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Cloning and analysis of authentic patient-derived HCV E1/E2 glycoproteins

Urbanowicz, Richard A.; Ball, Jonathan K.; Tarr, Alexander W.


Richard A. Urbanowicz

Professor of Molecular Virology


Mansun Law


Experimental characterization of the properties of authentic viruses circulating in infected individuals presents a problem when investigating RNA viruses with error-prone polymerases. The hepatitis C virus provides an extreme example of RNA virus genetic variability, as the nucleotide composition of HCV genomes can vary by more than 30% between strains. The envelope glycoproteins E1 and E2 in particular are able to tolerate a particularly high level of variation. They are under continual selection pressure from the host antibody response during chronic infection and can tolerate adaptive mutations, leading to great diversity in a single host. The diversity of E1/E2 in circulating viruses has hindered investigations of their function and development of a vaccine that will generate antibodies able to potently neutralize entry of genetically distinct strains.

Here we describe methods used in our laboratory to overcome the limitations of investigating the properties of the envelope glycoproteins representing only small numbers of HCV variants. Using a high-fidelity, limiting dilution (“endpoint”) PCR approach to amplify single E1/E2 cDNA templates, which can then generate recombinant model viral particles using retrovirus packaging/reporter constructs. These retroviral pseudoparticles (pseudotypes) facilitate investigation of the properties of authentic E1/E2 glycoproteins in a single-round infection assay. We also describe optimized methods for generation of infectious pseudoparticles from patient-isolated E1/E2 and methods for performing neutralization assays with both anti-virus and anti-host antibodies.

Acceptance Date Mar 22, 2018
Online Publication Date Dec 29, 2018
Publication Date Jan 1, 2019
Deposit Date Jan 25, 2019
Journal Methods in Molecular Biology; Hepatitis C Virus Protocols
Electronic ISSN 1940-6029
Publisher Springer Publishing Company
Pages 275-294
Series Title Methods in Molecular Biology
Series Number 1911
Book Title Hepatitis C Virus Protocols
Chapter Number 19
ISBN 9781493989751
Public URL