Liver Unit, Institut de Malalties Digestives, Departament de Medicina (IMD), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Hospital Clinic, University of Barcelona, Spain
Correspondence
Juan-Carlos Saiz
jcsaiz{at}inia.es
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the HCV sequences reported in this study are AY378328AY378694, AY378705AY379066, AY381300AY381643 and AY381644AY381994.
Present address: Departamento de Biotecnología, Instituto Nacional de Investigaciones Agrarias (INIA), Ctra Coruña km 7·5, 28040 Madrid, Spain.
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INTRODUCTION |
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The introduction of alpha interferon (IFN-) plus ribavirin combination therapy was an important breakthrough in the treatment of chronic HCV infection. However, the rate of sustained virological response is still unsatisfactory (Davis et al., 1998
; McHutchison et al., 1998
), particularly in patients infected with genotype 1 HCV (Poynard et al., 1998
), the most prevalent in our geographical area (López-Labrador et al., 1997
). Although viral genotype and viral load, as well as the chronology of serum HCV RNA clearance during therapy, are definitely related to response, further insight into viral factors involved in therapeutic responsiveness is still necessary.
Enomoto et al. (1995, 1996)
suggested that the genetic heterogeneity of a specific domain of the non-structural 5A (NS5A) region of HCV, termed the IFN sensitivity-determining region (ISDR), was related closely to response in Japanese patients with HCV genotype 1b infection. Similar observations were made in Spain (Sáiz et al., 1998
; Puig-Basagoiti et al., 2001
) and, although this is a controversial issue, analysis of the published information supports the hypothesis that a relationship exists between NS5A heterogeneity and response to therapy (Giménez-Barcons et al., 2001
; Witherell & Beineke, 2001
).
The NS5A region of HCV seems to play a role in virus replication and tumorigenesis, but other in vivo functions of this region remain largely unknown (Pawlotsky & Germanidis, 1999; Macdonald & Harris, 2004
). Recently, it has been suggested that the region of NS5A that is responsible for resistance to IFN includes the C-terminal 26 aa adjacent to the ISDR. This region has been termed the protein kinase-binding domain (PKR-bd) (Gale et al., 1997
). Cellular PKR is an IFN-inducible protein kinase that mediates the antiviral and antiproliferative effects of IFN by phosphorylating eukaryotic initiation factor 2
(Meurs et al., 1993
; Gale et al., 1997
). However, PKR-independent effects of NS5A in the IFN response have also been proposed (Macdonald & Harris, 2004
). In addition, mutations in the so-called V3 domain of NS5A (Inchauspe et al., 1991
), a region of unknown function that appears to be under strong selective pressure, have also been related to response to antiviral therapy (Duverlie et al., 1998
; Nousbaum et al., 2000
; Murphy et al., 2002
).
HCV replication is associated with a high rate of mutation that gives rise to a mixed and changing population of mutants, known as quasispecies (Martell et al., 1992; Domingo, 1996
). The quasispecies nature of HCV may have important implications concerning viral persistence, pathogenicity and resistance to antiviral agents (Domingo, 1996
; Forns & Bukh, 1999
; Forns et al., 1999
; Farci & Purcell, 2000
). However, previous work on the possible relationship between HCV quasispecies and response to therapy was mainly based on single-strand conformational polymorphism assays, which do not provide precise information about viral quasispecies, whilst more accurate studies based on analysis of cloned viral populations were reported less frequently. On the other hand, most of these studies were carried out in patients treated with IFN-
monotherapy and focused on hypervariable region 1 (HVR-1) of the E2 envelope protein of HCV (Forns et al., 1999
; Farci & Purcell, 2000
), whereas the NS5A quasispecies dynamics during combined IFN/ribavirin therapy have only been analysed in a small number of HCV-1b patients (Gerotto et al., 1999
; Nousbaum et al., 2000
).
We report here the results of an extensive analysis of the structure and evolution of NS5A quasispecies that was carried out in representative serial serum samples from patients with genotype 1b chronic hepatitis. Responders to IFN/ribavirin combination therapy, non-responders to this therapy and untreated patients were included in the study.
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METHODS |
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Detection and amplification of HCV RNA.
