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Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

  • Gabriella Kiss (a1), Xuemin Chen (a1), Melinda A. Brindley (a2), Patricia Campbell (a3), Claudio L. Afonso (a4), Zunlong Ke (a5), Jens M. Holl (a1), Ricardo C. Guerrero-Ferreira (a1), Lauren A. Byrd-Leotis (a3), John Steel (a3), David A. Steinhauer (a3), Richard K. Plemper (a1) (a2), Deborah F. Kelly (a6), Paul W. Spearman (a1) and Elizabeth R. Wright (a1)...


Electron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.


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Briggs, J.A., Grunewald, K., Glass, B., Forster, F., Krausslich, H.G. & Fuller, S.D. (2006a). The mechanism of HIV-1 core assembly: Insights from three-dimensional reconstructions of authentic virions. Structure 14, 1520.
Briggs, J.A., Johnson, M.C., Simon, M.N., Fuller, S.D. & Vogt, V.M. (2006b). Cryo-electron microscopy reveals conserved and divergent features of Gag packing in immature particles of Rous sarcoma virus and human immunodeficiency virus. J Mol Biol 355, 157168.
Butan, C., Winkler, D.C., Heymann, J.B., Craven, R.C. & Steven, A.C. (2008). RSV capsid polymorphism correlates with polymerization efficiency and envelope glycoprotein content: Implications that nucleation controls morphogenesis. J Mol Biol 376, 11681181.
Calder, L.J., Wasilewski, S., Berriman, J.A. & Rosenthal, P.B. (2010). Structural organization of a filamentous influenza A virus. Proc Natl Acad Sci USA 107, 1068510690.
Carlson, L.A., Briggs, J.A., Glass, B., Riches, J.D., Simon, M.N., Johnson, M.C., Muller, B., Grunewald, K. & Krausslich, H.G. (2008). Three-dimensional analysis of budding sites and released virus suggests a revised model for HIV-1 morphogenesis. Cell Host Microbe 4, 592599.
de Marco, A., Davey, N.E., Ulbrich, P., Phillips, J.M., Lux, V., Riches, J.D., Fuzik, T., Ruml, T., Krausslich, H.G., Vogt, V.M. & Briggs, J.A. (2010). Conserved and variable features of Gag structure and arrangement in immature retrovirus particles. J Virol 84, 1172911736.
Fodor, E., Devenish, L., Engelhardt, O.G., Palese, P., Brownlee, G.G. & Garcia-Sastre, A. (1999). Rescue of influenza A virus from recombinant DNA. J Virol 73, 96799682.
Fontana, J., Cardone, G., Heymann, J.B., Winkler, D.C. & Steven, A.C. (2012). Structural changes in influenza virus at low pH characterized by cryo-electron tomography. J Virol 86, 29192929.
Gias, E., Nielsen, S.U., Morgan, L.A. & Toms, G.L. (2008). Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient. J Virol Methods 147, 328332.
Gilmore, B.L., Showalter, S.P., Dukes, M.J., Tanner, J.R., Demmert, A.C., McDonald, S.M. & Kelly, D.F. (2013). Visualizing viral assemblies in a nanoscale biosphere. Lab Chip 13, 216219.
Hall, W.W., Martin, S.J. & Gould, E. (1974). Defective interfering particles produced during the replication of measles virus. Med Microbiol Immunol 160, 155164.
Hammonds, J., Chen, X., Zhang, X., Lee, F. & Spearman, P. (2007). Advances in methods for the production, purification, and characterization of HIV-1 Gag-Env pseudovirion vaccines. Vaccine 25, 80368048.
Harris, A., Cardone, G., Winkler, D.C., Heymann, J.B., Brecher, M., White, J.M. & Steven, A.C. (2006). Influenza virus pleiomorphy characterized by cryoelectron tomography. Proc Natl Acad Sci USA 103, 1912319127.
Heymann, J.B., Butan, C., Winkler, D.C., Craven, R.C. & Steven, A.C. (2008). Irregular and semi-regular polyhedral models for Rous sarcoma virus cores. Comput Math Methods Med 9, 197210.
Hicar, M.D., Chen, X., Briney, B., Hammonds, J., Wang, J.J., Kalams, S., Spearman, P.W. & Crowe, J.E. Jr. (2010). Pseudovirion particles bearing native HIV envelope trimers facilitate a novel method for generating human neutralizing monoclonal antibodies against HIV. J Acquir Immune Defic Syndr 54, 223235.
Keller, P.W., Adamson, C.S., Heymann, J.B., Freed, E.O. & Steven, A.C. (2011). HIV-1 maturation inhibitor bevirimat stabilizes the immature Gag lattice. J Virol 85, 14201428.
Kelly, D.F., Abeyrathne, P.D., Dukovski, D. & Walz, T. (2008a). The affinity grid: A pre-fabricated EM grid for monolayer purification. J Mol Biol 382, 423433.
