Cloning, expression, and functional analysis of patient-derived hepatitis C virus glycoproteins.
Hepatitis C virus (HCV) infection is a major cause of severe chronic liver disease including cirrhosis and hepatocellular carcinoma. HCV has been classified into six major genotypes that exhibit extensive genetic variability, particularly in the envelope glycoproteins E1 and E2. Knowledge of genotypic and quasispecies variation on viral glycoprotein properties is important in understanding the structure-function relationship of the proteins. Through their perceived role as components of the virion and mediators of virus attachment and entry, HCV glycoproteins are primary targets for the development of antiviral agents. In this chapter, we describe methods optimized to extract E1E2-encoding sequences of all the major genotypes from HCV-infected patient sera, and their amplification, cloning, expression, and biochemical characterization. Furthermore, we describe a method to generate retroviral nucleocapsid pseudotyped with HCV E1E2 of diverse genotypes (HCVpp) whereby infectivity of the retroviral particle is conferred by HCV glycoproteins. Finally, we show how the HCVpp can be used in an infection assay to determine the viral glycoprotein function at the level of the host-pathogen interface and subsequent events leading to virus infection.
Tarr
Alexander W
AW
The Institute of Infection, Immunity, and Inflammation, School of Molecular Medical Sciences, The University of Nottingham, Queen's Medical Centre, UK.
Owsianka
Ania M
AM
Szwejk
Alexandra
A
Ball
Jonathan K
JK
Patel
Arvind H
AH
D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D003001:Cloning, Molecular; D015870:Gene Expression; D014644:Genetic Variation; D005838:Genotype; D006023:Glycoproteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D016174:Hepacivirus / Q000235:genetics* / Q000378:metabolism; D019698:Hepatitis C, Chronic; D006801:Humans; D008103:Liver Cirrhosis / Q000235:genetics / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics*; D053585:Virus Attachment
C087035:E1 protein, Hepatitis C virus; D006023:Glycoproteins; D014759:Viral Envelope Proteins