Novel functional hepatitis C virus glycoprotein isolates identified using an optimized viral pseudotype entry assay

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.

Human Adaptation of Ebola Virus during the West African Outbreak

“…To determine whether these lineage-specific amino acid substitutions resulted in more efficient human cell infection, and hence increased fitness, we generated a panel of GP mutants based on the earliest sampled Makona isolate, Kissidougou-C15 (GenBank: KJ660346, sampled from Guinea in March 2014; Figure 2), and tested their ability to support entry into a range of human and bat cell lines using a lentiviral pseudotype assay (Urbanowicz et al., 2016). Although variants from both lineages A and B reached higher infectivity in human cells than Kissidougou-C15 (Figure 3), the A82V change appears to have set the GP on separate evolutionary pathways, as we observed distinct sets of additional amino acid changes in each lineage that increased entry efficacy in human cells.…”

“…Transfections were performed as previously described (Mohan et al., 2015, Urbanowicz et al., 2016). Briefly, 1.2x10 6 HEK293T cells were seeded overnight in a 10cm diameter Primaria coated dish (Corning) in 10mL of DMEM supplemented with non-essential amino acids and heat-inactivated FBS.…”

Theaflavins, polyphenols of black tea, inhibit entry of hepatitis C virus

“…Viral pseudoparticles are good models for entry inhibitor assay and also for screening against different viral genotypes (29). To confirm that theaflavins act at the entry step, HCVpp were produced and inoculated to Huh-7 cells in the presence of each theaflavins at active concentrations (EC 50 and 10 × EC 50 ).…”

Generation of MERS-CoV Pseudotyped Viral Particles for the Evaluation of Neutralizing Antibodies in Mammalian Sera

“…Lentiviral vectors are retroviruses, which are enveloped single-stranded RNA viruses, derived, for example, from human immunodeficiency virus type 1 (HIV-1). They have been used to develop pseudotype viral particles for many pathogenic viruses [3][4][5][6]. These replication-deficient vectors offer a number of advantages including that they do not replicate in mammalian cells, they infect dividing and nondividing cells, they can incorporate large transgenes derived from other pathogenic viruses as large as 9 kb, and they induce no or weak immune response [7][8][9].…”