Abstract
Observation of molecular processes inside living cells is fundamental to a deeper understanding of virus-host interactions in filoviral-infected cells. These observations can provide spatiotemporal insights into protein synthesis, protein-protein interaction dynamics, and transport processes of these highly pathogenic viruses. Thus, live-cell imaging provides the possibility for antiviral screening in real time and gives mechanistic insights into understanding filovirus assembly steps that are dependent on cellular factors, which then represent potential targets against this highly fatal disease. Here we describe analysis of living filovirus-infected cells under maximum biosafety (i.e., BSL4) conditions using plasmid-driven expression of fluorescently labeled viral and cellular proteins and/or viral genome-encoded expression of fluorescently labeled proteins. Such multiple-color and multidimensional time-lapse live-cell imaging analyses are a powerful method to gain a better understanding of the filovirus infection cycle.
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References
Hoenen T, Groseth A, Callison J, Takada A, Feldmann H (2013) A novel Ebola virus expressing luciferase allows for rapid and quantitative testing of antivirals. Antivir Res 99(3):207–213. doi:10.1016/j.antiviral.2013.05.017
Kondratowicz AS, Maury WJ (2012) Ebolavirus: a brief review of novel therapeutic targets. Future Microbiol 7(1):1–4. doi:10.2217/fmb.11.110
Dolnik O, Stevermann L, Kolesnikova L, Becker S (2015) Marburg virus inclusions: a virus-induced microcompartment and interface to multivesicular bodies and the late endosomal compartment. Eur J Cell Biol 94(7–9):323–331. doi:10.1016/j.ejcb.2015.05.006
Schmidt KM, Schumann M, Olejnik J, Krahling V, Muhlberger E (2011) Recombinant Marburg virus expressing EGFP allows rapid screening of virus growth and real-time visualization of virus spread. J Infect Dis 204(Suppl 3):S861–S870. doi:10.1093/infdis/jir308
Parhamifar L, Wu L, Andersen H, Moghimi SM (2014) Live-cell fluorescent microscopy platforms for real-time monitoring of polyplex-cell interaction: basic guidelines. Methods 68(2):300–307. doi:10.1016/j.ymeth.2014.02.004
Ettinger A, Wittmann T (2014) Fluorescence live cell imaging. Methods Cell Biol 123:77–94. doi:10.1016/B978-0-12-420138-5.00005-7
Kuhn JH, Andersen KG, Bao Y, Bavari S, Becker S, Bennett RS, Bergman NH, Blinkova O, Bradfute S, Brister JR, Bukreyev A, Chandran K, Chepurnov AA, Davey RA, Dietzgen RG, Doggett NA, Dolnik O, Dye JM, Enterlein S, Fenimore PW, Formenty P, Freiberg AN, Garry RF, Garza NL, Gire SK, Gonzalez JP, Griffiths A, Happi CT, Hensley LE, Herbert AS, Hevey MC, Hoenen T, Honko AN, Ignatyev GM, Jahrling PB, Johnson JC, Johnson KM, Kindrachuk J, Klenk HD, Kobinger G, Kochel TJ, Lackemeyer MG, Lackner DF, Leroy EM, Lever MS, Muhlberger E, Netesov SV, Olinger GG, Omilabu SA, Palacios G, Panchal RG, Park DJ, Patterson JL, Paweska JT, Peters CJ, Pettitt J, Pitt L, Radoshitzky SR, Ryabchikova EI, Saphire EO, Sabeti PC, Sealfon R, Shestopalov AM, Smither SJ, Sullivan NJ, Swanepoel R, Takada A, Towner JS, van der Groen G, Volchkov VE, Volchkova VA, Wahl-Jensen V, Warren TK, Warfield KL, Weidmann M, Nichol ST (2014) Filovirus RefSeq entries: evaluation and selection of filovirus type variants, type sequences, and names. Virus 6(9):3663–3682. doi:10.3390/v6093663
McElroy AK, Spiropoulou CF (2014) Biomarkers for understanding Ebola virus disease. Biomark Med 8(9):1053–1056. doi:10.2217/bmm.14.75
