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Departement de Microbiologie Medicale et Moleculaire EA 3856, Universite Franois Rabelais, Tours
Laboratoire de Bacteriologie et de Virologie, CHU d'Angers, Angers, France
ABSTRACT
The hepatitis C virus (HCV) envelope protein 2 (E2) interacts in vitro with the interferon alpha (IFN-)-inducible double-stranded RNA-activated protein kinase, suggesting a possible mechanism by which HCV may evade the antiviral effects of IFN-. Variability in the part of the HCV E2 gene encoding the carboxy-terminal part of the protein, which includes the interaction domain (E2-PePHD), was explored in 25 patients infected with HCV genotype 1b and receiving IFN- therapy. PCR products were generated and sequenced for 15 patients with a sustained response and for 10 patients with no virological response after treatment with IFN- and ribavirin. PePHD amino acid sequences were obtained for isolates from serum collected before and during treatment, after 2 months in responders, and after 6 months in nonresponders. Quasispecies analysis of the pretreatment PePHD region was performed for isolates from patients displaying amino acid substitutions in this domain on direct sequencing. The E2-PePHD sequence was highly conserved in both resistant and susceptible genotype 1b strains and was identical to the prototype HCV type J sequence. No significant emergence of PePHD mutants during therapy was observed in our clonal analysis, and sporadic mutations and treatment outcomes were not found to be correlated. The PePHD sequence before or during treatment cannot be used to predict reliably the outcome of treatment in HCV type 1b-infected patients.
INTRODUCTION
Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and hepatocellular carcinoma worldwide, with HCV inducing chronic infection in 50 to 70% of cases (3). Treatment with interferon alpha (IFN-) effectively reduces the viral load, but complete eradication of the virus is achieved in less than 20% of patients treated with IFN- alone and in 40 to 45% of patients treated with a combination of IFN- and ribavirin (12). Treatment outcome largely depends on the sensitivity of HCV to IFN-. Ribavirin increases the frequency of virus eradication in patients who initially respond to IFN- but has a limited effect in patients who do not respond to IFN-. The response to IFN- depends largely, but not exclusively, on virus-specific factors (6). The HCV genotype appears to be a major determinant, because patients infected with HCV genotype 2 (HCV-2) or HCV-3 have a higher rate of response to monotherapy and combination therapy than do those infected with HCV-1 (10). The mechanisms underlying the strong IFN- resistance observed in patients infected with HCV-1 are unclear. A direct interaction between viral proteins and the IFN- pathway was recently suggested when the HCV envelope protein 2 (E2) of HCV-1 isolates was shown to inhibit IFN--induced protein kinase (PKR) in vitro by means of a specific interaction with a 12-amino-acid domain: E2-PePHD (PKR-eIF2alpha phosphorylation homology domain). This domain may act as a pseudosubstrate for PKR, inhibiting its kinase activity and impairing the inhibition of viral protein synthesis mediated by this enzyme (19). The PePHD domain of HCV-1 has a sequence more similar to that of PKR autophosphorylation sites than do the sequences of the other genotypes. The relationship between E2-PePHD sequence conservation and resistance to antiviral treatments for HCV infection in vivo remains unclear.
In this study, we investigated the pretreatment pattern of mutations affecting the PePHD domain and flanking regions in 25 HCV-1b-infected patients with sustained virological responses or no virological response. We also analyzed changes in the sequence of the PePHD motif during therapy. As IFN--resistant strains might be selected or may emerge during IFN- therapy, we carried out clonal analysis to identify any possible emerging variants in pretreatment samples.
