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Previous Article | Next Article 
Journal of Virology, January 2000, p. 661-668, Vol. 74, No. 2
Laboratoire de
Virologie,1 Service
d'Hépato-Gastro-Entérologie,2 and
Service de Medecine Interne,3
Hôpital Purpan, CHU Toulouse, 31059 Toulouse Cédex, France
Received 11 June 1999/Accepted 14 October 1999
Hepatitis C virus (HCV) populations persist in vivo as a mixture of
heterogeneous viruses called quasispecies. The relationship between the
genetic heterogeneity of these variants and their responses to
antiviral treatment remains to be elucidated. We have studied 26 virus
strains to determine the influence of hypervariable region 1 (HVR-1) of
the HCV genome on the effectiveness of alpha interferon (IFN- Hepatitis C virus (HCV), the
causative agent of non-A, non-B hepatitis (1, 5), is a
positive-strand RNA virus that exists within its host as pools of
related genetic variants, referred to as quasispecies (19,
32). Its heterogeneous character is most evident in hypervariable
region 1 (HVR-1) of the envelope gene, which mutates over time in
response to host pressures (11, 18, 57). Recent data have
suggested that the heterogeneity of quasispecies is involved in viral
persistence (50), cellular tropism (48), the
pathogenesis of hepatic disease (16, 37), and response to
antiviral therapy (15, 31).
Alpha interferon (IFN- Several studies have suggested that the great heterogeneity of HVR-1
could be involved in the resistance to IFN- Patients and samples.
We retrospectively selected a group of
26 patients from the 136 patients given the standard IFN-
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Genetic Heterogeneity of Hypervariable Region 1 of
the Hepatitis C Virus (HCV) Genome and Sensitivity of HCV to Alpha
Interferon Therapy
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
)
therapy. Following PCR amplification, we cloned and sequenced HVR-1.
Pretreatment serum samples from 13 individuals with chronic hepatitis C
whose virus was subsequently eradicated by treatment were compared with
samples from 13 nonresponders matched according to the major factors
known to influence the response, i.e., sex, genotype, and pretreatment
serum HCV RNA concentration. The degree of virus variation was assessed
by analyzing 20 clones per sample and by calculating nucleotide
sequence entropy (complexity) and genetic distances (diversity). Types
of mutational changes were also determined by calculating nonsynonymous
substitutions per nonsynonymous site (Ka) and
synonymous substitutions per synonymous site
(Ks). The paired-comparison analysis of the
nucleotide sequence entropy and genetic distance showed no statistical
differences between responders and nonresponders. By contrast,
nonsynonymous substitutions were more frequent than synonymous
substitutions (P 
0.05) in responders, but there was
no significant difference in nonresponders. Nonsynonymous substitutions
tended to be more frequent than synonymous substitutions in women
(P = 0.06) but not in men. Nucleotide entropy and
genetic distances were significantly related to serum RNA concentration
(P 0.01). Our findings suggest that after
controlling for the major determinants of interferon response, neither
complexity nor diversity of the HVR-1 region is associated per se with
virus eradication. Because a higher proportion of nonsynonymous
substitutions than synonymous substitutions was found only in
responders, host anti-HCV-specific immune response rather than viral
factors may be playing an important role in the interferon response.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) is the first approved drug therapy for
hepatitis C virus infection (6, 20, 25). The standard treatment leads to a sustained clearance of HCV RNA in 15 to 20% of
patients (21). There is evidence that the amount of HCV RNA in the patient's serum and the genotype of the HCV are both indicators of a sustained clearance of HCV (17, 22, 33). However,
patients with the same genotype and similar RNA levels may respond
differently, indicating that particular viral strains have
characteristics conferring resistance or sensitivity to antiviral
therapy. Several Japanese studies have found a relationship between
mutations within the NS5A region of the HCV-1b genome and sensitivity
to IFN- (4, 9, 10, 13), but similar studies performed in
other parts of the world have not (26, 60). In vitro
experiments have shown that NS5A can interfere with IFN- signaling
pathways and cause resistance to therapy (14, 28, 54).
Pawlotsky et al. recently showed that no NS5A sequence was
intrinsically resistant or sensitive to IFN- (43), nor
does there appear to be any correlation between resistance to
interferon treatment in patients infected with HCV-3 and the rate of
mutation within the NS5A region (49, 53).
