Journal of Medical Virology 81:20212028 (2009)

NS5AISDR-V3 Region Genetic Variability of Tunisian HCV-1b Strains: Correlation With the Response to the Combined Interferon/Ribavirin Therapy
N. Bouzgarrou,1* E. Hassen,1 W. Mahfoudh,1 S. Gabbouj,1 E. Schvoerer,2 A. Ben Yahia,3 N. Ben Mami,4 H. Triki,3 and L. Chouchane5
1 2

Molecular Immuno-Oncology Laboratory, Faculty of Medicine, Monastir, Tunisia Institute of Virology, Strasbourg, France 3 Laboratory of Clinical Virology, Institut Pasteur de Tunis, Tunis-Belvedere, Tunisia 4 Gastroenterology B Unit, La Rabta Hospital, Tunis, Tunisia 5 Department of Genetic Medicine, Weill Cornell Medical College in Qatar, Doha, Qatar

In the non-structural protein 5A (NS5A) of hepatitis C virus (HCV), mutations within the interferon sensitivity-determining region (ISDR), the PKR-binding domain (PKR-BD), the variable region 3 (V3), and the interferon/ribavirin resistance-determining region (IRRDR) have been correlated with the IFN-based therapy response. In Tunisia, where a high prevalence of HCV-1b has been found, no data regarding the implication of NS5A in treatment response were available. The current study examined the relationship between the pre-treatment mutation number within ISDR, PKR-BD, V3, IRRDR, as well as in the entire ISDR-V3 region of NS5A (aa 22092379) and the response to the 48-week course of combined IFN plus ribavirin therapy in 15 HCV-1b-infected Tunisian patients. Referring to HCV-J sequence, a signicant high genetic variability was observed within PKR-BD in the sustained virological responder patients compared to non-responders (P 0.040). More importantly, when considering the entire region from ISDR to V3, referred to as NS5AISDR-V3, a clear difference in the mutation number was observed between sustained virological responders (19.6 3.16) and non-responders (15.0 1.41) (P 0.002). Additionally, a more detailed analysis of NS5AISDR-V3 region revealed an elevated degree of mutation rate within the region located between amino acids 2282 and 2308 (P 0.0006). Interestingly, an analysis of specic amino acid variations dened proline and serine at position 2300 as signature patterns for sensitive and resistant strains, respectively. The genetic variability within the NS5A region of HCV-1b strains was associated with the response to the combined IFN plus ribavirin therapy in our
2009 WILEY-LISS, INC.

Tunisian cohort. J. Med. Virol. 81:20212028, 2009. 2009 Wiley-Liss, Inc. KEY WORDS: hepatitis C virus; NS5A; treatment response; Tunisia

INTRODUCTION Hepatitis C virus (HCV) is the major cause of chronic viral hepatitis that can progress to cirrhosis and hepatocellular carcinoma over the course of 2030 years [Poynard et al., 1997; Alberti et al., 1999; Afdhal, 2004]. It is a single positive-strand RNA virus belonging to the Flaviviridae family [Miller and Purcell, 1990]. Based on a comparison of genome and polyprotein sequences of HCV isolates worldwide, six genotypes, HCV-1 to -6, each comprising multiple subtypes, such as HCV-1a and -1b, have been identied [Simmonds et al., 1994]. As in Japan, Taiwan, southern and eastern Europe, HCV subtypes 1b is the most common genotype in Tunisia with an average prevalence of 80% [Djebbi et al., 2003; Bouzgarrou et al., 2005; Mejri et al., 2005]. The current therapy consists of pegylated alpha interferon (PEGIFN-a) combined with ribavirin, a nucleoside analog, for

` Grant sponsor: Le Ministere de lEnseignement Superieur, de la Recherche scientique et de la Technologie (LR/05 Hepatites ` et maladies virales epidemiques); Grant sponsor: le Ministere de la Sante Publique de la Republique Tunisienne. *Correspondence to: N. Bouzgarrou, Molecular Immuno-Oncology Laboratory, Faculty of Medicine of Monastir, 5019 Monastir, Tunisia. E-mail: nadia.bouzgarrou@lycos.com Accepted 28 July 2009 DOI 10.1002/jmv.21641 Published online in Wiley InterScience (www.interscience.wiley.com)

2022

Bouzgarrou et al.

