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Determining the Importance of Peroxisomal Proteins for Viral Infections in Cultured Mammalian Cells

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Peroxisomes

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2643))

Abstract

Peroxisomes have recently been shown to play important roles in the context of viral infections. However, further and more detailed studies should be performed to unravel the specific mechanisms involved. The analysis of the relevance of particular peroxisomal components, such as peroxisomal proteins, for viral infections can be performed by comparing the production of new virus particles in the absence and presence of those specific components. Different methodologies are used to quantify the production of infectious virus particles, depending on the virus, cell type, and the specific characteristics of the viral infection to be analyzed. Here we provide a detailed protocol to study the importance of a putative peroxisomal protein on infection by viruses that induce the death of their host cells. We use the influenza A virus (IAV) infection in A549 cells as a model, and the quantification of the newly produced infectious virus particles is performed by a plaque assay.

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References

  1. Ferreira AR, Marques M, Ribeiro D (2019) Peroxisomes and innate immunity: antiviral response and beyond. Int J Mol Sci 20:1–352. https://doi.org/10.3390/ijms20153795

    Article  CAS  Google Scholar 

  2. Dixit E, Boulant S, Zhang Y et al (2010) Peroxisomes are signaling platforms for antiviral innate immunity. Cell 141:668–681. https://doi.org/10.1016/j.cell.2010.04.018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Sandalio LM, Rodríguez-Serrano M, Romero-Puertas MC, del Río LA (2013) Role of peroxisomes as a source of reactive oxygen species (ROS) signaling molecules. Subcell Biochem 69:231–255. https://doi.org/10.1007/978-94-007-6889-5_13

    Article  CAS  PubMed  Google Scholar 

  4. Ferreira AR, Marques M, Ramos B et al (2022) Emerging roles of peroxisomes in viral infections. Trends Cell Biol 32:124–139. https://doi.org/10.1016/j.tcb.2021.09.010

    Article  CAS  PubMed  Google Scholar 

  5. Racaniello VR, Skalka AM, Flint J, Rall GF (2015) Principles of virology, 4th edition

    Google Scholar 

  6. Payne S (2017) Methods to study viruses. Viruses 37–52. https://doi.org/10.1016/b978-0-12-803109-4.00004-0

  7. Matrosovich M, Matrosovich T, Garten W, Klenk HD (2006) New low-viscosity overlay medium for viral plaque assays. Virol J 3:63. https://doi.org/10.1186/1743-422X-3-63

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Baer A, Kehn-Hall K (2014) Viral concentration determination through plaque assays: using traditional and novel overlay systems. J Vis Exp 52065. https://doi.org/10.3791/52065

  9. Ryu W-S (2017) Diagnosis and methods. Mol Virol Hum Pathog Viruses 47–62. https://doi.org/10.1016/b978-0-12-800838-6.00004-7

  10. De Wit E, Spronken MIJ, Bestebroer TM et al (2004) Efficient generation and growth of influenza virus A/PR/8/34 from eight cDNA fragments. Virus Res 103:155–161. https://doi.org/10.1016/j.virusres.2004.02.028

    Article  CAS  PubMed  Google Scholar 

  11. Alenquer M, Vale-Costa S, Etibor TA et al (2019) Influenza A virus ribonucleoproteins form liquid organelles at endoplasmic reticulum exit sites. Nat Commun 10:1629. https://doi.org/10.1038/s41467-019-09549-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hollý J, Fogelová M, Jakubcová L et al (2017) Comparison of infectious influenza A virus quantification methods employing immuno-staining. J Virol Methods 247:107–113. https://doi.org/10.1016/j.jviromet.2017.06.004

    Article  CAS  PubMed  Google Scholar 

  13. Eisfeld AJ, Neumann G, Kawaoka Y (2014) Influenza A virus isolation, culture and identification. Nat Protoc 9:2663–2681. https://doi.org/10.1038/nprot.2014.180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Karakus U, Crameri M, Lanz C, Yángüez E (2018) Propagation and titration of influenza viruses. Methods Mol Biol 1836:59–88. https://doi.org/10.1007/978-1-4939-8678-1_4

    Article  CAS  PubMed  Google Scholar 

  15. Jorquera PA, Tripp RA (2016) Quantification of RSV infectious particles by plaque assay and immunostaining assay. Methods Mol Biol 1442:33–40. https://doi.org/10.1007/978-1-4939-3687-8_3

    Article  CAS  PubMed  Google Scholar 

  16. Caidi H, Harcourt JL, Haynes LM (2016) RSV growth and quantification by microtitration and qRT- PCR assays. Methods Mol Biol 1442:13–32. https://doi.org/10.1007/978-1-4939-3687-8_2

    Article  CAS  PubMed  Google Scholar 

  17. McKimm-Breschkin JL (2004) A simplified plaque assay for respiratory syncytial virus – direct visualization of plaques without immunostaining. J Virol Methods 120:113–117. https://doi.org/10.1016/j.jviromet.2004.02.020

