Ultrastructural analysis of hepatitis C virus particles (original) (raw)

2013, Proceedings of the National Academy of Sciences

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. enveloped virus | hepacivirus | lipoviral particle | virus structure | virus assembly H epatitis C virus (HCV) is an important human pathogen that infects the liver and establishes chronic infection in the majority of cases, leading to cirrhosis and hepatocellular carcinoma (HCC) over the course of many years. More than 170 million people, ∼3% of the world's population, have been infected with HCV. Each year, 4-5% of patients with HCVinduced cirrhosis develop HCC, making HCV infection the leading indicator for liver transplantation in many areas of the world (1). Surgery, however, does not provide a cure because the donor organ universally becomes reinfected. A prophylactic vaccine is not available and despite the recent addition of HCV-specific protease inhibitors to the pegylated (peg)-IFN and ribavirin regimen, which has increased the cure rate, better therapies are still needed to solve the emergence of resistant variants, severe side effects and suboptimal response rates in cirrhotic patients .