tetano
Editor, Senior Moderator
Microsc Microanal. 2013 Nov 26:1-11. [Epub ahead of print]
Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications.
Kiss G, Chen X, Brindley MA, Campbell P, Afonso CL, Ke Z, Holl JM, Guerrero-Ferreira RC, Byrd-Leotis LA, Steel J, Steinhauer DA, Plemper RK, Kelly DF, Spearman PW, Wright ER.
Source
Department of Pediatrics, School of Medicine, Division of Pediatric Infectious Diseases, Emory University, Children's Healthcare of Atlanta, Atlanta, GA 30322, USA.
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.
PMID:
24279992
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24279992
Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo-Electron Microscopy Applications.
Kiss G, Chen X, Brindley MA, Campbell P, Afonso CL, Ke Z, Holl JM, Guerrero-Ferreira RC, Byrd-Leotis LA, Steel J, Steinhauer DA, Plemper RK, Kelly DF, Spearman PW, Wright ER.
Source
Department of Pediatrics, School of Medicine, Division of Pediatric Infectious Diseases, Emory University, Children's Healthcare of Atlanta, Atlanta, GA 30322, USA.
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.
PMID:
24279992
[PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/24279992