(3, 25, 41),
but this issue is controversial (38). Most of this work has
suffered from an incomplete definition of the parameters studied (i.e.,
biochemical or virological responses), and the limited number of
molecular clones sequenced (n 10) has raised
concerns about sampling bias. In addition, viral factors such as
genotype and serum RNA concentration, which are known to influence the effectiveness of IFN-, have not been controlled. We have therefore performed a clonal analysis by sequencing more than 20 clones per
sample from two groups of individuals with chronic HCV infection to
determine more precisely the influence of pretreatment HVR-1 genetic
heterogeneity on the response to IFN-. The groups were matched
according to the major determinants of virological response, including
the HCV genotype and the serum HCV RNA concentration before treatment.
We compared the genetic complexity and diversity and measured the
proportions of synonymous and nonsynonymous mutations in the two groups.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-2b
treatment for chronic hepatitis C (3 MU three times per week for 6 to
12 months) and already enrolled in an observational study
(22). They comprised 13 patients with a sustained
virological response (R), defined by normal ALT activity and negative
HCV RNA by PCR analysis 6 months after IFN- withdrawal, and 13 non-sustained responders (NR), defined by elevated alanine
aminotransferase (ALT) activity and detectable HCV viremia 6 months
after IFN- was stopped. The matching criteria were three factors
independently associated with sustained virological response in a
multivariate analysis, i.e., sex, HCV genotype, and pretreatment serum
HCV RNA concentration (in each pair, 
RNA was less than 0.5 log
copies/ml). Interferon was given to 10 patients in each group for 12 months and to 3 patients for 6 months. There was no significant
difference in the estimated duration of infection for the responders
(129 months) and the nonresponders (131 months). The genetic
heterogeneity of the virus was established by cloning and sequencing
the HVR-1 region in samples taken just before the first dose of
IFN-. The demographic, histological (27), and virological
features of these 26 patients are indicated in Table
1.
TABLE 1.
Clinical, histological, and virological features of the
26 patients studied and characteristics of the HVR-1 quasispecies
before treatment
HCV RNA quantification. HCV RNA levels were measured in pretreatment sera by the standardized quantitative reverse transcription (RT)-PCR assay Amplicor HCV Monitor (Roche Molecular Systems, Branchburg, N.J.) according to the manufacturer's instructions.
HCV genotyping. HCV genotype was determined by the Inno-LiPA II HCV method (Innogenetics S.A., Gent, Belgium). After RT-PCR amplification, the amplified products were hybridized to immobilized probes specific for the different genotypes and subtypes.
Cloning and sequencing of HVR-1. (i) Primers.
Primers were
designed to amplify fragments containing the amino-terminal region of
the E2 gene (HVR-1). All the primer sequences are listed in Table
2.
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(ii) RNA extraction, RT, and nested-PCR amplification.
Virus
RNA was first extracted from 100 µl of serum by the guanidinium
thiocyanate-phenol-chloroform method. The RNA was eluted in 30 µl of
sterile water and stored at 
80°C. The RNA (10 µl) was then
reverse transcribed at 37°C for 60 min with 1 pmol of the outer
antisense primer in the presence of 20 U of Moloney murine leukemia
virus reverse transcriptase (Boehringer GmbH, Mannheim, Germany), and
nested PCR was performed. The first round was done with 5 pmol of outer
primers, and the second round was done with 5 pmol of inner primers.
The two rounds were performed with 2.5 U of Taq DNA
polymerase (AmpliTaq; Perkin-Elmer Cetus, Norwalk, Conn.) under the
same conditions, i.e., 5 min of denaturation at 95°C followed by 35 cycles of 95°C for 30 s, 55 to 60°C for 30 s, and 72°C
for 90 s, and then by a final extension at 72°C for 10 min. The
amplified products were analyzed by electrophoresis through a 2%
agarose gel (Gibco BRL, Paisley, Scotland) and staining with ethidium bromide.
(iii) Plasmid cloning. PCR products were purified with QIAamp columns (Qiagen, Courtaboeuf, France) as specified by the manufacturer. The purified products were quantified by spectrophotometry; 10 ng was directly ligated into 50 ng of PCR II vector (Original TA Cloning Kit, Invitrogen BV, Leek, The Netherlands) at 14°C overnight. Recombinant plasmids were used to transform Escherichia coli competent cells according to the manufacturer's protocol, and transformants were grown on ampicillin plates.
