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.

Sequence and Functional Analysis of the Envelope Glycoproteins of Hepatitis C Virus Variants Selectively Transmitted to a New Host

“…Genetic and phenotypic signatures associated with the transmission of true T/F E1E2 obtained by SGA were investigated, to the best of our knowledge, only in the previously cited study on experimental transmission of HCV in a chimeric mouse model (13). This study showed that the transmitted variants harbored key substitutions in E1E2, outside HVR1.…”

“…It remains unclear whether the transmission of T/F viruses results from a founder effect, with one or a small number of variants being transferred between hosts, or whether it is due to early evolutionary events, with larger numbers of variants undergoing a selective sweep due to differences in fitness constraints (10). In a recent report of the experimental transmission of HCV to chimeric SCID/Alb-uPA mice with transplanted human hepatocytes, the occurrence of selective sweeps was put forward to explain the finding that undetectable inoculum variants bearing an advantageous E1E2 motif became the major variants circulating in the infected mice (13). However, the presumed small inoculum resulting from needlestick accidents and the different proportions of T/F E1E2 sequences present in donor virus populations complicate the transposition of previously described scenarios to each of our transmission pairs.…”

“…Methods for accurately identifying T/F viruses and assessing genetic diversity at various stages of infection have also evolved with the development of SGA. This method is now the gold standard in the HIV field (14,(25)(26)(27) but has only recently been applied to HCV studies (12,13). We used SGA for the detailed investigation of HCV transmission in three cases of acute hepatitis acquired through needlestick accidents.…”

“…E1E2 SGA products selected for further phenotypic analyses were inserted into the pcDNA3.1 mammalian expression vector (Invitrogen). Lentiviral pseudoparticles were generated by the cotransfection of 293-T cells with pNL4-.Luc.R Ϫ E Ϫ and expression vectors encoding E1E2 glycoproteins derived from donors and recipients, vesicular stomatitis virus glycoprotein (VSV-G), the UKN1B5.23 HCV envelope, or the no-envelope (⌬E1E2) control, as previously reported (13,18,19). Viral supernatants were collected 48 h later and purified by passage through a filter with 0.45-m pores.…”

“…This results in the generation of a group of related, but genetically different, viral variants within each infected individual (9). The genetic bottleneck generally observed after transmission indicates that productive infections may be initiated by one or a small number of viral variants (10)(11)(12)(13). These variants are then subjected to constant immune pressure, and the role of the immune response in the clearance of HCV infection or the establishment of chronic hepatitis C has been thoroughly investigated.…”