Kelly, D.F., Dukovski, D. & Walz, T. (2008b). Monolayer purification: A rapid method for isolating protein complexes for single-particle electron microscopy. Proc Natl Acad Sci USA 105, 47034708.
Kelly, D.F., Dukovski, D. & Walz, T. (2010a). A practical guide to the use of monolayer purification and affinity grids. Methods Enzymol 481, 83107.
Kelly, D.F., Dukovski, D. & Walz, T. (2010b). Strategy for the use of affinity grids to prepare non-His-tagged macromolecular complexes for single-particle electron microscopy. J Mol Biol 400, 675681.
Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. (1996). Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116, 7176.
Lee, K.K. (2010). Architecture of a nascent viral fusion pore. EMBO J 29, 12991311.
Liljeroos, L., Huiskonen, J.T., Ora, A., Susi, P. & Butcher, S.J. (2011). Electron cryotomography of measles virus reveals how matrix protein coats the ribonucleocapsid within intact virions. Proc Natl Acad Sci USA 108, 1808518090.
Liljeroos, L., Krzyzaniak, M.A., Helenius, A. & Butcher, S.J. (2013). Architecture of respiratory syncytial virus revealed by electron cryotomography. Proc Natl Acad Sci USA 110, 1113311138.
Loney, C., Mottet-Osman, G., Roux, L. & Bhella, D. (2009). Paramyxovirus ultrastructure and genome packaging: Cryo-electron tomography of sendai virus. J Virol 83, 81918197.
Maeda, A., Suzuki, Y. & Matsumoto, M. (1978). Isolation and characterization of defective interfering particle of Newcastle disease virus. Microbiol Immunol 22, 775784.
Mastronarde, D.N. (2005). Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 3651.
Mbiguino, A. & Menezes, J. (1991). Purification of human respiratory syncytial virus: Superiority of sucrose gradient over percoll, renografin, and metrizamide gradients. J Virol Methods 31, 161170.
McGinnes, L.W., Pantua, H., Reitter, J. & Morrison, T.G. (2006). Newcastle disease virus: Propagation, quantification, and storage. Curr Protoc Microbiol Chapter 15, Unit 15F, 12.
Ono, N., Tatsuo, H., Hidaka, Y., Aoki, T., Minagawa, H. & Yanagi, Y. (2001). Measles viruses on throat swabs from measles patients use signaling lymphocytic activation molecule (CDw150) but not CD46 as a cellular receptor. J Virol 75, 43994401.
Plemper, R.K., Hammond, A.L. & Cattaneo, R. (2001). Measles virus envelope glycoproteins hetero-oligomerize in the endoplasmic reticulum. J Biol Chem 276, 4423944246.
Sugita, Y., Noda, T., Sagara, H. & Kawaoka, Y. (2011). Ultracentrifugation deforms unfixed influenza A virions. J Gen Virol 92, 24852493.
Tanner, J.R., Degen, K., Gilmore, B.L. & Kelly, D.F. (2012). Capturing RNA-dependent pathways for cryo-EM analysis. Comput Struct Biotech J 1, e201204003-1–6.
Valdovinos, M.R. & Gomez, B. (2003). Establishment of respiratory syncytial virus persistence in cell lines: Association with defective interfering particles. Intervirology 46, 190198.
Wickramasinghe, S.R., Kalbfuss, B., Zimmermann, A., Thom, V. & Reichl, U. (2005). Tangential flow microfiltration and ultrafiltration for human influenza A virus concentration and purification. Biotechnol Bioeng 92, 199208.
Wright, E.R., Schooler, J.B., Ding, H.J., Kieffer, C., Fillmore, C., Sundquist, W.I. & Jensen, G.J. (2007). Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells. EMBO J 26, 22182226.
Yamaguchi, M., Danev, R., Nishiyama, K., Sugawara, K. & Nagayama, K. (2008). Zernike phase contrast electron microscopy of ice-embedded influenza A virus. J Struct Biol 162, 271276.
Zhang, L., Hernan, R. & Brizzard, B. (2001). Multiple tandem epitope tagging for enhanced detection of protein expressed in mammalian cells. Mol Biotechnol 19, 313321.


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Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications

  • Gabriella Kiss (a1), Xuemin Chen (a1), Melinda A. Brindley (a2), Patricia Campbell (a3), Claudio L. Afonso (a4), Zunlong Ke (a5), Jens M. Holl (a1), Ricardo C. Guerrero-Ferreira (a1), Lauren A. Byrd-Leotis (a3), John Steel (a3), David A. Steinhauer (a3), Richard K. Plemper (a1) (a2), Deborah F. Kelly (a6), Paul W. Spearman (a1) and Elizabeth R. Wright (a1)...


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