HCV detection (ELISA-HCV third generation; Ortho Diagnostics Systems) and qualitative determination and quantification of HCV RNA (Amplicor HCV-RNA and COBAS Amplicor HCV Monitor test 2.0; Roche Diagnostics) were carried out with commercial reagents. HCV RNA extraction and specific amplification of the 5' non-coding (5'NC) and NS5A regions by RT-PCR were carried out as described previously (Sáiz et al., 1997, 1998
; Giménez-Barcons et al., 2001
) using a proof-reading polymerase (Expand; Roche Molecular Biochemicals). To minimize experimental error and under-representation of minor variants within a given quasispecies, three independent RT-nested PCRs were carried out on each sample. HCV genotype was determined by restriction fragment length polymorphism analysis of the 5'NC region, as described previously (López-Labrador et al., 1997
).
Cloning and sequencing.
RT-PCR-amplified products of 762 bp encompassing the PKR-bd, which included the ISDR, and V3 regions of the NS5A protein were purified, ligated to the TA-cloning plasmid pGEM-T Easy (Promega) and cloned independently, as described previously (Sánchez-Fueyo et al., 2001). On average, 20 (1725) randomly selected clones from each serum sample were analysed (775 clones, 5·5x105 nt). Clones were sequenced with a DyeDeoxy Terminator cycle sequencing kit (Perkin Elmer Applied Biosystems) in a 310 DNA sequencer (Applied Biosystems). Electropherograms were analysed by using Sequence Navigator software (Perkin Elmer).
Sequences of all clones were aligned with the CLUSTAL W 1.6 program (Thompson et al., 1994) and the within-consensus sequence of each sample (WTH-CONS) and the between-consensus sequence of all analysed clones (BTW-CONS) were obtained.
Statistical and phylogenetic analyses.
Shannon entropy (Pawlotsky et al., 1998), which is defined as the different sequences or clusters of sequences that appear at a given time point, was calculated at the nucleotide and amino acid levels as follows: S=
(pi ln pi), where pi is the frequency of each sequence in the viral population. Entropy was normalized (Sn) as Sn=S/ln n, where n is the total number of sequences analysed in each sample.
Genetic distance between pairs of sequences and phylogenetic trees were computed with the PHYLIP package (Felsenstein, 1993). Ka and Ks, where Ks is the frequency of synonymous substitutions per synonymous site and Ka is the frequency of non-synonymous substitutions per non-synonymous site, were calculated by pairwise comparison of every single sequence using the MEGA program (Kumar et al., 1994
), which was also applied to calculate the mutation rate (no. nucleotide substitutions per nucleotide site per year) for each isolate.
Comparisons between groups were made by using 2 or Fischer's tests for categorical variables and the Wilcoxon test (paired samples) or the MannWhitney test (unpaired samples) for quantitative variables. Values of P<0·05 were considered significant.
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RESULTS |
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Fig. 1 shows a schematic representation of the deduced amino acid changes that were detected in the different samples analysed. Mutations were scattered throughout the NS5A region and no specific amino acids or motifs related to a particular type of response to therapy were observed in any of the regions analysed.
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Analyses of these variables in samples taken serially during therapy showed a trend similar to that observed in baseline samples. In non-responders, genetic distances and Ks and Ka values were higher than in responders and remained stable or tended to increase during therapy, whereas a trend to decrease was observed in responders. Differences between responders and non-responders were statistically significant at several time points, particularly in the V3 domain, where genetic distances and Ks and Ka values were, in general, significantly lower in responder patients throughout the study period (Fig. 4).
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The deduced mutation rate was higher in the V3 domain than in other regions (Table 2). No differences between non-responders (between 5x103 and 10x103 substitutions nt1 y1) and untreated patients (between 4·6x103 and 15x103 substitutions nt1 y1) were observed. In contrast, the median rate of fixation of mutations observed in serial isolates from responders (26·546·1 substitutions nt1 y1) was higher than in other patients. The only exception was observed in non-responder patient 10 (between 2·5x102 and 4x102 substitutions nt1 y1), in whom the imposition of a minor variant was detected during follow-up (Fig. 3
).
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DISCUSSION |
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In the present study, and in agreement with previous observations (Enomoto et al., 1996; Duverlie et al., 1998
; Sáiz et al., 1998
; Pawlotsky & Germanidis, 1999
; Nousbaum et al., 2000
; Puig-Basagoiti et al., 2001
; Murphy et al., 2002
), no single amino acid position or motif was associated with different responses to therapy in any of the NS5A regions analysed. Furthermore, phylogenetic analyses did not show clustering of NS5A variants in relation to different responses to therapy.
