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.

Fig. 1.

Experimental setup for HCV pseudotype production in this study. Transfections were prepared with a fixed amount of the reporter plasmid pTG126, encoding an RNA transcript with a packaging signal (ψ) and a luciferase reporter gene under the control of a CMV IE promoter, with flanking MLV long terminal repeats (LTRs). This plasmid was co-transfected with varying quantities of both the E1/E2-encoding plasmid (derived from pcDNA3.1) and the MLV Gag/Pol-encoding packaging plasmid (phCMV5349) to create a matrix of transfections for each patient-derived clone. HEK293T cells were transfected with the mixture of plasmids, and the pseudotypes generated were used to infect HuH7 cells in a 96-well format. Infectivity was determined as RLU from the expressed reporter gene in the HuH7 cells.

Fig. 2.

Frequency of recovery of functional HCV glycoproteins using an MLV-based retroviral pseudoparticle assay. Patient isolates were screened using standard assay parameters as previously described (Tarr et al., 2007). (a) From a total of 63 patients, 118 E1/E2 clones were defined as functional in this assay (Urbanowicz et al., 2015). The majority of E1/E2 clones (76 %) were non-functional in this assay. The total number of clones screened for each genotype is noted above each bar. The proportion of functional clones (green) and the proportion of non-functional clones (blue) is displayed. When segregated by genotype, the proportion of functional genotype 3 and genotype 5 clones was lower than genotypes 1, 2 or 6. (b) Frequency of recovery of functional HCV glycoproteins from HCV-infected individuals. The total number of individuals infected with each genotype is highlighted above each bar. Again, the proportion of patients with functional clones (green) was lower for genotype 3 and genotype 5.

Fig. 3.

Plasmid quantity affects the infectivity of pseudoparticles generated with standard reference HCV envelope glycoproteins. (a and b) Transfection matrices were prepared for H77 (Owsianka et al., 2006) and clone UKN2A1.2 (Lavillette et al., 2005) with the resulting pseudoparticle preparations used to infect HuH7 cells. Graphs are presented as x-y contour plots of plasmid concentrations, with increasing normalized luciferase signal indicated by darkening blue colour. Signals were normalized against the maximal signal achieved for either H77 (~10 000 RLU), UKN2A1.2 (~5000 RLU) or that achieved with the negative control preparation (ΔE1/E2; ~100 RLU). Shades of blue represent the normalized infectivity data stratified into 20 % increments. HCVpp preparations with high signal were selected for neutralization assays (red, yellow and green points). (c and d) Neutralization of HCVpp prepared with different plasmid concentrations. Preparations were incubated with defined concentrations of the HCV-neutralizing mouse monoclonal antibody AP33. No differences were observed in the neutralization sensitivity of the particles.

Fig. 4.

HCVpp infection mediated by E1/E2 genes of closely related members of an HCV quasispecies. (a) The E1/E2 coding region was amplified by RT-PCR from a genotype 1a-infected patient (equivalent to amino acids 171–746 of the reference H77c clone). Maximum likelihood phylogenetic analysis was performed using a WAG model as implemented in MEGA 6.0. Bootstrap analysis of phylogeny using 1000 replicates was performed and values >80 are highlighted. (b) MLV-based HCVpp each possessing an E1/E2 clone were screened for infectivity in HuH7 cells, using standard plasmid concentrations for HCVpp genesis. Seven of the 16 isolated clones (●) were defined as functional (greater than 3× background signal generated with pseudotypes in the absence of glycoprotein, labelled as ΔE1/E2), although relative infectivity values varied widely. (c) Alignment of the E1/E2 coding regions of patient-derived clones UKN1A14.38, UKN1A14.40, UKN1A14.42, UKN1A14.43 and UKN1A14.44. The hypervariable region 1 (HVR1) is highlighted in yellow, the HVR2 is highlighted in red. Amino acid polymorphisms are highlighted between the closely related strains. The protease cleavage sites between Core, E1 signal peptide, E1 and E2 regions are highlighted with arrows.

Fig. 5.

HCVpp infectivity and protein incorporation into pseudoparticles. (a) Infectivity of patient clone UKN1A14.40. Pseudoparticles were made with a matrix of varying quantities of the plasmids encoding packaging vector and E1/E2 glycoproteins. Relative infectivity was normalized to the greatest signal observed (~10 000 RLU) and the signal achieved with a negative control preparation (ΔE1/E2). The graph is presented as an x-y contour plot of Gag- and E1/E2-encoding plasmid concentrations. Greater infectivity is presented as darker blue, stratified into 20 % increments, normalized to maximal infectivity. (b) Western blotting of transfected cell lysates of cells producing UKN1A14.40 HCVpp, using anti-E2 mAbs AP33 and ALP98 (upper panel), and pelleted pseudoparticles with AP33/ALP98 (middle panel) or anti-MLV CA (lower panel). The relative infectivity for each combination of plasmids is displayed as the shade of blue corresponding to the scale presented in panel (a). Very little incorporation of E2 was observed, while capsids were detected when using greater concentrations of the packaging vector. (c) HCVpp infectivity pseudoparticles representing patient clone UKN1A14.38. Pseudoparticles were made with a matrix of varying quantities of the plasmids encoding packaging vector and E1/E2 glycoproteins. Relative infectivity was normalized to the greatest signal observed (~10 000 RLU). Shades of blue represent the normalized infectivity data in 20 % increments. (d) Western blotting of transfected cell lysates producing UKN1A14.38 HCVpp, using anti-E2 antibodies AP33 and ALP98 (upper panel), and pelleted pseudoparticles with AP33/ALP98 (middle panel) or anti-MLV CA (lower panel). The relative infectivity for each combination of plasmids is displayed as the shade of blue corresponding to the scale presented in panel (c). Incorporation of E2 was observed when either 8 or 1.6 µg of packaging vector was used, while capsids were detected at all concentrations of the packaging vector. Greater concentrations of glycoprotein plasmid resulted in reduced expression of capsid.

