Ultrastructural analysis of hepatitis C virus particles
Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this repor...
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Published in | Proceedings of the National Academy of Sciences - PNAS Vol. 110; no. 23; pp. 9505 - 9510 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
National Academy of Sciences
04.06.2013
National Acad Sciences |
Subjects | |
Online Access | Get full text |
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Summary: | Hepatitis C virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people infected worldwide. Low yields, poor stability, and inefficient binding to conventional EM grids have posed significant challenges to the purification and structural analysis of HCV. In this report, we generated an infectious HCV genome with an affinity tag fused to the E2 envelope glycoprotein. Using affinity grids, previously described to isolate proteins and macromolecular complexes for single-particle EM, we were able to purify enveloped particles directly from cell culture media. This approach allowed for rapid in situ purification of virions and increased particle density that were instrumental for cryo-EM and cryoelectron tomography (cryo-ET). Moreover, it enabled ultrastructural analysis of virions produced by primary human hepatocytes. HCV appears to be the most structurally irregular member of the Flaviviridae family. Particles are spherical, with spike-like projections, and heterogeneous in size ranging from 40 to 100 nm in diameter. Exosomes, although isolated from unfractionated culture media, were absent in highly infectious, purified virus preparations. Cryo-ET studies provided low-resolution 3D structural information of highly infectious virions. In addition to apolipoprotein (apo)E, HCV particles also incorporate apoB and apoA-I. In general, host apolipoproteins were more readily accessible to antibody labeling than HCV glycoproteins, suggesting either lower abundance or masking by host proteins. |
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Bibliography: | http://dx.doi.org/10.1073/pnas.1307527110 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Author contributions: M.T.C., R.J.K., and C.M.R. designed research; M.T.C., K.U., M.K., T.J.E., L.A., and W.J.R. performed research; M.T.C. and L.A. contributed new reagents/analytic tools; M.T.C., K.U., M.K., T.J.E., L.A., W.J.R., M.S., R.J.K., and C.M.R. analyzed data; and M.T.C. and C.M.R. wrote the paper. 1Present address: Amgen, Seattle, WA 98119. Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved April 26, 2013 (received for review March 31, 2013) 2Present address: Center for Structural Biology, Vanderbilt University, Nashville, TN 37232. |
ISSN: | 0027-8424 1091-6490 |
DOI: | 10.1073/pnas.1307527110 |