Hepatitis C virus envelope glycoprotein fitness defines virus population composition following transmission to a new host

Abstract

Genetic variability is a hallmark of RNA virus populations. However, transmission to a new host often results in a marked decrease in population diversity. This genetic bottlenecking is observed during hepatitis C virus (HCV) transmission and can arise via a selective sweep or through the founder effect. To model HCV transmission, we utilized chimeric SCID/Alb-uPA mice with transplanted human hepatocytes and infected them with a human serum HCV inoculum. E1E2 glycoprotein gene sequences in the donor inoculum and recipient mice were determined following single-genome amplification (SGA). In independent experiments, using mice with liver cells grafted from different sources, an E1E2 variant undetectable in the source inoculum was selected for during transmission. Bayesian coalescent analyses indicated that this variant arose in the inoculum pretransmission. Transmitted variants that established initial infection harbored key substitutions in E1E2 outside HVR1. Notably, all posttransmission E1E2s had lost a potential N-linked glycosylation site (PNGS) in E2. In lentiviral pseudoparticle assays, the major posttransmission E1E2 variant conferred an increased capacity for entry compared to the major variant present in the inoculum. Together, these data demonstrate that increased envelope glycoprotein fitness can drive selective outgrowth of minor variants posttransmission and that loss of a PNGS is integral to this improved phenotype. Mathematical modeling of the dynamics of competing HCV variants indicated that relatively modest differences in glycoprotein fitness can result in marked shifts in virus population composition. Overall, these data provide important insights into the dynamics and selection of HCV populations during transmission.

Fig 1

Combined phylogenetic reconstruction of donor and recipient viral E1E2 populations. Shown is a maximum-likelihood tree (HKY + Γ) derived using SGA and bulk E1E2 sequences present in the KP donor inoculum and in chimeric mice posttransmission. Red circles, KP inoculum; green squares, mouse 594 time point 1; green triangles, mouse 594 time point 2; black squares, mouse 666 time point 1; black triangles, mouse 666 time point 2; blue squares, mouse 714 time point 1; blue triangles, mouse 714 time point 2; pink squares, mouse 931 time point 1; pink triangles, mouse 931 time point 2; turquoise circles, bulk amplified sequences. The scale bar is proportional to the genetic distance and represents 0.0005 nucleotide substitution per site.

Fig 2

Stability of HVR1 sequences upon transmission. (A) Alignment of HVR1 amino acid sequence variants (A to G) present in donor/recipient populations. (B) Frequencies of HVR1 variants circulating within donor/recipient hosts.

Fig 3

Phylogenetic trees and patterns of substitution in sequences derived during experimental HCV transmission from the KP inoculum to chimeric mice 594, 666, 714, and 931. Chimeric mouse 594 (A), 666 (B), 714 (C), and 931 (D)-derived sequences were analyzed via phylogenetic reconstructions (left panels) and Highlighter plots (right panels) with pretransmission KP donor sequences included. The ML trees are rooted on the KP consensus (KP_con) sequence, which is the master sequence in corresponding Highlighter plots. The Highlighter plots depict the relative locations of synonymous (green vertical bars) and nonsynonymous (red vertical bars) substitutions in each E1E2 amplicon compared to the pretransmission master sequence. A schematic representation of the E1E2 gene, depicting the locations of PNG sites and the E1/E2 boundary, is provided above the Highlighter plots for the purposes of positional referencing. The scale bar is proportional to the genetic distance and represents 0.0005 nucleotide substitution per site.

Fig 4

Identification of key residues involved in HCV transmission and the frequencies of key residue combinations in donor and recipient viral E1E2 populations. (A) Schematic E1E2 diagram depicting the locations of four key residues involved in transmission. The positions of conserved potential N-glycosylation sites are indicated above the E1E2 protein. The coordinates given are relative to the homologous positions in the H77 reference strain polyprotein (accession no. NC_004102). The colored vertical columns located below key residues indicate consensus amino acids present in the E1E2 SGA populations of donor and four recipient chimeric (top), as well as consensus, amino acid sequences derived from bulk amplified RNA obtained from eight other transmission experiments using the same KP inoculum (bottom). (B) Frequencies of key residue combinations circulating within donor/recipient hosts. TP1, time point 1; TP2, time point 2.

Fig 5

Identification of the number of transmitted lineages for each experimental infection event. Shown are donor/recipient pairs KP/594 (A), KP/666 (B), KP/714 (C), and KP/931 (D). The lineages are color coded according to key residue combinations: red, SNHV; green, SDHV; yellow, TDYD; orange, SDHV; blue, TDYV/A. The scale bar located below each tree is proportional to the time. The units correspond to days, with 0 representing the final sampling date for each individual chimeric mouse and the dotted line representing the time of experimental infection. Lineages/variants present in the KP inoculum are indicated by asterisks.

Fig 6

Locations of reported T-cell epitopes restricted by the donor KP's HLA class I alleles in the E1E2 sequence. The mouse consensus E1E2 amino acid sequence is shown, with the four amino acid substitutions associated with establishment of productive infection following HCV transmission highlighted in pink. Sequences corresponding to the locations of reported T-cell epitopes restricted by the donor KP's HLA class I alleles are highlighted in different colors, with the peptide sequence(s) to which T-cell responses were demonstrated shown above them.

Fig 7

Infectivities of HCVpp bearing donor and recipient variant E1E2s in hepatoma cells and primary hepatocytes. (A and B) HCVpp infectivities conferred by the major inoculum E1E2 variant (SNHV), the major recipient E1E2 variant (TDYV), and two minor variants present in both the inoculum and recipient mice (SDHV and TDYD). Mean infectivity values are expressed as percentages of the infectivity conferred by H77c E1E2 in Huh7.5 cells (A) and PHs (B). Infectivity assays were performed in the presence or absence of anti-CD81 monoclonal antibody (MAb) 2s131, and the values presented are means for six replicates from two independent experiments with associated error bars (standard deviations). The associated significance values indicate that the TDYV variant confers more efficient entry than the SDHV, TDYD, and SNHV variants. Additionally, both the SDHV and TDYD variants confer improved capacity for entry compared to the SNHV variant. Differences in the mean infectivities conferred by each HCV E1E2 were assessed using analysis of variance (ANOVA) with infectivity: ***, P < 0.001. (C) p24 concentrations for each of the variant HCVpp preparations used to infect Huh7.5 cells or PHs.

Fig 8

Mathematical modeling of HCV population expansion following transmission. The virus populations were modeled for two hypothetical variants, 1 and 2. If γ is equal to 1, the two variants have identical rates of infection of target cells; increasing γ gives variant 2 a fitness advantage. “ε = 0.1” is used to represent immunodeficient mice (A to D), while “ε = 1” represents mice with a functioning immune system (E to H). The y axes are plotted on a logarithmic scale.