2448 weeks. It was associated with a high long-term response rate in patients infected with genotypes 2 and 3, whereas genotype 1 patients showed only 46% of response rates [Manns et al., 2001; Fried et al., 2002; Zeuzem et al., 2004]. Some clinical and molecular studies have identied other host-related and virusrelated factors linked to the response to IFN-based therapy. Indeed, gender, age, liver histology, viral load, quasi-species complexity, and viral kinetics within 4 weeks of treatment have been retained as predictive factors [Hayashi et al., 1998; Pawlotsky et al., 1998; Myers et al., 2003; Zeuzem et al., 2006; Kau et al., 2008; Yu et al., 2008]. In addition, the non-structural protein 5A (NS5A), a hydrophilic phosphoprotein, is believed to inuence the treatment response. A large number of amino acid mutations within NS5A protein have been associated previously with an effective response to IFN-based therapy. Enomoto et al. [1995, 1996] rst described a signicant correlation between sequence variations in the so-called IFN sensitivity-determining region (ISDR) (amino acids 22092248) and the response to IFN-a monotherapy in HCV-1b Japaneseinfected patients. Later, other Japanese studies conrmed the same association between ISDR and treatment response [Watanabe et al., 2005; Murayama et al., 2007]. However, conicting results have been reported in studies from Europe [Duverlie et al., 1998; McKechnie et al., 2000; Veillon et al., 2004; Kmieciak et al., 2006] and the United States [Nousbaum et al., 2000; Murphy et al., 2002]. Some in vitro studies indicated that HCV resistance to IFN-a is mediated partially through the interaction between NS5A and the RNA-dependent protein kinase (PKR), one of the most important host antiviral proteins [Gale et al., 1997]. This interaction requires 26 additional amino acids carboxy terminal to the ISDR [Gale et al., 1998]. This region located between amino acids 2209 and 2274 was termed PKR-binding domain (PKRBD). A signicant association between mutations within HCV-1 PKR-BD and sustained virological response to IFN-a or IFN-a/PEG-IFN-a plus ribavirin has been reported [Berg et al., 2000; Sarrazin et al., 2002; Macquillan et al., 2004; Munoz de Rueda et al., 2008]. It has also been demonstrated that the NS5A protein inhibits IFN-a activity through a PKR-independent mechanism in liver-derived cells [Podevin et al., 2001]. Furthermore, some additional studies on the full NS5A sequences have suggested that a high degree of sequence variation in the variable region 3 (V3; aa 23562379) or in the V3 region plus its N-terminally anking region, called interferon/ribavirin resistancedetermining region (IRRDR; aa 23342379), may correlate with an early and sustained virological response to IFN-a and ribavirin [Veillon et al., 2004, 2007; El-Shamy et al., 2007, 2008]. As the association between NS5A heterogeneity and the response to IFN-based therapy remains controversial, the aim of the current study was to determine whether the pre-treatment number of amino acid substitutions within NS5A protein was correlated with
J. Med. Virol. DOI 10.1002/jmv

the response to combined IFN and ribavirin therapy in Tunisian HCV-1b-infected patients. The analysis focused on ISDR, PKR-BD, IRRDR, and V3 regions as well as the entire ISDR-V3 region of NS5A (NS5AISDR-V3). Amino acid signature patterns for sensitive and resistant Tunisian strains were also investigated. PATIENTS AND METHODS Patients The current study included 21 Tunisian patients infected chronically with HCV-1b (10 men, 11 women; mean age: 50.6 6.09) treated at the Gastroenterology B Unit of La Rabta Hospital. The diagnosis of all patients was conrmed by biochemical and molecular assays, including the detection of anti-HCV antibodies using the third-generation commercial enzyme immunoassays (INNOTEST HCV Ab III, Innogenetics-Belgium and Murex anti-HCV, Murex Diagnostics, Chatillon, France) and the detection of HCV-RNA in serum (Amplicor HCV assay, Roche Diagnostics, Mannheim, Germany). All patients were negative for hepatitis B surface antigen and antibodies to human immunodeciency virus 1 and 2. They were treated with a 48-week course of a combination therapy of the standard IFN-a (n 4) or pegylated IFN-a (n 17) with ribavirin. The follow-up of patients consisted of alanine aminotransferase (ALT) determination and serum HCV-RNA quantitation at months 3 and 12, and 6 months after the end of therapy (Amplicor HCV assay, Roche Diagnostics). Sustained virological responders were dened on the basis of ALT normalization and undetectable RNA 6 months after the end of therapy. Nonresponders corresponded to patients who had never responded to the treatment (>2 log10 decrease in the viral load at month 3), those who were HCV-RNA positive at month 12, and those who relapsed and redeveloped HCV-RNA 6 months after the end of treatment. In 15 patients (8 sustained virological responders, 7 non-responders), for whom sequential pre-treatment sera were available, the NS5AISDR-V3 sequences (aa 22092379) for the dominant variant of HCV-1b were determined by direct sequencing. Approval for the study was given by the National Ethical Committee and informed consent was obtained from each participant. NS5A Amplication Total RNA was extracted from 140 ml of pre-treatment sera using QIAamp viral RNA mini kits (Qiagen, Hilden, Germany) according to the manufacturers recommendations. After reverse transcription using Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), a nested-polymerase chain reaction (nested-PCR) was performed by the outer primers, E1 and E2, and the inner primers, I3 and I4, previously described by Duverlie et al. [1998]. Briey, the

NS5A Genetic Variability and Treatment Response

2023

rst-round PCR was conducted using 10 ml of cDNA added to 40 ml of a reaction mixture containing 1 Gold Tad buffer, 2.5 mM MgCl2, 10 mM dNTP, 0.8 mM E1 and E2, and 2.5 U of Gold Taq DNA polymerase. After denaturation at 948C for 5 min, 30 cycles of amplication were carried out. Each cycle consisted of denaturation at 908C for 90 sec, annealing at 508C for 90 sec, and extension at 728C for 2.30 min. These were followed by an extension at 728C for 7 min. For the second-round PCR, 5 ml of the rst reaction mixture was further amplied over 35 cycles with 0.8 mM of the inner primers, I3 and I4. Reaction conditions were as follows: denaturation at 908C for 2 min, annealing at 558C for 60 sec, and extension at 728C for 60 sec, followed by an extension at 728C for 7 min. The resulting amplication products were analyzed in 1% agarose gel stained with ethidium bromide. NS5AISDR-V3 Direct Sequencing and Sequence Analysis The nested-PCR products were puried from agarose gel using QIAquick Gel Extraction Kit (Qiagen). The puried products were sequenced directly according to the manufacturers instructions with the internal forward primers, S534, S775, S1033, and the internal reverse primers, AS818 and AS1174, also described by Duverlie et al. [1998] using ABI PRISM BigDye terminator v3.1 Ready Reaction Cycle Sequencing Kit (Applied Biosystem, Foster City, CA). After purication, an electrophoresis was performed in the ABI PRISM 310 Genetic Analyser (Applied Biosystems). The obtained sequences (GenBank accession numbers: FJ828844 FJ828858) were aligned with the HCV-J prototype (GenBank accession number: D90208) for genotype 1b [Kato et al., 1990] using the BioEdit program. Signature Pattern Analysis The VESPA program [Korber and Myers, 1992] was used to detect signature patterns (specic amino acids) for sensitive and resistant strains. Statistical Analysis The MannWhitney test was used to compare the baseline clinical parameters and the number of