(iv) Nucleotide sequencing. Twenty independently isolated cDNA clones from PCR products were selected. Plasmid DNAs containing HVR-1 inserts were prepared and sequenced on both strands by the dideoxy chain termination method (PRISM Ready Reaction AmpliTaq Fs and Dye Deoxy primers; Applied Biosystems, Paris, France) on a model 377 automated DNA sequencer (Applied Biosystems, Foster City, Calif.). Electropherogram data were analyzed by the Sequence Navigator program.
(v) Calculation of genetic complexity and diversity.
Nucleotide sequences were aligned with the CLUSTAL W program version
1.5. We quantified the complexity of the HCV strain in the region of
interest by calculating the Shannon entropy as follows: S = 
i
(pi ln pi), where
pi is the frequency of each sequence in the
viral quasispecies (58). The normalized entropy, Sn, was calculated at the nucleotide level as
follows: Sn = S/ln N,
where N is the total number of sequences analyzed. It was
calculated at both the nucleotide and amino acid levels. We quantified
diversity as the mean genetic distance calculated for all pairs of
nucleotide sequences by using the DNADIST module in the PHYLIP package
version 3.572. The calculation was based on a Kimura two-parameter
distance matrix with a transition-to-transversion ratio of 2.0. The
mean and standard error of the mean (SEM) within-sample genetic
distances were calculated for the quasispecies in each of the 26 patients before treatment. The numbers of synonymous
(Ks) and nonsynonymous (Ka) substitutions per synonymous and
nonsynonymous site, respectively, were calculated with the Jukes-Cantor
correction for multiple substitutions (24) by using the MEGA
program (30).
Statistical analysis. Comparisons between the responders and nonresponders were performed by using Student's paired t test. Correlations among quantitative variables were computed with Pearson's rank correlation test, and linear regression was used to illustrate these correlations. P values of less than 0.05 were considered to be significant.
Nucleotide sequence accession numbers. The sequences have been submitted to EMBL with accession no. AF 166548 to AF 166589.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Patient characteristics.
The patients' demographic and
biological characteristics are indicated in Table 1. No significant
differences in matching criteria (i.e., sex, genotype, and pretreatment
HCV RNA levels) were found between responders (4.98 ± 0.20 log
copies/ml) and nonresponders (5.15 ± 0.25 log copies/ml). There
was also no significant difference between the Knodell histological
scores of the two groups. By contrast, responders were significantly
younger (34 ± 2 years) than nonresponders (43 ± 3 years;
P 0.05).
Analysis of HCV quasispecies distribution.
The quasispecies
distribution of HVR-1 was analyzed by cloning and sequencing the PCR
products of pretreatment samples from the 26 selected patients (Table
1). A total of 515 pretreatment HVR-1 clones were generated and
sequenced. Amino acid sequences and their relative frequencies in the
virus population are shown in Fig.
1. The complexity of the
HVR-1 region was estimated by normalized nucleotide sequence entropy in
the 26 pretreatment samples on the basis of the first 20 clones
isolated. The normalized nucleotide entropy was 0.522 ± 0.044. The normalized amino acid entropy was 0.435 ± 0.046. The
diversity of HVR-1 was evaluated as the average genetic distance within
the quasispecies. The mean within-sample genetic distance in the 26 patients was 0.0733 ± 0.0124. Complexity and diversity were
significantly related to each other (r = 0.74;
P 0.001). The proportion of nonsynonymous substitutions (0.0837 ± 0.0146) was significantly higher than the
proportion of synonymous substitutions (0.0677 ± 0.0149;
P 0.05), suggesting that HVR-1 mutations were more
products of an immune selection pressure than of random genetic drift.
|
Correlation between HCV quasispecies heterogeneity and response to
IFN-.
We have compared HVR-1 genetic heterogeneity in
responders and nonresponders. We first analyzed the repertoire sizes of
the two groups. The mean nucleotide sequence entropy of responders (0.452 ± 0.050) did not differ significantly from that of
nonresponders (0.592 ± 0.069) (Fig.