Although the number of cases analysed was relatively small, there were no significant differences in the mutation rate of the different regions analysed between non-responder and untreated patients. In fact, in these patients, mutation rates were closely similar to those observed previously in a large number of untreated patients with chronic hepatitis C of variable severity seen at our institution (Soguero et al., 2000; Giménez-Barcons et al., 2001
; Franco et al., 2003
). In contrast, the median rate of fixation of mutations observed in sustained responders was 3070 times greater, depending on the region evaluated, and led to a high number of amino acid changes during therapy, suggesting that, in responder patients, treatment exerts a selective pressure over this region.
Overall, the NS5A region remained quite stable during combined antiviral therapy and no specific amino acid or combination of amino acid changes could be related to the type of response to treatment.
In the PKR-bd, imposition of amino acid changes was quite infrequent during antiviral therapy and, when detected, they were observed exclusively in a minority of isolates from sustained responders. Thus, accumulation of nucleotide changes in the NS5A region, including the PKR-bd and the ISDR, was not usually reflected in the imposition of amino acid changes, indicating that these domains remain mainly stable during antiviral therapy. Similar observations have been described in untreated patients during the natural course of infection (Franco et al., 2003). Previous studies have indicated that strains bearing an ISDR with a so-called wild-type sequence (Enomoto et al., 1996
) similar to that of prototype HCV-J (Kato et al., 1990
) represent the sequence with the highest affinity to cellular PKR (Gale et al., 1997
); however, in our study, whilst one of the strains that arose during treatment (patient 7) presented a sequence with a putative higher capacity to interact with PKR, the other (patient 4) showed a more divergent sequence. Thus, our data indicate that amino acid changes in the PKR-bd alone are not necessarily responsible for the different sensitivities to antiviral therapy. In fact, it has recently been described that not only mutations in this domain, but also changes located outside the PKR-bd, or a combination of both, affect NS5APKR interaction (Macdonald & Harris, 2004
) and it is thus possible that mutations in NS5A regions other than the PKR-bd also contribute to decreasing the capacity of different HCV strains to interact with PKR or with some other cellular pathway, making the arising viral population more susceptible to drugs. In any case, our study could not assign any mutation or combination of mutations to such a capacity.
On the other hand, and in contrast to other regions of the NS5A, in the V3 domain, mutations were more common and occurred in isolates from responders and non-responders. Even more, analysis of sequential samples showed that, overall, the rate of fixation of mutations during therapy was higher in V3 than in other viral regions, supporting the hypothesis that this domain has a higher capacity to accumulate mutations (Inchauspe et al., 1991; Nousbaum et al., 2000
). However, until the biological function of the V3 domain is completely elucidated, it is not possible to establish whether the mutations that arose during antiviral therapy were merely a reflection of the high capacity of this domain to accept amino acid changes with preservations of its functional role, or whether they represent a direct consequence of the pressure exerted on the V3 domain by antiviral therapy.
Our results also indicate that, in treated patients, mutations were always detected during the first week of therapy and were due to the imposition of minor variants that were already present prior to treatment, strongly supporting the hypothesis that antiviral drugs do exert pressure over specific HCV NS5A variants, as suggested previously (Pawlotsky et al., 1998; Polyak et al., 1998
; Sáiz et al., 1998
; Nousbaum et al., 2000
). For instance, in patient 6, a patient co-infected with two different HCV-1b strains, the variant represented less at baseline disappeared after 1 week of treatment. However, it should be noted that clearance of one of the co-infecting variants has also been observed during the natural course of the disease in the absence of antiviral therapy (Franco et al., 2003
) and, thus, imposition of minor variants and/or strains cannot be attributed exclusively to the action of the drugs in treated patients.
The pre-therapy complexity and diversity of NS5A quasispecies observed in this study were higher in non-responders than in responders, particularly in the V3 domain. Differences became more apparent during therapy, as the diversity and complexity of the quasispecies remained stable or tended to increase in non-responders, but tended to decrease in sustained responders. It is therefore possible that greater complexity and diversity of quasispecies offer the virus a better chance of having subpopulations that are able to elude antiviral therapy.
In summary, according to the observations made in the current study, fixation of mutations in the NS5A region induced by IFN and ribavirin combination therapy is uncommon, although it is more frequent in the V3 domain. Fixation of mutations occurs very early during therapy and is due to the imposition of minor variants that are already present prior to therapy. Overall, the complexity and diversity of quasispecies are lower and tend to decrease in sustained responders, whereas they tend to remain stable in non-responders. These differences are more marked in the V3 domain. Therefore, the quasispecies composition and dynamics of HCV NS5A seem to be related to responsiveness to combined antiviral therapy of patients with chronic hepatitis C.
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ACKNOWLEDGEMENTS |
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Received 13 August 2004;
accepted 13 December 2004.
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