mutations within ISDR, PKR-BD, IRRDR, and NS5AISDR-V3 sequences between sustained virological responders and non-responder patients. The comparison of the qualitative variables between the two groups of patients was performed using Fishers exact probability test. A P-value <0.05 was considered statistically signicant. RESULTS Baseline Characteristics of Chronic HCV-1b Patients According to Their Response to Combination Therapy Patients baseline characteristics are shown in Table I. Eleven patients (52.4%) achieved a sustained virological response with undetectable HCV-RNA 6 months after the end of therapy, whereas 10 (47.6%) had no response. There were no signicant differences between the two groups regarding pre-treatment clinical variables including age, gender, ALT, AST, GGT, PAL, HCV-RNA level, and stage of brosis. Comparison of Pre-Treatment Sequences Variability of the NS5A Region Obtained From Sustained Virological Responder and Non-Responder Patients Treated With Combination Therapy First, pre-treatment serum from 15 treated patients was analyzed for ISDR (22092248), PKR-BD (2209 2274), IRRDR (23342379), and V3 (23562379) amino acid sequences. As shown in Figure 1, amino acid sequences were aligned and compared to HCV-1b prototype (HCV-J) sequence. According to Enomoto et al. [1995] classication, 4 of 15 patients (26.67%) were infected with the wild-type ISDR strains (0 mutations), 10 of 13 patients (66.67%) were infected with the intermediate-type ISDR strains (13 mutations) and only one patient was infected with the mutant-type strain (!4 mutations) (7.66%). No statistically signicant associations were observed between the mean number of amino acid substitutions in ISDR and treatment response (1.62 1.85 for sustained virological responders vs. 0.57 0.53 for non-responders; P 0.151). However, the mean number of PKR-BD

TABLE I. Comparison of Baseline Characteristics of Tunisian HCV-1b-Infected Patients in Relation to Their Response to the Combination Therapy Sustained virological responders Number of patients (%) Gender (male/female) Age, years (M SD) ALT (M SD) (UI/ml) AST (M SD) (UI/ml) GGT (M SD) (UI/ml) PAL (M SD) (UI/ml) RNA (M SD) (UI/ml) Fibrosis score F2 F3 F4 11/21 (52.4) 6/5 51.5 5.43 135 62.7 87.4 34.9 73.0 74.2 217 57.8 6.805E05 8.048E05 3 4 4 Non-responders 10/21 (47.6) 4/6 49.6 6.90 126 101 98.7 68.9 86.1 99.3 306 148 7.645E05 9.180E05 4 3 3 P-value 0.669 0.653 0.349 0.756 0.917 0.061 0.496 0.826

J. Med. Virol. DOI 10.1002/jmv

2024

Bouzgarrou et al.

Fig. 1. Pre-treatment amino acid sequences of ISDR-V3 region of NS5A (22092379). Each sequence was compared to the HCV-J prototype sequence.

amino acid substitutions was signicantly high in patients with a sustained virological response than in those who did not respond to antiviral therapy (6.50 1.85 vs. 4.86 0.69, respectively; P 0.040). As for IRRDR and V3 regions, amino acid sequence analyses demonstrated a similar variability rate in sustained virological responder and non-responder patients. Next, when considering the whole carboxy-terminal region from ISDR to V3 (NS5AISDR-V3), the comparison of pre-treatment sequence variability showed a signicant association between the mean number of mutations within NS5AISDR-V3 region (22092379) and the response to combination therapy (P 0.002) (Table II). More interestingly, a great difference in mutation number was observed within the region between amino acids 2282 and 2308 (NS5A22822308) (P 0.0006).

Correlation Between Pre-Treatment-Specic Mutations Within NS5AISDR-V3 and Response to Combination Therapy Considering the previous studies [Enomoto et al., 1995; Watanabe et al., 2001; Aslan et al., 2004; Kohashi et al., 2006; Di Lello et al., 2008; El-Shamy et al., 2008], amino acid substitutions at positions 2209, 2216, 2217, 2218, 2227, 2360, and 2378 were compared between sustained virological responder and non-responder patients. Referring to HCV-J sequence, no patients presented a substitution in amino acid 2209. Only one responder patient presented a substitution in amino acid 2216. For a second responder patient the substitution in amino acid was observed at position 2217 and for another one, at 2227. Similarly, no amino acid substitutions were observed at position 2360. A higher

TABLE II. Comparison of Baseline Amino Acid Mutation Number in the NS5A Region According to the Response to the Combination Therapy Sustained virological responders (n 8) ISDR22092248 PKR-BD22092274 NS5A22822308 V323562379 IRRDR23342379 NS5AISDR-V3 1.62 1.85 6.5 1.85 3.25 0.70 7 1.69 7.50 1.77 19.6 3.16
a

Non-responders (n 7) 0.57 0.53 4.86 0.69 1.29 0.48 5.86 0.37 6.29 0.48 15.0 1.41

P-value 0.151 0.040 0.0006 0.189 0.151 0.002

IRRDR, interferon/ribavirine resistance-determining region; ISDR, interferon sensitivity-determining region; NS5A, non-structural 5A protein; PKR-BD, PKR-binding domain; V3, variable region 3. a Expressed as mean SD.

