Transmission of Clonal Hepatitis C Virus Genomes Reveals the Dominant but Transitory Role of CD8⁺T Cells in Early Viral Evolution

Abstract

The RNA genome of the hepatitis C virus (HCV) diversifies rapidly during the acute phase of infection, but the selective forces that drive this process remain poorly defined. Here we examined whether Darwinian selection pressure imposed by CD8؉ T cells is a dominant force driving early amino acid replacement in HCV viral populations. This question was addressed in two chimpanzees followed for 8 to 10 years after infection with a well-defined inoculum composed of a clonal genotype 1a (isolate H77C) HCV genome. Detailed characterization of CD8 ؉ T cell responses combined with sequencing of recovered virus at frequent intervals revealed that most acute-phase nonsynonymous mutations were clustered in class I epitopes and appeared much earlier than those in the remainder of the HCV genome. Moreover, the ratio of nonsynonymous to synonymous mutations, a measure of positive selection pressure, was increased 50-fold in class I epitopes compared with the rest of the HCV genome. Finally, some mutation of the clonal H77C genome toward a genotype 1a consensus sequence considered most fit for replication was observed during the acute phase of infection, but the majority of these amino acid substitutions occurred slowly over several years of chronic infection. Together these observations indicate that during acute hepatitis C, virus evolution was driven primarily by positive selection pressure exerted by CD8 ؉ T cells. This influence of immune pressure on viral evolution appears to subside as chronic infection is established and genetic drift becomes the dominant evolutionary force.Upon transmission to a new host, the hepatitis C virus (HCV) persists in approximately 65 to 80% of infected patients. HCV circulates in infected individuals as a swarm of genetically different but closely related viral variants known as a quasispecies. Heterogeneity of the HCV genomes is the result of the high virion production rate in the liver (35) and the lack of proofreading activity of the viral RNA-dependent RNA polymerase. These characteristics facilitate rapid adaptation of HCV to the host, in part through selection of preexisting minor variants from the quasispecies population (43). Viral evolution is governed by the positive or negative selection of mutations and the accumulation of neutral substitutions (genetic drift) (43). The rate of HCV evolution has been reported to be much higher during the acute phase than the chronic phase of infection (7,26). It is assumed that this process is driven by the adaptive immune response. Neutralizing antibodies contribute to early diversification of the HCV envelope genes through positive selection of escape mutations (15), a process that is maintained during the chronic phase of infection (49). CD4 ϩ T cells appear to exert limited selection pressure against the virus because mutations in major histocompatibility complex (MHC) class II epitopes are uncommon (19,21). Mutational escape from CD8 ϩ T cells is well established (8,16,23,24,45,46). In chimpanzees, the only animal model of human HCV infect...

Evolution of a Cell Culture-Derived Genotype 1a Hepatitis C Virus (H77S.2) during Persistent Infection with Chronic Hepatitis in a Chimpanzee

“…Thus, the H77S.2 G1202 residue may serve as a natural adaptive mutation that enhances H77S.2 replication in Huh-7 cells but reduces its fitness in chimpanzees. Consistent with the latter notion, G1202E substitutions have also been noted in two chimpanzees infected by intrahepatic inoculation of synthetic H77c RNA in other studies (Table 3) (39).…”

“…Of these 8, 4 (L546M, I1568M, I1641V, and A1997T) appeared before week 62, a time frame associated with the highest rate of nonsynonymous substitution (Fig. 6B) and CD8 ϩ T cell selection pressure (39). S1358P occurred in the same time frame, but this substitution reconstitutes an epitope rather than providing escape from one.…”

“…Both are reversions of cell culture-adaptive mutations in the H77S.2 clone. Interestingly, G1202 and I1655, as well as S1358 (NS3-332), which represents a change from the wild-type H77c sequence to the consensus genotype 1a sequence (39), are also located within known T cell epitopes that are potentially relevant in this animal (see the following section). Two nonsynonymous substitutions occurred within that part of NS5A domain I for which structural models are available.…”

“…Immune selection pressure has generally been considered to be the dominant force driving increased nonsynonymous substitutions in HCV genomes during the acute phase of infection (7). Nonsynonymous substitutions in nonstructural proteins are concentrated in class I epitopes during the first year of infection and then subside, presumably as CD8 ϩ T cell effector functions fail (39,41). Because this pattern of substitutions was observed in 4x0193, we sought to determine whether any amino acid changes fell within known class I epitopes.…”

Functionally conserved architecture of hepatitis C virus RNA genomes

“…For example, two recent chimpanzee studies monitored chronic infection by H77c viruses and independently detected a silent mutation, A7586G, that arose early and was subsequently maintained during infection (42,43). In the context of our H77c structure model, this mutation yields an impressive 6.6 kcal/mol stabilization of a structural element in the H7430 region (Fig.…”