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

• Type of Material: Article
• Authors: Gabriella Kiss, Emory University; Xuemin Chen, Emory University; Melinda A. Brindley, Georgia State University; Patricia Campbell, Emory University; Claudio L. Afonso, USDA ARS; Zunlong Ke, Georgia Institute of Technology; Jens M. Holl, Emory University; Ricardo C. Guerrero-Ferreira, Emory University; Lauren A. Byrd-Leotis, Emory University; John Steel, Emory University; David Steinhauer, Emory University; Richard Karl Plemper, Emory University; Deborah F. Kelly, Virginia Tech; Paul Spearman, Emory University; Elizabeth Wright, Emory University
• Language: English
• Date: 2014-02-01
• Publisher: Cambridge University Press (CUP): STM Journals
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © Microscopy Society of America 2014.
• Final Published Version (URL): http://dx.doi.org/10.1017/S1431927613013937
• Title of Journal or Parent Work: Microscopy and Microanalysis
• ISSN: 1431-9276
• Volume: 20
• Issue: 1
• Start Page: 164
• End Page: 174
• Grant/Funding Information: This work was supported in part by Emory University, Children’s Healthcare of Atlanta, the Center for AIDS Research at Emory University (P30 AI050409), and the Georgia Research Alliance to E.R.W.; NSF grant 0923395 to E.R.W; and public health service grants AI101775 to E.R.W, AI058828 to P.W.S., and AI083402 to R.K.P. from the NIH/NIAID; USDA_ARS CRIS project number 66612-32000-064 to C.L.A.; and Centers for Excellence in Influenza Research and Surveillance (CEIRS) contract number HHSN266200700006C to J.S. and D.A.S.
• Keywords: cryo-electron microscopy (cryo-EM); cryo-electron tomography (cryo-ET); TOMOGRAPHY; PARTICLES; ULTRACENTRIFUGATION; Technology; affinity grid (AG); INFLUENZA-A VIRUS; Microscopy; Science & Technology; enveloped virus; ROUS-SARCOMA-VIRUS; Materials Science, Multidisciplinary; CRYOTOMOGRAPHY; ARCHITECTURE; REVEALS; Materials Science; RESPIRATORY SYNCYTIAL VIRUS; MONOLAYER PURIFICATION
• Research Categories: Health Sciences, Immunology; Biology, Virology

Abstract

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.