J. Med. Virol. DOI 10.1002/jmv

NS5A Genetic Variability and Treatment Response

TABLE III. Comparison of Pre-Treatment-Specic Mutation Rate Within NS5AISDR-V3 Between Sustained Virological Responder and Non-Responder Patients Substitution rate (n) Amino acid position 2209 2216 2217 2218 2227 2287 2300 2303 2349 2353 2360 2378a
a

2025

Sustained virological responders (n 8) 0 1 1 3 1 4 6 4 4 4 0 3

Non-responders (n 7) 0 0 0 1 0 1 0 1 0 1 0 0

With respect to the consensus sequence.

substitution rate was observed in sustained virological responder patients at positions 2218 (3 out of 8, 37.5%) and 2378 (3 out of 8; 37.5%). However, the statistical analysis did not reveal any signicant differences compared with non-responder patients (1 out of 7 (14.3%) and 0 out of 7 (0%), respectively). On the other hand, when the specic amino acid mutations within the whole 22092379 region of NS5A were compared, frequent amino acid substitutions at positions 2287, 2300, and 2303 within NS5A22822308, and at positions 2349 and 2353 within IRRDR were observed in sustained virological responder patients (Table III). The VESPA analysis revealed that P2300 and S2300 dened the signature patterns for sensitive and resistant strain sequences, respectively (P 0.006). DISCUSSION This is the rst study investigating the NS5A genetic variability of Tunisian HCV-1b strains and its links with treatment response. The sustained virological response rate, as assessed by the lack of detectable HCV-RNA in sera 6 months after the end of therapy, was 52.4%. This result is consistent with previous studies on HCV-1b response to PEG-IFN plus ribavirin therapy [El-Shamy et al., 2008; Munoz de Rueda et al., 2008; Yen et al., 2008].

The current study is particularly focused on NS5A genetic variability as a predictive factor to IFN therapy outcome. Amino acid sequences of ISDR, PKR-BD, IRRDR, and V3 obtained from 15 HCV-1b-infected patients (8 sustained virological responders and 7 nonresponders) were compared to HCV-J as these regions have been suggested as predictors of IFN therapy response. A positive correlation between mutations in the ISDR was reported previously in several Japanese studies [Enomoto et al., 1996; Watanabe et al., 2005; Murayama et al., 2007]. Other studies from Europe and the USA failed to demonstrate such an association [Duverlie et al., 1998; McKechnie et al., 2000; Nousbaum et al., 2000; Veillon et al., 2004; Kmieciak et al., 2006]. However, interestingly, a meta-analysis conducted on 525 European patients using the MEDLINE database revealed a clear positive association between the sustained virological rate and the number of mutations within ISDR [Pascu et al., 2004]. Furthermore, Munoz de Rueda et al. [2008] recently reported a signicant association between the high mutation rate within ISDR and the response to PEG-IFN/ribavirin therapy. However, like other studies from Taiwan [Yang et al., 2003], Turkey [Aslan et al., 2004], and Australia [Macquillan et al., 2004], no associations between the number of mutations in ISDR and treatment response were found in our study. Nevertheless, the percentage of the wild-, intermediate-, and mutant-type strains in our population (26.67%, 66.67%, and 7.66%, respectively) was more or less similar to that reported in the meta-analysis from the European cohort (24.8%, 63.4%, and 11.8%, respectively) [Pascu et al., 2004]. It was concluded that the observed discrepancy regarding the implication of ISDR variability in treatment response could be explained by the under representation of the mutant-type strains in the study populations. Indeed, another study from Taiwan included an adequate sample of the mutanttype strains and conrmed the relation between ISDR variability and treatment response (Table IV) [Hung et al., 2003]. Accordingly, any reliable ISDR mutation analysis should consider representative samples of the different types of strains (wild, intermediate, mutant) in order to draw the appropriate conclusions. NS5A and PKR interaction is a potential mechanism of IFN treatment resistance. This interaction requires ISDR plus 26 additional amino acids at its C-terminal

TABLE IV. Wild/Intermediate/Mutant ISDR Strain Frequency and Association Between Amino Acid Mutations Within ISDR Region and Treatment Outcome Wild (%) Australia (n 37) China (n 20) Europe (n 525) Spain (n 60) Taiwan (n 57) Taiwan (n 30) Tunisia (n 15) Turkey (n 39) 13.5 45 24.8 37 38.6 83.33 26.67 38.5 Intermediate (%) 81 35 63.4 57 45.6 16.67 66.67 61.5 Mutant (%) 5.4 20 11.8 7 15.8 0 7.66 0 P-value NS 0.02 0.001 0.005 0.038 NS NS NS Refs. Macquillan et al. [2004] Shen et al. [2007] Pascu et al. [2004] Munoz de Rueda et al. [2008] Hung et al. [2003] Yang et al. [2003] Our study Aslan et al. [2004]
J. Med. Virol. DOI 10.1002/jmv

2026

Bouzgarrou et al.

