期刊
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
卷 110, 期 23, 页码 9505-9510出版社
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1307527110
关键词
enveloped virus; hepacivirus; lipoviral particle; virus structure; virus assembly
资金
- National Institutes of Health (NIH) [AI072613, AI075099]
- Greenberg Medical Research Institute
- Starr Foundation
- Rockefeller University Women and Science Fellowship
- National Center for Research Resources [C06 RR017528-01-CEM]
- NIH
- NIH [S10 RR17291]
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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