2). The mean amino acid sequence
entropies of responders (0.372 ± 0.047) and nonresponders
(0.498 ± 0.077) also did not differ. The average within-sample
genetic distances were similar in responders (0.0704 ± 0.0194)
and in nonresponders (0.0762 ± 0.0163) (Fig. 2). We then analyzed
the types of mutational changes. The proportion of synonymous and
nonsynonymous substitutions in responders (0.0509 ± 0.0187 and
0.0848 ± 0.0241, respectively) did not differ significantly from
those observed in nonresponders (0.0838 ± 0.0229 and 0.0826 ± 0.0177). The Ka/Ks ratio was
higher in responders (Ka/Ks = 2.4)
than in nonresponders (Ka/Ks = 1.5), but the difference did not reach statistical significance. By contrast,
nonsynonymous substitutions were significantly more frequent than
synonymous substitutions in the responders (P 0.05)
while there was no significant difference in the nonresponders. As
highlighted in Fig. 1, the nonsynonymous changes occurred throughout the HVR-1 domain.
|
Relationship between HVR-1 genetic heterogeneity and clinical,
histological, and virological features.
Nucleotide sequence
entropy did not differ significantly between women (0.571 ± 0.063) and men (0.464 ± 0.060), but the genetic distances were
higher in women (0.0974 ± 0.0187) than in men (0.0453 ± 0.0121) (P 0.05). The proportion of nonsynonymous
substitutions in women (0.1117 ± 0.0227) was significantly higher
than in men (0.0511 ± 0.0129; P 0.05). The
Ka/Ks ratio was higher in women (Ka/Ks = 2.2) than in men
(Ka/Ks = 1.6), although the
difference was not significant. Lastly, nonsynonymous substitutions
tended to be more frequent than synonymous substitutions in women
(P = 0.06) but not in men. Nucleotide and amino acid
entropy, genetic distances, the proportion of synonymous and
nonsynonymous substitutions, and
Ka/Ks ratios did not differ among
the different genotypes. None of the parameters used to assess HVR-1
heterogeneity correlated with age. However, we did find positive
correlations between pretreatment HCV RNA concentrations and nucleotide
sequence entropy (r = 0.64; P 0.01)
or genetic distance (r = 0.60; P 0.01) (Fig. 3). The proportions of
synonymous and nonsynonymous substitutions were also related to HCV RNA
concentrations (rKs = 0.57;
rKa = 0.56; P 0.01).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Many attempts have been made to establish a correlation between
the genetic heterogeneity of HCV RNA and the sensitivity of HCV to
IFN-, but published studies have given conflicting results. This may
be because the authors have used different techniques with different
performances to assess virus heterogeneity, such as single-strand
conformational polymorphism analysis (29, 36, 44),
heteroduplex analysis (HDA) (45), and combined HDA and single-strand conformational polymorphism analysis (56). For this study, we have cloned and sequenced a sufficiently large number of
molecular clones per sample (more than 20) to prevent sample bias
(8, 15). We have also checked for virus factors known to
influence the effectiveness of IFN- by analyzing the HVR-1 sequences
in two groups of subjects, matched for sex, HCV genotype, and
pretreatment serum HCV RNA concentration. To the best of our knowledge,
this is the first case-control study designed to assess the influence
of the genetic heterogeneity of HVR-1 on virus sensitivity to IFN-.
Genetic variation in responders and nonresponders was characterized by
complexity and diversity. The complexity, the distribution of variants
in the population, was estimated by calculating the Shannon entropy.
Diversity was measured as the mean genetic distance calculated for all
pairs of sequences. Our data show no significant difference between
responders and nonresponders for either measure of HVR-1 genetic
heterogeneity. A similar observation was also reported by Polyak et al.
with HDA (45). By contrast, other studies based on cloning
and sequencing techniques have reported that patients with
heterogeneous virus populations are less responsive than patients with
homogeneous virus populations (3, 25, 41). However, the
number of molecular clones analyzed per sample was less than 10 and the
influence of HCV RNA concentration on the response to IFN- was not
controlled. We found that sustained response was significantly
associated with youth, whereas previous multivariate analyses have
found the predictive value of age to be less than those of virus
characteristics (22). Thus, by checking for the influence of
major factors, genotype and HCV RNA concentration, we could show the
impact of other factors such as age and therefore could measure the
effect of virus genetic heterogeneity on the sustained response to
IFN-. We found a correlation among complexity, diversity, and serum
HCV RNA concentrations, in contrast to the absence of a relationship
between quasispecies distribution and virus eradication. This supports
the possibility that viral quasispecies arise as a consequence of the
limited fidelity of HCV replication and the highly dynamic process of
virus production (40).
