Abstract
Retrovirus pseudotypes are a highly tractable model used to study the entry pathways of enveloped viruses. This model has been extensively applied to the study of the hepatitis C virus (HCV) entry pathway, preclinical screening of antiviral antibodies and for assessing the phenotype of patient-derived viruses using HCV pseudoparticles (HCVpp) possessing the HCV E1 and E2 glycoproteins. However, not all patient-isolated clones produce particles that are infectious in this model. This study investigated factors that might limit phenotyping of patient-isolated HCV glycoproteins. Genetically related HCV glycoproteins from quasispecies in individual patients were discovered to behave very differently in this entry model. Empirical optimization of the ratio of packaging construct and glycoprotein-encoding plasmid was required for successful HCVpp genesis for different clones. The selection of retroviral packaging construct also influenced the function of HCV pseudoparticles. Some glycoprotein constructs tolerated a wide range of assay parameters, while others were much more sensitive to alterations. Furthermore, glycoproteins previously characterized as unable to mediate entry were found to be functional. These findings were validated using chimeric cell-cultured HCV bearing these glycoproteins. Using the same empirical approach we demonstrated that generation of infectious ebolavirus pseudoviruses (EBOVpv) was also sensitive to the amount and ratio of plasmids used, and that protocols for optimal production of these pseudoviruses are dependent on the exact virus glycoprotein construct. These findings demonstrate that it is crucial for studies utilizing pseudoviruses to conduct empirical optimization of pseudotype production for each specific glycoprotein sequence to achieve optimal titres and facilitate accurate phenotyping.
Highlights
Chronic hepatitis C virus (HCV) infection affects approximately 3 % of the world’s population
Some studies identified high proportions of infectious patient-isolated E1/E2 clones (Dowd et al, 2009; Fafi-Kremer et al, 2010), while others recovered lower frequencies of virus clones that are infectious in HCV pseudoparticles (HCVpp) assay (Lavillette et al, 2005; McKeating et al, 2004; Urbanowicz et al, 2015)
The data presented here offers a potential explanation for the discrepancies in the recovery frequency of infectious patient-isolated clones between studies, and develops approaches to expand the pool of functional HCVpp clones
Summary
Chronic hepatitis C virus (HCV) infection affects approximately 3 % of the world’s population. Infection can result in fibrosis, decompensated liver disease and hepatocellular carcinoma. Genetic variability is a key feature of HCV, with viral isolates classified into six major genotypes that differ by up to 30 % at the nucleotide level. The genomes of isolates within a genotype can differ by up to 15 %. The virus exists as a quasispecies population in an infected host, with approximately 1 % of the genome varying between strains (Nasu et al, 2011). This extreme variability contributes to the persistent
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