Abstract
This chapter describes the methods for the study of binding and entry of the two different forms of vaccinia virus (VV)—the intracellular mature virus (IMV) and extracellular enveloped virus (EEV)—using immunofluorescent staining and confocal microscopy. After binding to or penetration of the cells, IMV, EEV, and virus cores are distinguished by different antibodies. Bound virus or penetrated cores are visualized and recorded by confocal microscopy and can be accurately counted. Although specific antibodies to IMV, EEV, and virus cores are required, this method is highly quantitative and also allows the recognition of virus aggregates, which would not be possible using other techniques, such as flow cytometry and radiolabeling of virus particles. Furthermore, this method bypasses the need for EEV purification that may damage the EEV membrane and release an IMV particle.
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References
Smith, G. L., Vanderplasschen, A., and Law, M. (2002) The formation and function of extracellular enveloped vaccinia virus. J. Gen. Virol. 83, 2915–2931.
McIntosh, A. A. and Smith, G. L. (1996) Vaccinia virus glycoprotein A34R is required for infectivity of extracellular enveloped virus. J. Virol. 70, 272–281.
Ichihashi, Y. (1996) Extracellular enveloped vaccinia virus escapes neutralization. Virology 217, 478–485.
Wolffe, E. J., Katz, E., Weisberg, A., and Moss, B. (1997) The A34R glycoprotein gene is required for induction of specialized actin-containing microvilli and efficient cell-to-cell transmission of vaccinia virus. J. Virol. 71, 3904–3915.
Vanderplasschen, A. and Smith, G. L. (1997) A novel virus binding assay using confocal microscopy: demonstration that the intracellular and extracellular vaccinia virions bind to different cellular receptors. J. Virol. 71, 4032–4041.
Postlethwaite, R. (1960) A plaque technique for the titration of vaccinia virus in chick embryo cells and some features of vaccinial infection in this system. Virology 10, 466–482.
Payne, L. G. and Norrby, E. (1978) Adsorption and penetration of enveloped and naked vaccinia virus particles. J. Virol. 27, 19–27.
Dales, S. (1962) An electron microscope study of the early association between two mammalian viruses and their hosts. J. Cell Biol. 13, 303–321.
Granados, R. R. (1973) Entry of an insect poxvirus by fusion of the virus envelope with the host cell membrane. Virology 52, 305–309.
Armstrong, J. A., Metz, D. H., and Young, M. R. (1973) The mode of entry of vaccinia virus into L cells. J. Gen. Virol. 21, 533–537.
Chang, A. and Metz, D. H. (1976) Further investigations on the mode of entry of vaccinia virus into cells. J. Gen. Virol. 32, 275–282.
Krijnse-Locker, J., Kuehn, A., Schleich, S., Rutter, G., Hohenberg, H., Wepf, R., and Griffiths, G. (2000) Entry of the two infectious forms of vaccinia virus at the plasma membrane is signaling-dependent for the IMV but not the EEV. Mol. Biol. Cell. 11, 2497–2511.
Hügin, A. W. and Hauser, C. (1994) The epidermal growth factor receptor is not a receptor for vaccinia virus. J. Virol. 68, 8409–8412.
Vanderplasschen, A., Hollinshead, M., and Smith, G. L. (1998) Intracellular and extracellular vaccinia virions enter cells by different mechanisms. J. Gen. Virol. 79, 877–887.
Allison, A. C. and Valentine, R. C. (1960) Virus particle adsorption III. Adsorption of viruses by cell monolayers and effects of some variables on adsorption. Biochim. Biophys. Acta 40, 400–410.
Janeczko, R. A., Rodriguez, J. F., and Esteban, M. (1987) Studies on the mechanism of entry of vaccinia virus in animal cells. Arch. Virol. 92, 135–150.
Doms, R. W., Blumenthal, R., and Moss, B. (1990) Fusion of intra-and extracellular forms of vaccinia virus with the cell membrane. J. Virol. 64, 4884–4892.
Rodriguez, D., Rodriguez, J. R., Ojakian, G. K., and Esteban, M. (1991) Vaccinia virus preferentially enters polarized epithelial cells through the basolateral surface. J. Virol. 65, 494–498.
Lai, C. F., Gong, S. C., and Esteban, M. (1991) The 32-kilodalton envelope protein of vaccinia virus synthesized in Escherichia coli binds with specificity to cell surfaces. J. Virol. 65, 499–504.
Chang, W., Hsiao, J. C., Chung, C. S., and Bair, C. H. (1995) Isolation of a monoclonal antibody which blocks vaccinia virus infection. J. Virol. 69, 517–522.
Chung, C. S., Hsiao, J. C., Chang, Y. S., and Chang, W. (1998) A27L protein mediates vaccinia virus interaction with cell surface heparan sulfate. J. Virol. 72, 1577–1585.
Hsiao, J. C., Chung, C. S., and Chang, W. (1999) Vaccinia virus envelope D8L protein binds to cell surface chondroitin sulfate and mediates the adsorption of intracellular mature virions to cells. J. Virol. 73, 8750–8761.
