Hypervariable Region 1 Sequence Stability during Hepatitis C Virus Replication in Chimpanzees

Abstract

The putative envelope 2 (E2) gene of hepatitis C virus (HCV) contains a highly variable region referred to as hypervariable region 1 (HVR1). We hypothesized that this genetic variability is driven by immune selection pressure, rather than representing the accumulation of random mutations in a region with relatively little functional constraint. To test this hypothesis, we examined the E2 sequence of a human inoculum that was passaged through eight chimpanzees, which appear to have a replicative rate (opportunity for chance mutation) similar to that of humans. Acute-phase plasma samples from a human (the inoculum) and six of eight serially infected chimpanzees were studied. For each, 33 cloned cDNAs were examined by a combined heteroduplex-single-stranded conformational polymorphism assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns (clonotypes) for sequencing. The sequence diversity of HCV was significantly lower in the chimpanzees than in the humans, and during eight serial passages there was no change in the sequence of the majority clonotype from each animal examined. Similarly, the rates of protein sequence altering (nonsynonymous) substitution were lower in the chimpanzees than in the humans. These findings demonstrate that nonsynonymous mutations indicate selection pressure rather than being an incidental result of HCV replication.An estimated 170 million people worldwide are infected with hepatitis C virus (HCV) (4). More than 80% of infections result in persistent viremia, which may be associated with chronic hepatitis, cirrhosis, liver failure, and hepatocellular cancer (3,41,44). HCV-associated disease is responsible for more than 10,000 deaths each year in the United States alone, and this mortality is expected to rise (8). Because of limitations of in vitro replication systems, studies of HCV pathogenesis have been limited to observation of natural infection of humans and experimental infection of chimpanzees.The chimpanzee is the best experimental model of HCV infection. HCV does not grow efficiently in tissue culture, and the only other candidate model is a tree shrew that is too small to permit adequate sampling with current technology (52). In chimpanzees, HCV replication has been demonstrated within days of experimental infection, and the potential for reinfection has been demonstrated (14,15,37,51). Serum HCV RNA levels and the extent of hepatic HCV involvement are similar in chimpanzees and humans, suggesting that replication rates are similar (1). The chimpanzee has also been used to test the infectivity of HCV clones and vaccine candidates. However, Bassett et al. recently demonstrated that the natural history of HCV infection in a cohort of experimentally infected chimpanzees differed from what is typically found in humans (5, 6). These chimpanzees had a higher rate of clearance of viremia, lower rate of antibody production to envelope proteins, and lower rate of envelope amino acid change. Similarly, others have reported little change in...

Hepatitis C Virus Envelope Glycoprotein Fitness Defines Virus Population Composition following Transmission to a New Host

“…In our transmission experiments, the major HVR1 amino acid variants pre-and posttransmission were identical, which is in agreement with other transmission studies (34,37,54,60). HVR1 is often used as a surrogate marker for entire E1E2 genetic variation, and the HVR1 clonotype has been used as a basis for choosing E1E2 clones for downstream analyses (20).…”

“…A number of studies have been performed using samples obtained during acute infection of humans and chimpanzees, and the restricted diversity observed posttransmission indicates that productive infection is initiated by a single or a limited number of viral variants (8,34,36,54,60,67). However, there are few studies that have tracked the entire transmission process from donor to recipient, and those that have, have focused on analysis of the first hypervariable region (HVR1) of E2 in the chimpanzee model (54,60). Other regions of the envelope glycoproteins contain important functional and neutralizing determinants (16, 26, 27, 32, 43, 48-50, 52, 63), and the natural histories of HCV infection in chimpanzees and humans differ.…”

Monoclonal Antibodies with Broad Specificity for Hepatitis C Virus Hypervariable Region 1 Variants Can Recognize Viral Particles

“…In agreement with the weak anti-HVR1 responses detected in chimpanzees, HVR1 sequence diversity was significantly lower in this setting compared with those in humans (12) That being stated, the biological relevance of HVR1 is still unclear. Several studies identified binding sites for allegedly neutralizing Ab in this region (13,14), and it is therefore conceivable that HVR1 expressed on integral HCV particles could represent a major target for immune responses that may play an important role in the outcome of HCV infection.…”

Human Immunodeficiency Virus Seroconversion and Evolution of the Hepatitis C Virus Quasispecies

“…We and others have shown that HCV replication alone is not sufficient to produce nonsynonymous substitutions in chimpanzees sampled prior to a measurable adaptive immune response (26). That d N represents immune pressure was recently shown by Evans and coworkers in a study of simian immunodeficiency virus-infected macaques (8).…”

“…Nucleotide substitutions that do not affect the protein structure (synonymous substitutions, or d S ) accumulate in a quasispecies over time, reflecting the rate of viral replication among other factors. In contrast, changes in the amino acid composition of the HCV quasispecies (nonsynonymous substitutions, or d N ) typically reflect immunologic pressure and are not predicted by replication alone (8,26).…”