An important finding of this study is that pretreatment nonsynonymous
substitutions in HVR-1 were significantly more frequent than synonymous
substitutions in patients who had cleared their HCV after IFN-
therapy, suggesting a stronger selective pressure for changes in amino
acids in these patients. It has been demonstrated that the
27-amino-acid segment located in the N-terminal portion of the HCV
envelope protein, HVR-1, contains linear neutralizing B-cell epitopes
(11, 50, 51, 61). There is recent evidence that HVR-1 is
also a helper T-cell recognition site (52) and may be
responsible for antagonism to T cells by influencing the priming of a
CD4+-T-cell response toward HVR-1 immunogenic variants
(12). Thus, the selection pressure driving the genetic
variations of the virus may come from the T cells themselves or from
neutralizing antibodies whose production depends on T cells specific
for this region. A collaboration between helper T cells and cytotoxic T
lymphocytes (CTLs) is a common feature of many virus infections, and it
has been reported that intrahepatic HCV-specific CTL activity may affect the subsequent response to IFN- therapy (39). In
vitro experiments have shown that the T-cell response to HCV peptides and recombinant core protein during IFN- treatment was significantly more vigorous in patients infected with genotype 2c than in those infected with genotype 1b (35). The authors suggest that
this could be one of the factors contributing to the different
susceptibilities of genotype 1 and genotype 2 to IFN- treatment.
Different humoral responses to the HVR-1 region in HCV-1b- and
HCV-2c-infected patients have also been reported (59). The
antiviral effect of IFN- results from both inhibition of viral
replication and modulation of the immune response to viral epitopes. As
recently suggested by mathematical models, the major initial effect of
IFN- is to block virion production or release (40), but
the subsequent second-phase decline is thought to reflect the death
rate of productively infected cells, where HCV-specific CTL activity
could be a major contributor. Other recent studies demonstrated that
IFN- therapy was associated with an increased rate of fixation of
mutations in the HVR-1 region compared to the same region in untreated
patients, supporting the idea that IFN- acts partially via
immunomodulation (42, 46). Thus, in addition to genotype and
HCV RNA concentration, the pretreatment host immune status could
influence IFN--stimulated immune responses and ultimately virus
eradication. Interestingly, women had a higher proportion of
nonsynonymous substitutions than did men. It has previously been
suggested that the greater response of women to IFN- could be a
consequence of their being given relatively higher doses because of
their lower body weight (2, 23). Differences in the
immunological status of men and women may represent another factor.
In summary, in our case control study, which included a majority of HCV
genotype 2- and 3-infected patients, the complexity and diversity of
HVR-1 were correlated with the serum HCV RNA concentration but were not
associated per se with virus eradication after IFN- therapy. By
contrast, the higher proportion of nonsynonymous substitutions found in
responders suggests that the HCV-specific immune response is involved
in the clearing of HCV by IFN-. Pretreatment immune status, and
especially the intensity and the quality of the anti-HCV immune
responses of individuals, could be a major determinant of HCV RNA
clearance. The beneficial effect of combining ribavirin with IFN- to
treat HCV infection (7, 34, 47) could be due to the
modulation of several immune cell functions, but the influence of the
envelope sequence, apart from HVR-1, on the early antiviral effect of
IFN- cannot be excluded. It has been suggested recently that a short
amino acid sequence in the E2 C terminus region containing
phosphorylation sites interacts with PKR (55). Studies
combining the characterization of HCV quasispecies heterogeneity and
anti-HCV immune responses should be useful for determining how
therapeutic agents act and for further optimizing the treatment of
chronic hepatitis C.
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ACKNOWLEDGMENTS |
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We thank Jean-Paul Charlet (Service d'Epidémiologie Hôtel-Dieu, Toulouse, France) for help with statistical analysis. We thank Corine Tourne-Peteilh for technical assistance. We also thank Eliane Coutanceau for secretarial assistance.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Virologie, CHU Toulouse, 31059 Toulouse Cédex, France. Phone: (33) 5 61 77 24 63. Fax: (33) 5 61 77 25 42. E-mail: izopet{at}cict.fr.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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