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Proc Natl Acad Sci U S A. 2013 Jun 4;110(23):9505-10. doi: 10.1073/pnas.1307527110. Epub 2013 May 20.

Ultrastructural analysis of hepatitis C virus particles.

Author information

1
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065, USA.

Abstract

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.

KEYWORDS:

enveloped virus; hepacivirus; lipoviral particle; virus assembly; virus structure

PMID:
23690609
PMCID:
PMC3677472
DOI:
10.1073/pnas.1307527110
[Indexed for MEDLINE]
Free PMC Article
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