Lalani, A. S., Masters, J., Zeng, W., Barrett, J., Pannu, R., Everett, H., Arendt, C. W., and McFadden, G. (1999) Use of chemokine receptors by poxviruses. Science 286, 1968–1971.
Lin, C. L., Chung, C. S., Heine, H. G., and Chang, W. (2000) Vaccinia virus envelope H3L protein binds to cell surface heparan sulfate and is important for intracellular mature virion morphogenesis and virus infection in vitro and in vivo. J. Virol. 74, 3353–3365.
Rodger, G. and Smith, G. L. (2002) Replacing the SCR domains of vaccinia virus protein B5R with EGFP causes a reduction in plaque size and actin tail formation but enveloped virions are still transported to the cell surface. J. Gen. Virol. 83, 323–332.
Geada, M. M., Galindo, I., Lorenzo, M. M., Perdiguero, B., and Blasco, R. (2001) Movements of vaccinia virus intracellular enveloped virions with GFP tagged to the F13L envelope protein. J. Gen. Virol. 82, 2747–2760.
Ward, B. M. and Moss, B. (2001) Vaccinia virus intracellular movement is associated with microtubules and independent of actin tails. J. Virol. 75, 11651–11663.
Vanderplasschen, A. and Smith, G. L. (1999) Using confocal microscopy to study virus binding and entry into cells. Methods Enzymol. 307, 591–607.
Law, M. and Smith, G. L. (2001) Antibody neutralization of the extracellular enveloped form of vaccinia virus. Virology 280, 132–142.
Czerny, C. P. and Mahnel, H. (1990) Structural and functional analysis of orthopoxvirus epitopes with neutralizing monoclonal antibodies. J. Gen. Virol. 71, 2341–2352.
Parkinson, J. E. and Smith, G. L. (1994) Vaccinia virus gene A36R encodes a M(r) 43-50 K protein on the surface of extracellular enveloped virus. Virology 204, 376–390.
Schmelz, M., Sodeik, B., Ericsson, M., Wolffe, E. J., Shida, H., Hiller, G., and Griffiths, G. (1994) Assembly of vaccinia virus: the second wrapping cisterna is derived from the trans-Golgi network. J. Virol. 68, 130–147.
Roos, N., Cyrklaff, M., Cudmore, S., Blasco, R., Krijnse-Locker, J., and Griffiths, G. (1996) A novel immunogold cryoelectron microscopic approach to investigate the structure of the intracellular and extracellular forms of vaccinia virus. EMBO J. 15, 2343–2355.
Sanderson, C. M., Parkinson, J. E., Hollinshead, M., and Smith, G. L. (1996) Overexpression of the vaccinia virus A38L integral membrane protein promotes Ca2+ influx into infected cells. J. Virol. 70, 905–914.
Payne, L. G. (1980) Significance of extracellular enveloped virus in the in vitro and in vivo dissemination of vaccinia. J. Gen. Virol. 50, 89–100.
Payne, L. G. (1979) Identification of the vaccinia hemagglutinin polypeptide from a cell system yielding large amounts of extracellular enveloped virus. J. Virol. 31, 147–155.
Boulter, E. A. and Appleyard, G. (1973) Differences between extracellular and intracellular forms of poxvirus and their implications. Prog. Med. Virol. 16, 86–108.
Joklik, W. K. (1962) The purification of four strains of poxvirus. Virology 18, 9–18.
Law, M., Hollinshead, R., and Smith, G. L. (2002) Antibody-sensitive and antibody-resistant cell-to-cell spread by vaccinia virus: role of the A33R protein in antibody-resistant spread. J. Gen. Virol. 83, 209–222.
Cudmore, S., Blasco, R., Vincentelli, R., Esteban, M., Sodeik, B., Griffiths, G., and Krijnse Locker, J. (1996) A vaccinia virus core protein, p39, is membrane associated. J. Virol. 70, 6909–6921.
Eppstein, D. A., Marsh, Y. V., Schreiber, A. B., Newman, S. R., Todaro, G. J., and Nestor, J. J., Jr. (1985) Epidermal growth factor receptor occupancy inhibits vaccinia virus infection. Nature 318, 663–665.
Masters, J., Hinek, A. A., Uddin, S., Platanias, L. C., Zeng, W., McFadden, G., and Fish, E. N. (2001) Poxvirus infection rapidly activates tyrosine kinase signal transduction. J. Biol. Chem. 276, 48371–48375.
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Law, M., Smith, G.L. (2004). Studying the Binding and Entry of the Intracellular and Extracellular Enveloped Forms of Vaccinia Virus. In: Isaacs, S.N. (eds) Vaccinia Virus and Poxvirology. Methods in Molecular Biology, vol 269. Humana Press. https://doi.org/10.1385/1-59259-789-0:187
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DOI: https://doi.org/10.1385/1-59259-789-0:187
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