extremity [Gale et al., 1998]. It results in a disruption of PKR dimerization and, consequently, an inhibition of PKR-mediated eIF-2alpha phosphorylation. Several studies have found that mutations within the corresponding region termed PKR-BD of HCV-1 are associated with a sustained virological response to IFN-abased therapy [Berg et al., 2000; Nousbaum et al., 2000; Sarrazin et al., 2000; Macquillan et al., 2004]. Recently, Munoz de Rueda et al. [2008] suggested that the presence of more than four mutations within PKRBD was an independent factor associated with a sustained virological response to combination therapy. In line with the previous studies, higher amino acid substitutions in PKR-BD were observed in sustained virological responders in comparison to non-responders in our population (mean of 6.50 1.85 vs. 4.86 0.69, respectively; P 0.040). However, as the correlation was weak, this result will need to be conrmed by a large-scale study. Full-length NS5A analysis studies demonstrated that the V3 region presents a high capacity to accumulate mutations [Inchauspe et al., 1991; Nousbaum et al., 2000; Puig-Basagoiti et al., 2005; Veillon et al., 2007; Jardim et al., 2009]. Accordingly, several reports have observed a signicant association between the amino acid substitution number within V3 or IRRDR and the treatment outcome [Duverlie et al., 1998; Murphy et al., 2002; Sarrazin et al., 2002; Veillon et al., 2004, 2007; Puig-Basagoiti et al., 2005; El-Shamy et al., 2008]. In the present study, despite the high variability in V3 or IRRDR, no signicant correlation was observed between the number of mutations within these regions and the treatment response. Nevertheless, our results are in accordance with a large study performed by Munoz de Rueda et al. [2008] on 60 HCV-1 patients receiving PEGIFN and ribavirin combination therapy. This does not exclude the implication of V3 region in IFN-a-based therapy response. In fact, the deletion of V3 region abrogated its anti-IFN activity signicantly. More importantly, exchanging V3 region between 1b-NS5A and 2a-NS5A resulted in a partial or complete switch of their anti-IFN activity. ISDR and PKR-BD deletions also demonstrated their importance in anti-IFN effects [Tsai et al., 2008]. Therefore, we considered it was essential the entire ISDR-V3 region of NS5A regarding combination therapy outcome should be analyzed. A greater variability among responder patients was observed in comparison to non-responder patients (mean of 19.6 3.16 vs. 15.0 1.41; P 0.002). Interestingly, as reported by Nousbaum et al. [2000], a higher number of mutations was accumulated within NS5A22822308 region in sustained virological responder patients (P 0.0006). Using the HCV-1b subgenomic replicon system, Tellinghuisen et al. [2008] demonstrated that deletions of amino acids from positions 22862294 and amino acids from positions 22952309 are lethal for HCV-RNA replication in Huh 7.5 cells. However, further investigations on the importance of NS5A22822308 region in the blockage of IFN function are needed.
J. Med. Virol. DOI 10.1002/jmv

Many studies have reported that a particular type of amino acid substitutions is also associated with treatment responsiveness. In particular, NS5A amino acid substitutions at positions 2209, 2216, 2217, 2218, 2227, 2360, and 2378 were correlated with a high rate of response [Enomoto et al., 1995; Watanabe et al., 2001; Aslan et al., 2004; Kohashi et al., 2006; Di Lello et al., 2008; El-Shamy et al., 2008]. Other studies identied HCV core amino acid substitutions at positions 70 and 91 as reliable predictive factors of the treatment response. Arginine 70 and leucine 91 were associated with the sustained virological response to PEG-IFN/ ribavirin therapy [Akuta et al., 2007a,b,c; Okanoue et al., 2009]. As NS5A, HCV core protein seems to be an antagonist with IFN-a function probably via inhibiting the Jak-STAT signaling cascade [Blindenbacher et al., 2003; Bode et al., 2003]. In our study, no amino acid mutations at the mentioned NS5A amino acid positions were correlated with a sustained virological response. However, frequent amino acid substitutions at positions 2287, 2300, 2303, 2349, and 2353 were observed in sustained virological responder patients (Table III). The commonest single amino acid substitution within the NS5AISDR-V3 region occurred at position 2300. Serine mutated to proline in the majority of responders (6 out of 8; 75%) but in no nonresponders (0 out of 7; 0%) (P 0.006). The VESPA analysis further demonstrated that P2300 and S2300 may dene the signature patterns for sensitive and resistant strain sequences, respectively. Knowing that proline breaks the alpha helix in the protein structure, we think that this nding may be relevant and is worth a deeper analysis based on amino acid substitution models. In summary, a distribution of wild/intermediate/ mutant strains similar to that reported in Europe was observed in Tunisia. This study demonstrated a signicant correlation between the number of amino acid substitutions within PKR-BD, but more importantly within NS5AISDR-V3 (aa 22092379) and combination therapy response. A high degree of sequence variation within the NS5A region from amino acids 22822308 and the presence of proline at position 2300 appeared to be predictive factors for a sustained virological response to combination therapy in Tunisian HCV-1b-infected patients. In the light of these results, cloning and sequence analysis of NS5AISDR-V3 region will be in use in order to investigate quasi-species genetic variability and its evolution during the combined IFN or PEG-IFN plus ribavirin therapy in relation to treatment response. Furthermore, a functional study on the impact of P2300 on HCV cycle, its interaction with interferon function, and more NS5A analyses with regard to response to treatment in the Mediterranean populations deserve a further exploration. ACKNOWLEDGMENTS We would like to thank Mr. Adel Rdissi for English revision.

