A Hepatitis C virus genotype 1b post-transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo
Open Access
- 28 June 2022
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLoS Pathogens
- Vol. 18 (6), e1010472
- https://doi.org/10.1371/journal.ppat.1010472
Abstract
Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes and isolates, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, likely due to cell-to-cell transmission, which appeared to substantially contribute to spreading of other isolates as well. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is an efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines. Chronic HCV infections remain an important global health issue, despite the availability of highly efficient therapies. So far no protective vaccine is available, which is in part due to the high divergence of HCV variants and the limited possibility to mirror this genetic diversity in cell culture. It has been proven particularly difficult to grow infectious virus in cell culture, requiring extensive adaptation with multiple mutations, which in turn affect infectivity of the adapted variants in vivo. Here we have isolated a genotype 1b variant from a very high titer serum of a patient after liver transplantation (German Liver Transplant 1, GLT1), showing an outstanding genome replication efficiency in cultured hepatoma cells. We were able to adapt this isolate to production of infectious virus, therefore generating a novel full-replication cycle cell culture model for the highly prevalent HCV genotype 1b. Despite multiple mutations required, adapted GLT1 was still infectious in vivo. GLT1 therefore is an important development facilitating future efforts in vaccine development.Keywords
Funding Information
- Deutsche Forschungsgemeinschaft (278191845)
- Deutsche Forschungsgemeinschaft (272983813)
- Deutsche Forschungsgemeinschaft (314905040)
- Deutsches Zentrum für Infektionsforschung (TTU 05.821)
- Deutsche Forschungsgemeinschaft (272983813)
- Deutsche Forschungsgemeinschaft (314905040)
- Deutsches Zentrum für Infektionsforschung (TTU 05.821)
- Deutsches Zentrum für Infektionsforschung (TTU 05.712)
- Medical Research Council (MC_UU12014/2)
- Deutsche Forschungsgemeinschaft (158989968)
- Chica and Heinz Schaller Foundation
- Universiteit Gent (UGent BOF)
- Research Foundation – Flanders
This publication has 83 references indexed in Scilit:
- Highly efficient full-length hepatitis C virus genotype 1 (strain TN) infectious culture systemProceedings of the National Academy of Sciences of the United States of America, 2012
- ViPR: an open bioinformatics database and analysis resource for virology researchNucleic Acids Research, 2011
- Recruitment and Activation of a Lipid Kinase by Hepatitis C Virus NS5A Is Essential for Integrity of the Membranous Replication CompartmentCell Host & Microbe, 2011
- Real-time imaging of hepatitis C virus infection using a fluorescent cell-based reporter systemNature Biotechnology, 2010
- Immunocytochemical techniques reveal multiple, distinct cellular pools of PtdIns4P and PtdIns(4,5)P2Biochemical Journal, 2009
- CD81 is dispensable for hepatitis C virus cell-to-cell transmission in hepatoma cellsJournal of General Virology, 2009
- Hepatitis C virus JFH-1 strain infection in chimpanzees is associated with low pathogenicity and emergence of an adaptive mutationJournal of Hepatology, 2008
- A Conserved Gly436-Trp-Leu-Ala-Gly-Leu-Phe-Tyr Motif in Hepatitis C Virus Glycoprotein E2 Is a Determinant of CD81 Binding and Viral EntryJournal of Virology, 2006
- Construction and characterization of infectious intragenotypic and intergenotypic hepatitis C virus chimerasProceedings of the National Academy of Sciences of the United States of America, 2006
- Production of infectious hepatitis C virus in tissue culture from a cloned viral genomeNature Medicine, 2005