Sanchez A, Geisbert T, Feldmann H (2007) Filoviridae – Marburg and Ebola viruses. In: Knipe D (ed) Fields virology, vol 1, 5th edn. Lippincott Williams and Wilkins, Philadelphia, PA, pp 1410–1448
Towner JS, Khristova ML, Sealy TK, Vincent MJ, Erickson BR, Bawiec DA, Hartman AL, Comer JA, Zaki SR, Stroher U, Gomes da Silva F, del Castillo F, Rollin PE, Ksiazek TG, Nichol ST (2006) Marburgvirus genomics and association with a large hemorrhagic fever outbreak in Angola. J Virol 80(13):6497–6516. doi:10.1128/JVI.00069-06
Krahling V, Dolnik O, Kolesnikova L, Schmidt-Chanasit J, Jordan I, Sandig V, Gunther S, Becker S (2010) Establishment of fruit bat cells (Rousettus aegyptiacus) as a model system for the investigation of filoviral infection. PLoS Negl Trop Dis 4(8):e802. doi:10.1371/journal.pntd.0000802
Enterlein S, Volchkov V, Weik M, Kolesnikova L, Volchkova V, Klenk HD, Muhlberger E (2006) Rescue of recombinant Marburg virus from cDNA is dependent on nucleocapsid protein VP30. J Virol 80(2):1038–1043. doi:10.1128/JVI.80.2.1038-1043.2006
Volchkov VE, Volchkova VA, Muhlberger E, Kolesnikova LV, Weik M, Dolnik O, Klenk HD (2001) Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity. Science 291(5510):1965–1969. doi:10.1126/science.1057269
Kuhn JH, Bao Y, Bavari S, Becker S, Bradfute S, Brister JR, Bukreyev AA, Chandran K, Davey RA, Dolnik O, Dye JM, Enterlein S, Hensley LE, Honko AN, Jahrling PB, Johnson KM, Kobinger G, Leroy EM, Lever MS, Muhlberger E, Netesov SV, Olinger GG, Palacios G, Patterson JL, Paweska JT, Pitt L, Radoshitzky SR, Saphire EO, Smither SJ, Swanepoel R, Towner JS, van der Groen G, Volchkov VE, Wahl-Jensen V, Warren TK, Weidmann M, Nichol ST (2013) Virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family Filoviridae. Arch Virol 158(1):301–311. doi:10.1007/s00705-012-1454-0
Schudt G, Kolesnikova L, Dolnik O, Sodeik B, Becker S (2013) Live-cell imaging of Marburg virus-infected cells uncovers actin-dependent transport of nucleocapsids over long distances. Proc Natl Acad Sci U S A 110(35):14402–14407. doi:10.1073/pnas.1307681110
Ebihara H, Theriault S, Neumann G, Alimonti JB, Geisbert JB, Hensley LE, Groseth A, Jones SM, Geisbert TW, Kawaoka Y, Feldmann H (2007) In vitro and in vivo characterization of recombinant Zaire ebolavirus expressing eGFP. J Infect Dis 196(Suppl 2):313–322
Hoenen T, Shabman RS, Groseth A, Herwig A, Weber M, Schudt G, Dolnik O, Basler CF, Becker S, Feldmann H (2012) Inclusion bodies are a site of ebolavirus replication. J Virol 86(21):11779–11788. doi:10.1128/JVI.01525-12
Dolnik O, Kolesnikova L, Welsch S, Strecker T, Schudt G, Becker S (2014) Interaction with Tsg101 is necessary for the efficient transport and release of nucleocapsids in marburg virus-infected cells. PLoS Pathog 10(10):e1004463. doi:10.1371/journal.ppat.1004463
Schudt G, Dolnik O, Kolesnikova L, Biedenkopf N, Herwig A, Becker S (2015) Transport of Ebolavirus nucleocapsids is dependent on actin polymerization: live-cell imaging analysis of Ebolavirus-infected cells. J Infect Dis 212(Suppl 2):S160–S166. doi:10.1093/infdis/jiv083
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Schudt, G., Dolnik, O., Becker, S. (2017). Live-Cell Imaging of Filoviruses. In: Hoenen, T., Groseth, A. (eds) Ebolaviruses. Methods in Molecular Biology, vol 1628. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7116-9_15
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DOI: https://doi.org/10.1007/978-1-4939-7116-9_15
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