NS5A Genetic Variability and Treatment Response

2027
Hoffman J, Yu J. 2002. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 347:975982. Gale M, Jr., Korth MJ, Tang NM, Tan SL, Hopkins DA, Dever TE, Polyak SJ, Gretch DR, Katze MG. 1997. Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230:217227. Gale M, Jr., Blakely CM, Kwieciszewski B, Tan SL, Dossett M, Tang NM, Korth MJ, Polyak SJ, Gretch DR, Katze MG. 1998. Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: Molecular mechanisms of kinase regulation. Mol Cell Biol 18:5208 5218. Hayashi J, Kishihara Y, Ueno K, Yamaji K, Kawakami Y, Furusyo N, Sawayama Y, Kashiwagi S. 1998. Age-related response to interferon alfa treatment in women vs men with chronic hepatitis C virus infection. Arch Intern Med 158:177181. Hung CH, Lee CM, Lu SN, Lee JF, Wang JH, Tung HD, Chen TM, Hu TH, Chen WJ, Changchien CS. 2003. Mutations in the NS5A and E2-PePHD region of hepatitis C virus type 1b and correlation with the response to combination therapy with interferon and ribavirin. J Viral Hepat 10:8794. Inchauspe G, Zebedee S, Lee DH, Sugitani M, Nasoff M, Prince AM. 1991. Genomic structure of the human prototype strain H of hepatitis C virus: Comparison with American and Japanese isolates. Proc Natl Acad Sci USA 88:1029210296. Jardim AC, Yamasaki LH, de Queiroz AT, Bittar C, Pinho JR, Carareto CM, Rahal P, Mello IM. 2009. Quasispecies of hepatitis C virus genotype 1 and treatment outcome with peginterferon and ribavirin. Infect Genet Evol 9:689698. Kato N, Hijikata M, Ootsuyama Y, Nakagawa M, Ohkoshi S, Sugimura T, Shimotohno K. 1990. Molecular cloning of the human hepatitis C virus genome from Japanese patients with non-A, non-B hepatitis. Proc Natl Acad Sci USA 87:95249528. Kau A, Vermehren J, Sarrazin C. 2008. Treatment predictors of a sustained virologic response in hepatitis B and C. J Hepatol 49: 634651. Kmieciak D, Kruszyna , Migdalski P, acinski M, Juszczyk J, Trzeciak WH. 2006. Mutations within protein kinase R-binding domain of NS5A protein of hepatitis C virus (HCV) and specicity of HCV antibodies in pretreatment sera of HCV-chronically infected patients and their effect on the result of treatment. Jpn J Infect Dis 59:9299. Kohashi T, Maekawa S, Sakamoto N, Kurosaki M, Watanabe H, Tanabe Y, Chen CH, Kanazawa N, Nakagawa M, Kakinuma S, Yamashiro T, Itsui Y, Koyama T, Enomoto N, Watanabe M. 2006. Site-specic mutation of the interferon sensitivity-determining region (ISDR) modulates hepatitis C virus replication. J Viral Hepat 13:582590. Korber B, Myers G. 1992. Signature pattern analysis: A method for assessing viral sequence relatedness. AIDS Res Hum Retroviruses 8:15491560. Macquillan GC, Niu X, Speers D, English S, Garas G, Harnett GB, Reed WD, Allan JE, Jeffrey GP. 2004. Does sequencing the PKRBD of hepatitis C virus NS5A predict therapeutic response to combination therapy in an Australian population? J Gastroenterol Hepatol 19:551557. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. 2001. Peginterferon alfa-2b plus ribavirin compared with interferon alfa2b plus ribavirin for initial treatment of chronic hepatitis C: A randomised trial. Lancet 358:958965. McKechnie VM, Mills PR, McCruden EA. 2000. The NS5a gene of hepatitis C virus in patients treated with interferon-alpha. J Med Virol 60:367378. Mejri S, Salah AB, Triki H, Alaya NB, Djebbi A, Dellagi K. 2005. Contrasting patterns of hepatitis C virus infection in two regions from Tunisia. J Med Virol 76:185193. Miller RH, Purcell RH. 1990. Hepatitis C virus shares amino acid sequence similarity with pestiviruses and aviviruses as well as members of two plant virus supergroups. Proc Natl Acad Sci USA 87:20572061. Munoz de Rueda P, Casado J, Paton R, Quintero D, Palacios A, Gila A, Quiles R, Leon J, Ruiz-Extremera A, Salmeron J. 2008. Mutations in E2-PePHD, NS5A-PKRBD, NS5A-ISDR, and NS5A-V3 of hepatitis C virus genotype 1 and their relationships to pegylated interferon-ribavirin treatment responses. J Virol 82:66446653. Murayama M, Katano Y, Nakano I, Ishigami M, Hayashi K, Honda T, Hirooka Y, Itoh A, Goto H. 2007. A mutation in the interferon