    Article  CAS  PubMed  Google Scholar 

  18. Mendoza EJ, Manguiat K, Wood H, Drebot M (2020) Two detailed plaque assay protocols for the quantification of infectious SARS-CoV-2. Curr Protoc Microbiol 57:cpmc105. https://doi.org/10.1002/cpmc.105

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Case JB, Bailey AL, Kim AS et al (2020) Growth, detection, quantification, and inactivation of SARS-CoV-2. Virology 548:39–48. https://doi.org/10.1016/J.VIROL.2020.05.015

    Article  CAS  PubMed  Google Scholar 

  20. Despres HW, Mills MG, Shirley DJ et al (2022) Measuring infectious SARS-CoV-2 in clinical samples reveals a higher viral titer:RNA ratio for Delta and Epsilon vs. Alpha variants. Proc Natl Acad Sci U S A 119:e2116518119. https://doi.org/10.1073/pnas.2116518119

    Article  PubMed  PubMed Central  Google Scholar 

  21. Jureka AS, Silvas JA, Basler CF (2020) Propagation, inactivation, and safety testing of SARS-CoV-2. Viruses 12:622. https://doi.org/10.3390/v12060622

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yi MK (2010) Hepatitis C virus: propagation, quantification, and storage. Curr Protoc Microbiol Chapter 15. https://doi.org/10.1002/9780471729259.mc15d01s19

  23. Stewart H, Bartlett C, Ross-Thriepland D et al (2015) A novel method for the measurement of hepatitis C virus infectious titres using the IncuCyte ZOOM and its application to antiviral screening. J Virol Methods 218:59–65. https://doi.org/10.1016/j.jviromet.2015.03.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Dangsagul W, Ruchusatsawat K, Tawatsin A et al (2021) Zika virus isolation, propagation, and quantification using multiple methods. PLoS One 16:e0255314. https://doi.org/10.1371/journal.pone.0255314

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Brien JD, Hassert M, Stone ET et al (2019) Isolation and quantification of zika virus from multiple organs in a mouse. J Vis Exp 2019:e59632. https://doi.org/10.3791/59632

    Article  CAS  Google Scholar 

  26. Carneiro PH, Mendes-Monteiro L, Mohana-Borges R (2022) Virus propagation and plaque assay for dengue virus. Methods Mol Biol 2409:3–9. https://doi.org/10.1007/978-1-0716-1879-0_1

    Article  CAS  PubMed  Google Scholar 

  27. Payne AF, Binduga-Gajewska I, Kauffman EB, Kramer LD (2006) Quantitation of flaviviruses by fluorescent focus assay. J Virol Methods 134:183–189. https://doi.org/10.1016/j.jviromet.2006.01.003

    Article  CAS  PubMed  Google Scholar 

  28. Nadgir SV, Hensler HR, Knowlton ER et al (2013) Fifty percent tissue culture infective dose assay for determining the titer of infectious human herpesvirus 8. J Clin Microbiol 51:1931–1934. https://doi.org/10.1128/JCM.00761-13

    Article  PubMed  PubMed Central  Google Scholar 

  29. Kalser J, Adler B, Mach M et al (2017) Differences in growth properties among two human cytomegalovirus glycoprotein O genotypes. Front Microbiol 8:1609. https://doi.org/10.3389/fmicb.2017.01609

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

The authors acknowledge support from the European Union through the Horizon 2020 program (H2020-WIDESPREAD-2020-5 ID-952373) and from the Portuguese Foundation for Science and Technology (FCT) (PTDC/BIA-CEL/31378/2017; CEECIND/03747/2017; SFRH/BD/ 137851/2018 and UIDB/04501/2020).

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Authors and Affiliations

  1. Institute of Biomedicine (iBiMED) and Department of Medical Sciences, University of Aveiro, Aveiro, Portugal

    Mariana Marques, Ana Rita Ferreira & Daniela Ribeiro

Authors
  1. Mariana Marques
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  2. Ana Rita Ferreira
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  3. Daniela Ribeiro
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Corresponding author

Correspondence to Daniela Ribeiro .

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Editors and Affiliations

  1. Faculty of Health and Life Sciences, Biosciences, University of Exeter, Exeter, Devon, UK

    Michael Schrader

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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature

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Marques, M., Ferreira, A.R., Ribeiro, D. (2023). Determining the Importance of Peroxisomal Proteins for Viral Infections in Cultured Mammalian Cells. In: Schrader, M. (eds) Peroxisomes. Methods in Molecular Biology, vol 2643. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3048-8_21

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  • DOI: https://doi.org/10.1007/978-1-0716-3048-8_21

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3047-1

  • Online ISBN: 978-1-0716-3048-8

  • eBook Packages: Springer Protocols

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