Visualizing the Internalization of Marburg Viruslike Particles into Living Cells

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2024 Nov 25

PMID 39585615

Authors

Asuka Nanbo

Miako Sakaguchi

Wakako Furuyama

Affiliations

Soon will be listed here.

Abstract

Viral entry into cells is a pivotal stage of the infection process and, therefore, a prime target for the development of antiviral therapeutics. Here, we describe a system to monitor the internalization of lipophilic dye-labeled Marburg viruslike particles (VLPs) into living cells. Using cells stably expressing fluorescent protein-fused markers for specific cell organelles, the VLP entry process can be visualized. This procedure enables the characterization of the entry process by visualizing individual steps using specific bio-probes. Additionally, when combined with image analysis, this method allows for the quantification of the efficiencies of individual entry steps including particle adsorption, uptake by endocytosis, and membrane fusion. Finally, this method can be used for antiviral drug screening.

References

Liu D, Pan L, Zhai H, Qiu H, Sun Y . Virus tracking technologies and their applications in viral life cycle: research advances and future perspectives. Front Immunol. 2023; 14:1204730. PMC: 10272434. DOI: 10.3389/fimmu.2023.1204730. View

Liu S, Wang Z, Xie H, Liu A, Lamb D, Pang D . Single-Virus Tracking: From Imaging Methodologies to Virological Applications. Chem Rev. 2020; 120(3):1936-1979. PMC: 7075663. DOI: 10.1021/acs.chemrev.9b00692. View

Li Q, Li W, Yin W, Guo J, Zhang Z, Zeng D . Single-Particle Tracking of Human Immunodeficiency Virus Type 1 Productive Entry into Human Primary Macrophages. ACS Nano. 2017; 11(4):3890-3903. DOI: 10.1021/acsnano.7b00275. View

Furuyama W, Shifflett K, Feldmann H, Marzi A . The Ebola virus soluble glycoprotein contributes to viral pathogenesis by activating the MAP kinase signaling pathway. PLoS Pathog. 2021; 17(9):e1009937. PMC: 8478236. DOI: 10.1371/journal.ppat.1009937. View

Isono M, Furuyama W, Kuroda M, Kondoh T, Igarashi M, Kajihara M . A biaryl sulfonamide derivative as a novel inhibitor of filovirus infection. Antiviral Res. 2020; 183:104932. PMC: 11075116. DOI: 10.1016/j.antiviral.2020.104932. View

Nanbo A, Maruyama J, Imai M, Ujie M, Fujioka Y, Nishide S . Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles. PLoS Pathog. 2018; 14(1):e1006848. PMC: 5786336. DOI: 10.1371/journal.ppat.1006848. View

Furuyama W, Marzi A, Nanbo A, Haddock E, Maruyama J, Miyamoto H . Discovery of an antibody for pan-ebolavirus therapy. Sci Rep. 2016; 6:20514. PMC: 4748290. DOI: 10.1038/srep20514. View

Furuyama W, Marzi A, Carmody A, Maruyama J, Kuroda M, Miyamoto H . Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection. PLoS Pathog. 2016; 12(12):e1006139. PMC: 5231391. DOI: 10.1371/journal.ppat.1006139. View

Nanbo A, Imai M, Watanabe S, Noda T, Takahashi K, Neumann G . Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog. 2010; 6(9):e1001121. PMC: 2944813. DOI: 10.1371/journal.ppat.1001121. View

10.

Hao X, Shang X, Wu J, Shan Y, Cai M, Jiang J . Single-particle tracking of hepatitis B virus-like vesicle entry into cells. Small. 2011; 7(9):1212-8. DOI: 10.1002/smll.201002020. View

11.

Chen C, Zhuang X . Epsin 1 is a cargo-specific adaptor for the clathrin-mediated endocytosis of the influenza virus. Proc Natl Acad Sci U S A. 2008; 105(33):11790-5. PMC: 2504482. DOI: 10.1073/pnas.0803711105. View

12.

Lakadamyali M, Rust M, Zhuang X . Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell. 2006; 124(5):997-1009. PMC: 2660893. DOI: 10.1016/j.cell.2005.12.038. View

13.

Rust M, Lakadamyali M, Zhang F, Zhuang X . Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat Struct Mol Biol. 2004; 11(6):567-73. PMC: 2748740. DOI: 10.1038/nsmb769. View

14.

van der Schaar H, Rust M, Chen C, van der Ende-Metselaar H, Wilschut J, Zhuang X . Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells. PLoS Pathog. 2008; 4(12):e1000244. PMC: 2592694. DOI: 10.1371/journal.ppat.1000244. View

15.

van der Schaar H, Rust M, Waarts B, van der Ende-Metselaar H, Kuhn R, Wilschut J . Characterization of the early events in dengue virus cell entry by biochemical assays and single-virus tracking. J Virol. 2007; 81(21):12019-28. PMC: 2168764. DOI: 10.1128/JVI.00300-07. View

16.

Coller K, Berger K, Heaton N, Cooper J, Yoon R, Randall G . RNA interference and single particle tracking analysis of hepatitis C virus endocytosis. PLoS Pathog. 2009; 5(12):e1000702. PMC: 2790617. DOI: 10.1371/journal.ppat.1000702. View

17.

Jha N, Latinovic O, Martin E, Novitskiy G, Marin M, Miyauchi K . Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion. PLoS Pathog. 2011; 7(1):e1001260. PMC: 3024281. DOI: 10.1371/journal.ppat.1001260. View

18.

Miyauchi K, Kim Y, Latinovic O, Morozov V, Melikyan G . HIV enters cells via endocytosis and dynamin-dependent fusion with endosomes. Cell. 2009; 137(3):433-44. PMC: 2696170. DOI: 10.1016/j.cell.2009.02.046. View

19.

McDonald D, Vodicka M, Lucero G, Svitkina T, Borisy G, Emerman M . Visualization of the intracellular behavior of HIV in living cells. J Cell Biol. 2002; 159(3):441-52. PMC: 2173076. DOI: 10.1083/jcb.200203150. View

20.

Ayala-Nunez N, Wilschut J, Smit J . Monitoring virus entry into living cells using DiD-labeled dengue virus particles. Methods. 2011; 55(2):137-43. DOI: 10.1016/j.ymeth.2011.07.009. View