REFERENCES
Afdhal NH. 2004. The natural history of hepatitis C. Semin Liver Dis 24:S3S8. Akuta N, Suzuki F, Kawamura Y, Yatsuji H, Sezaki H, Suzuki Y, Hosaka T, Kobayashi M, Kobayashi M, Arase Y, Ikeda K, Kumada H. 2007a. Predictive factors of early and sustained responses to peginterferon plus ribavirin combination therapy in Japanese patients infected with hepatitis C virus genotype 1b: Amino acid substitutions in the core region and low-density lipoprotein cholesterol levels. J Hepatol 46:403410. Akuta N, Suzuki F, Kawamura Y, Yatsuji H, Sezaki H, Suzuki Y, Hosaka T, Kobayashi M, Kobayashi M, Arase Y, Ikeda K, Miyakawa Y, Kumada H. 2007b. Prediction of response to pegylated interferon and ribavirin in hepatitis C by polymorphisms in the viral core protein and very early dynamics of viremia. Intervirology 50:361 368. Akuta N, Suzuki F, Kawamura Y, Yatsuji H, Sezaki H, Suzuki Y, Hosaka T, Kobayashi M, Kobayashi M, Arase Y, Ikeda K, Kumada H. 2007c. Predictors of viral kinetics to peginterferon plus ribavirin combination therapy in Japanese patients infected with hepatitis C virus genotype 1b. J Med Virol 79:16861695. ` Alberti A, Chemello L, Benvegnu L. 1999. Natural history of hepatitis C. J Hepatol 31:S17S24. Aslan N, Bozdayi AM, Cetinkaya H, Sarioglu M, Turkay C, Bozkaya H, lu Karayalcin S, Yurtaydin C, Uzunalimog O. 2004. The mutations in ISDR of NS5A gene are not associated with response to interferon treatment in Turkish patients with chronic hepatitis C virus genotype 1b infection. Turk J Gastroenterol 15:2126. Berg T, Mas Marques A, Hohne M, Wiedenmann B, Hopf U, Schreier E. 2000. Mutations in the E2-PePHD and NS5A region of hepatitis C virus type 1 and the dynamics of hepatitis C viremia decline during interferon alfa treatment. Hepatology 32:13861395. Blindenbacher A, Duong FH, Hunziker L, Stutvoet ST, Wang X, Terracciano L, Moradpour D, Blum HE, Alonzi T, Tripodi M, La Monica N, Heim MH. 2003. Expression of hepatitis C virus proteins inhibits interferon alpha signaling in the liver of transgenic mice. Gastroenterology 124:14651475. Bode JG, Ludwig S, Ehrhardt C, Albrecht U, Erhardt A, Schaper F, ussinger D. 2003. IFN-alpha antagonistic activity Heinrich PC, Ha of HCV core protein involves induction of suppressor of cytokine signaling-3. FASEB J 17:488490. Bouzgarrou N, Fodha I, Othman SB, Achour A, Grattard F, Trabelsi A, Pozzetto B. 2005. Evaluation of a total core antigen assay for the diagnosis of hepatitis C virus infection in hemodialysis patients. J Med Virol 77:502508. Di Lello F, Garcia G, Kott V, Sookoian S, Campos R. 2008. Diversity of hepatitis C virus genotype 1b in Buenos Aires, Argentina: Description of a new cluster associated with response to treatment. J Med Virol 80:619627. Djebbi A, Triki H, Bahri O, Cheikh I, Sadraoui A, Ben Ammar A, Dellagi K. 2003. Genotypes of hepatitis C virus circulating in Tunisia. Epidemiol Infect 130:501505. Duverlie G, Khorsi H, Castelain S, Jaillon O, Izopet J, Lunel F, Eb F, Penin F, Wychowski C. 1998. Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity. J Gen Virol 79:13731381. El-Shamy A, Sasayama M, Nagano-Fujii M, Sasase N, Imoto S, Kim SR, Hotta H. 2007. Prediction of efcient virological response to pegylated interferon/ribavirin combination therapy by NS5A sequences of hepatitis C virus and anti-NS5A antibodies in pretreatment sera. Microbiol Immunol 51:471482. El-Shamy A, Nagano-Fujii M, Sasase N, Imoto S, Kim SR, Hotta H. 2008. Sequence variation in hepatitis C virus nonstructural protein 5A predicts clinical outcome of pegylated interferon/ribavirin combination therapy. Hepatology 48:3847. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Izumi N, Marumo F, Sato C. 1995. Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest 96:224230. Enomoto N, Sakuma I, Asahina Y, Kurosaki M, Murakami T, Yamamoto C, Ogura Y, Izumi N, Marumo F, Sato C. 1996. Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 334:7781. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL, Jr., Haussinger D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A,

J. Med. Virol. DOI 10.1002/jmv

2028
sensitivity-determining region is associated with responsiveness to interferon-ribavirin combination therapy in chronic hepatitis patients infected with a Japan-specic subtype of hepatitis C virus genotype 1B. J Med Virol 79:3540. Murphy MD, Rosen HR, Marousek GI, Chou S. 2002. Analysis of sequence congurations of the ISDR, PKR-binding domain, and V3 region as predictors of response to induction interferon-alpha and ribavirin therapy in chronic hepatitis C infection. Dig Dis Sci 47:11951205. Myers RP, Patel K, Pianko S, Poynard T, McHutchison JG. 2003. The rate of brosis progression is an independent predictor of the response to antiviral therapy in chronic hepatitis C. J Viral Hepat 10:1622. Nousbaum J, Polyak SJ, Ray SC, Sullivan DG, Larson AM, Carithers RL, Jr., Gretch DR. 2000. Prospective characterization of fulllength hepatitis C virus NS5A quasispecies during induction and combination antiviral therapy. J Virol 74:90289038. Okanoue T, Itoh Y, Hashimoto H, Yasui K, Minami M, Takehara T, Tanaka E, Onji M, Toyota J, Chayama K, Yoshioka K, Izumi N, Akuta N, Kumada H. 2009. Predictive values of amino acid sequences of the core and NS5A regions in antiviral therapy for hepatitis C: A Japanese multi-center study. J Gastroenterol. DOI 10.1007/s00535-009-0087.x (published online 11 June 2008). Pascu M, Martus P, Hohne M, Wiedenmann B, Hopf U, Schreier E, Berg T. 2004. Sustained virological response in hepatitis C virus type 1b infected patients is predicted by the number of mutations within the NS5A-ISDR: A meta-analysis focused on geographical differences. Gut 53:13451351. Pawlotsky JM, Pellerin M, Bouvier M, Roudot-Thoraval F, Germanidis G, Bastie A, Darthuy F, Remire J, Soussy CJ, Dhumeaux D. 1998. Genetic complexity of the hypervariable region 1 (HVR1) of hepatitis C virus (HCV): Inuence on the characteristics of the infection and responses to interferon alfa therapy in patients with chronic hepatitis C. J Med Virol 54:256264. Podevin P, Sabile A, Gajardo R, Delhem N, Abadie A, Lozach PY, Beretta L, Brechot C. 2001. Expression of hepatitis C virus NS5A natural mutants in a hepatocytic cell line inhibits the antiviral effect of interferon in a PKR-independent manner. Hepatology 33:15031511. Poynard T, Bedossa P, Opolon P. 1997. Natural history of liver brosis progression in patients with chronic hepatitis C. The OBSVIRC, METAVIR, CLINIVIR, and DOSVIRC groups. Lancet 349:825 832. Puig-Basagoiti F, Forns X, Furcic I, Ampurdanes S, Gimenez-Barcons M, Franco S, Sanchez-Tapias JM, Saiz JC. 2005. Dynamics of hepatitis C virus NS5A quasispecies during interferon and ribavirin therapy in responder and non-responder patients with genotype 1b chronic hepatitis C. J Gen Virol 86:10671075. Sarrazin C, Berg T, Lee JH, Ruster B, Kronenberger B, Roth WK, Zeuzem S. 2000. Mutations in the protein kinase-binding domain of the NS5A protein in patients infected with hepatitis C virus type 1a are associated with treatment response. J Infect Dis 181:432441. Sarrazin C, Herrmann E, Bruch K, Zeuzem S. 2002. Hepatitis C virus nonstructural 5A protein and interferon resistance: A new model for testing the reliability of mutational analyses. J Virol 76:11079 11090. Shen C, Hu T, Shen L, Gao L, Xie W, Zhang J. 2007. Mutations in ISDR of NS5A gene inuence interferon efcacy in Chinese patients with