Fig. 5.

HCVpp infectivity and protein incorporation into pseudoparticles. (a) Infectivity of patient clone UKN1A14.40. Pseudoparticles were made with a matrix of varying quantities of the plasmids encoding packaging vector and E1/E2 glycoproteins. Relative infectivity was normalized to the greatest signal observed (~10 000 RLU) and the signal achieved with a negative control preparation (ΔE1/E2). The graph is presented as an x-y contour plot of Gag- and E1/E2-encoding plasmid concentrations. Greater infectivity is presented as darker blue, stratified into 20 % increments, normalized to maximal infectivity. (b) Western blotting of transfected cell lysates of cells producing UKN1A14.40 HCVpp, using anti-E2 mAbs AP33 and ALP98 (upper panel), and pelleted pseudoparticles with AP33/ALP98 (middle panel) or anti-MLV CA (lower panel). The relative infectivity for each combination of plasmids is displayed as the shade of blue corresponding to the scale presented in panel (a). Very little incorporation of E2 was observed, while capsids were detected when using greater concentrations of the packaging vector. (c) HCVpp infectivity pseudoparticles representing patient clone UKN1A14.38. Pseudoparticles were made with a matrix of varying quantities of the plasmids encoding packaging vector and E1/E2 glycoproteins. Relative infectivity was normalized to the greatest signal observed (~10 000 RLU). Shades of blue represent the normalized infectivity data in 20 % increments. (d) Western blotting of transfected cell lysates producing UKN1A14.38 HCVpp, using anti-E2 antibodies AP33 and ALP98 (upper panel), and pelleted pseudoparticles with AP33/ALP98 (middle panel) or anti-MLV CA (lower panel). The relative infectivity for each combination of plasmids is displayed as the shade of blue corresponding to the scale presented in panel (c). Incorporation of E2 was observed when either 8 or 1.6 µg of packaging vector was used, while capsids were detected at all concentrations of the packaging vector. Greater concentrations of glycoprotein plasmid resulted in reduced expression of capsid.

Fig. 6.

Effect of pseudoparticle backbone on HCVpp infectivity. (a) Nine patient-derived E1/E2 clones representing genotypes 1–6 which were previously selected for function using the MLV Gag/Pol packaging vector were tested using either an MLV packaging vector (grey bars) or an HIV packaging vector (black bars). A VSV-G pseudovirus preparation was performed in parallel. The threshold for a positive signal was set at three times the signal achieved with a negative (Δglycoprotein) (dotted line). Three of the nine clones were infectious using an MLV packaging vector, but non-infectious using an HIV packaging vector. (b) The two clones investigated from patient 1A14 (UKN1A14.38, UKN1A14.40) were compared with both MLV and HIV packaging vectors. UKN1A14.40 was non-functional in the context of both packaging vectors, although the signal was higher when using the HIV-1-based packaging construct. UKN1A14.38 was functional in both vectors.

Fig. 7.

HCVcc chimeras possessing the E1/E2 glycoproteins of patient-isolated clones are infectious. Infectivity assays were performed with cell-culture supernatants of HuH7.5 cells electroporated with RNA representing H77/JFH1-luc reporter viruses possessing the E1/E2 genes of H77c (H77.20), UKN1A14.38 or UKN1A14.40. Filtered cell supernatants sampled at 24 h and 96 h were used to infect naïve Huh7.5 cells and luciferase readout measured at 24 h and 96 h after infection. Data from a mock transfected virus preparation (~800 RLU) were subtracted from each of these values.

Fig. 8.

Infectivity of pseudoviruses generated with GP_1,2 glycoproteins representing the Zaire Mayinga, Zaire Makona and Reston ebolaviruses. Transfections were performed with matrices of plasmids similar to that performed with HCV glycoproteins, using an MLV packaging construct. Infectivity was assessed by relative luminescence, and normalized to maximal signal for each glycoprotein (a, Mayinga: 30 000 RLU; b, Makona: 300 000 RLU; c, Reston: 75 000 RLU). (d) Mayinga EBOVpv, Makona EBOVpv and RESTVpv were tested using either an MLV packaging vector (grey bars) or an HIV packaging vector (black bars). While all three strains of EBOV performed similarly in the HIV-1-based vector, RESTVpv were significantly less infectious in the MLV-based pseudoviruses.