Calu-3 cells and kidney organoids. This highlighted a role for ARF6 in multiple cell contexts. Together, these experiments point to ARF6 as a putative target to develop antiviral strategies against SARS-CoV-2.

Received: 29/06/2022
Accepted: 12/06/2023
Published Online: 21/06/2023

Keyword(s): ARF6 , endocytic pathway , entry , SARS-CoV-2 and therapeutic targets

Funding

This study was supported by the:

Pharmacological Sciences Training Program T32 (Award GM007767)
- Principle Award Recipient: J.W.Wotring
National Center for Advancing Translational Sciences Grant (Award UL1TR002240)
- Principle Award Recipient: J.Z.Sexton
NIH-NIDDK (Award R01DK120623)
- Principle Award Recipient: J.Z.Sexton
Marie Skłodowska-Curie Actions (Award GA 841247)
- Principle Award Recipient: C.Mirabelli
University of Michigan (Award UniveBiological Science Scholars Program)
- Principle Award Recipient: C.E. Wobus

This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.

Article metrics loading...

/content/journal/jgv/10.1099/jgv.0.001868

2023-06-21

2024-05-08

Full text loading...

/deliver/fulltext/jgv/104/6/jgv001868.html?itemId=/content/journal/jgv/10.1099/jgv.0.001868&mimeType=html&fmt=ahah

References

Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 2020; 181:271–280 [View Article] [PubMed]
[Google Scholar]
Jackson CB, Farzan M, Chen B, Choe H. Mechanisms of SARS-CoV-2 entry into cells. Nat Rev Mol Cell Biol 2022; 23:3–20 [View Article] [PubMed]
[Google Scholar]
Mercer J, Schelhaas M, Helenius A. Virus entry by endocytosis. Annu Rev Biochem 2010; 79:803–833 [View Article] [PubMed]
[Google Scholar]
Bayati A, Kumar R, Francis V, McPherson PS. SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosis. J Biol Chem 2021; 296:100306 [View Article] [PubMed]
[Google Scholar]
Van Acker T, Tavernier J, Peelman F. The small GTPase Arf6: an overview of its mechanisms of action and of its role in host- pathogen interactions and innate immunity.. Int J Mol Sci 2019; 20:2209 [View Article] [PubMed]
[Google Scholar]
Gordon DE, Jang GM, Bouhaddou M, Xu J, Obernier K et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 2020; 583:459–468 [View Article] [PubMed]
[Google Scholar]
Chen F, Shi Q, Pei F, Vogt A, Porritt RA et al. A systems-level study reveals host-targeted repurposable drugs against SARS-CoV-2 infection. Mol Syst Biol 2021; 17:e10239 [View Article] [PubMed]
[Google Scholar]
Harder JL, Menon R, Otto EA, Zhou J, Eddy S et al. Organoid single cell profiling identifies a transcriptional signature of glomerular disease. JCI Insight 2019; 4:e122697 [View Article] [PubMed]
[Google Scholar]
Mirabelli C, Wotring JW, Zhang CJ, McCarty SM, Fursmidt R et al. Morphological cell profiling of SARS-CoV-2 infection identifies drug repurposing candidates for COVID-19. Proc Natl Acad Sci 2021; 118:e2105815118 [View Article] [PubMed]
[Google Scholar]
Sanjana NE, Shalem O, Zhang F. Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 2014; 11:783–784 [View Article] [PubMed]
[Google Scholar]
Emmer BT, Sherman EJ, Lascuna PJ, Graham SE, Willer CJ et al. Genome-scale CRISPR screening for modifiers of cellular LDL uptake. PLoS Genet 2021; 17:e1009285 [View Article] [PubMed]
[Google Scholar]
Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C et al. Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 2006; 10:839–850 [View Article] [PubMed]
[Google Scholar]
Sviridov D, Miller YI, Ballout RA, Remaley AT, Bukrinsky M. Targeting lipid rafts-a potential therapy for COVID-19. Front Immunol 2020; 11:574508 [View Article] [PubMed]
[Google Scholar]
Li X, Zhu W, Fan M, Zhang J, Peng Y et al. Dependence of SARS-CoV-2 infection on cholesterol-rich lipid raft and endosomal acidification. Comput Struct Biotechnol J 2021; 19:1933–1943 [View Article] [PubMed]
[Google Scholar]
Mayor S, Parton RG, Donaldson JG. Clathrin-independent pathways of Endocytosis. Cold Spring Harb Perspect Biol 2014; 6(6): [View Article]
[Google Scholar]
Shang J, Wan Y, Luo C, Ye G, Geng Q et al. Cell entry mechanisms of SARS-CoV-2. Proc Natl Acad Sci U S A 2020; 117:11727–11734 [View Article] [PubMed]
[Google Scholar]
Pia L, Rowland-Jones S. Omicron entry route. Nat Rev Immunol 2022; 22:144 [View Article] [PubMed]
[Google Scholar]
Kim Y, Jang G, Lee D, Kim N, Seon JW et al. Trypsin enhances SARS-CoV-2 infection by facilitating viral entry. Arch Virol 2022; 167:441–458 [View Article] [PubMed]
[Google Scholar]
Wang H, Yang P, Liu K, Guo F, Zhang Y et al. SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway. Cell Res 2008; 18:290–301 [View Article] [PubMed]
[Google Scholar]
Zhou Y-Q, Wang K, Wang X-Y, Cui H-Y, Zhao Y et al. SARS-CoV-2 pseudovirus enters the host cells through spike protein-CD147 in an Arf6-dependent manner. Emerg Microbes Infect 2022; 11:1135–1144 [View Article] [PubMed]
[Google Scholar]
Rosenberg EM, Jian X, Soubias O, Yoon H-Y, Yadav MP et al. The small molecule inhibitor NAV-2729 has a complex target profile including multiple ADP-ribosylation factor regulatory proteins. J Biol Chem 2023; 299:102992 [View Article] [PubMed]
[Google Scholar]
Koch J, Uckeley ZM, Doldan P, Stanifer M, Boulant S et al. TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells. EMBO J 2021; 40:e107821 [View Article] [PubMed]
[Google Scholar]

http://instance.metastore.ingenta.com/content/journal/jgv/10.1099/jgv.0.001868

ARF6 is a host factor for SARS-CoV-2 infection in vitro

J. Gen. Virol. 104, 001868 (2023); https://doi.org/10.1099/jgv.0.001868

/content/journal/jgv/10.1099/jgv.0.001868

Data & Media loading...

Supplements

Volume 104, Issue 6

Research Article

Open Access

ARF6 is a host factor for SARS-CoV-2 infection in vitro

Abstract

Funding

Supplementary material 1

Most read this month

Most cited Most Cited RSS feed

A tale of two clades: monkeypox viruses

Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5

Updated classification of norovirus genogroups and genotypes

ICTV Virus Taxonomy Profile: Parvoviridae

Characteristics of the Microplate Method of Enzyme-Linked Immunosorbent Assay for the Detection of Plant Viruses

ICTV Virus Taxonomy Profile: Picornaviridae

ICTV Virus Taxonomy Profile: Flaviviridae

ICTV Virus Taxonomy Profile: Hepeviridae 2022

ICTV Virus Taxonomy Profile: Adenoviridae 2022

Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’