Bouzgarrou et al.
chronic hepatitis C virus genotype 1b infection. J Gastroenterol Hepatol 22:18981903. Simmonds P, Smith DB, McOmish F, Yap PL, Kolberg J, Urdea MS, Holmes EC. 1994. Identication of genotypes of hepatitis C virus by sequence comparisons in the core, E1 and NS-5 regions. J Gen Virol 75:10531061. Tellinghuisen TL, Foss KL, Treadaway JC, Rice CM. 2008. Identication of residues required for RNA replication in domains II and III of the hepatitis C virus NS5A protein. J Virol 82:1073 1083. Tsai YH, Kuang WF, Lu TY, Kao JH, Lai MY, Liu CJ, Chen PJ, Hwang LH. 2008. The non-structural 5A protein of hepatitis C virus exhibits genotypic differences in interferon antagonism. J Hepatol 49:899907. Veillon P, Payan C, Gaudy C, Goudeau A, Lunel F. 2004. Mutation analysis of ISDR and V3 domains of hepatitis C virus NS5A region before interferon therapy with or without ribavirin. Pathol Biol 52:505510. Veillon P, Payan C, Le Guillou-Guillemette H, Gaudy C, Lunel F. 2007. Quasispecies evolution in NS5A region of hepatitis C virus genotype 1b during interferon or combined interferon-ribavirin therapy. World J Gastroenterol 13:11951203. Watanabe H, Enomoto N, Nagayama K, Izumi N, Marumo F, Sato C, Watanabe M. 2001. Number and position of mutations in the interferon (IFN) sensitivity-determining region of the gene for nonstructural protein 5A correlate with IFN efcacy in hepatitis C virus genotype 1b infection. J Infect Dis 183:1195 1203. Watanabe K, Yoshioka K, Yano M, Ishigami M, Ukai K, Ito H, Miyata F, Mizutani T, Goto H. 2005. Mutations in the nonstructural region 5B of hepatitis C virus genotype 1b: Their relation to viral load, response to interferon, and the nonstructural region 5A. J Med Virol 75:504512. Yang SS, Lai MY, Chen DS, Chen GH, Kao JH. 2003. Mutations in the NS5A and E2-PePHD regions of hepatitis C virus genotype 1b and response to combination therapy of interferon plus ribavirin. Liver Int 23:426433. Yen YH, Hung CH, Hu TH, Chen CH, Wu CM, Wang JH, Lu SN, Lee CM. 2008. Mutations in the interferon sensitivity-determining region (nonstructural 5A amino acid 2209-2248) in patients with hepatitis C-1b infection and correlating response to combined therapy of pegylated interferon and ribavirin. Aliment Pharmacol Ther 27:7279. Yu ML, Dai CY, Huang JF, Chiu CF, Yang YH, Hou NJ, Lee LP, Hsieh MY, Lin ZY, Chen SC, Hsieh MY, Wang LY, Chang WY, Chuang WL. 2008. Rapid virological response and treatment duration for chronic hepatitis C genotype 1 patients: A randomized trial. Hepatology 47:18841893. Zeuzem S, Hultcrantz R, Bourliere M, Goeser T, Marcellin P, SanchezTapias J, Sarrazin C, Harvey J, Brass C, Albrecht J. 2004. Peginterferon alfa-2b plus ribavirin for treatment of chronic hepatitis C in previously untreated patients infected with HCV genotypes 2 or 3. J Hepatol 40:993999. Zeuzem S, Buti M, Ferenci P, Sperl J, Horsmans Y, Cianciara J, Ibranyi E, Weiland O, Noviello S, Brass C, Albrecht J. 2006. Efcacy of 24 weeks treatment with peginterferon alfa-2b plus ribavirin in patients with chronic hepatitis C infected with genotype 1 and low pretreatment viremia. J Hepatol 44:97103.

J. Med. Virol. DOI 10.1002/jmv