MATERIALS AND METHODS
Study samples. We studied HCV-positive, human immunodeficiency virus-negative subjects who had not previously been treated and who had volunteered to take part in a clinical trial comparing two treatments: IFN- alone (6 x 106 U three times per week for 12 months) and a combination of IFN- and ribavirin (1.2 g/day for 9 months). Ribavirin treatment was initiated if HCV RNA was detected by reverse transcription (RT)-PCR 2 months (M2) after the initiation of IFN- therapy. The dual therapy was maintained for 9 months (9). We selected 25 patients infected with HCV-1b for this study. Fifteen of these patients (5 receiving IFN- alone and 10 receiving dual therapy) had a sustained virological response to antiviral therapy, defined as normal alanine aminotransferase activity and an absence of HCV RNA detection by PCR 6 months after the end of treatment. Ten patients did not respond to the dual therapy. Pretreatment viral loads ranged from 1.5 x 104 to 6.9 x 106 IU/ml (median, 4.8 x 105 IU/ml) among the responders and from 8.1 x 104 to 4.1 x 106 IU/ml (median, 8.6 x 105 IU/ml) among the nonresponders. Pretreatment serum samples were collected in the 8 weeks preceding the initiation of antiviral therapy. Mutations that affected the PePHD domain and that occurred during treatment were analyzed at M2 for responders to dual therapy and at 6 months (M6) after the initiation of therapy for nonresponders.
Amplification of the PePHD domain and its flanking regions. We used a classical nested RT-PCR method, including an initial RT step followed by two amplification steps. Virus RNA was extracted from patient serum (300 μl) with RNA columns (QIAamp viral RNA mini kit; Qiagen). RNA extracts were reverse transcribed with a specific primer (5'-CAGACGCGCGCGTCCGC-3'; nucleotide positions 2808 to 2824 according to the numbering for HCV-J). RT was carried out at 42°C for 30 min in the presence of 12.5 U of Moloney murine leukemia virus reverse transcriptase (Invitrogen). The primers used for partial amplification of the region of HCV-1b E2, which corresponds to the carboxy terminus of the protein, have been described before (17). The first round of PCR was carried out with the RT primer described above as the outer antisense primer (20 pmol) and with primer 5'-GGGCCCTGGTTGACACC-3' (nucleotide positions 2127 to 2143) as the outer sense primer (20 pmol). The first round of amplification consisted of 35 cycles, as follows: denaturation at 94°C for 15 s, primer annealing at 55°C for 30 s, and primer extension at 72°C for 3 min, followed by a final prolonged extension step at 72°C for 7 min. The second step was performed with the inner sense primer 5'-GACTACCCATACAGGCTCTGG-3' (20 pmol; nucleotide positions 2157 to 2177) and the inner antisense primer 5'-GCGTCCGCCAGGAGGAGGAA-3' (20 pmol; nucleotide positions 2496 to 2515). The conditions for the second round were identical to those for the first round. Negative controls, in which distilled water replaced the RNA or cDNA, were included at each of the three steps to ensure that the RT and PCR mixtures were not contaminated. HCV-negative serum samples were used to check the specificity of the assay. Amplified products were subjected to electrophoresis in 1% agarose gels (Gibco BRL) and stained with ethidium bromide. A 357-bp PCR product 146 nucleotides upstream and 175 nucleotides downstream from the HCV PePHD domain was generated.
Direct sequencing of PePHD and its flanking regions. For direct sequencing of the E2 region, 50 μl of the second-round PCR product was purified by centrifugation on silica columns (Microcon PCR; Millipore). The positive and negative strands were sequenced with a Big Dye Terminator kit (version 3.0; Applied Biosystems) on an automated 3100 AVANT DNA sequencer (Applied Biosystems). Sequence data were analyzed with the Sequencing Analysis program (version 3.7; Applied Biosystems), and nucleotide sequences were aligned with BIOEDIT software (Department of Microbiology, North Carolina State University [http://www.mbio.ncsu.edu/BioEdit/bioedit.html]).
The PePHD protein sequences (corresponding to codons 659 to 670) of samples collected before treatment and at M2 (for responders to dual therapy) or M6 (for nonresponders) were aligned and compared with the HCV-J PePHD consensus sequence RSELSPLLLSTT. If mutations were detected in PePHD at M2 or M6, quasispecies analysis was carried out with pretreatment samples from the corresponding patients. Six pretreatment samples were used for cloning; these samples were from four nonresponders and two responders. Purified products were ligated into 50 ng of PCR 2.1 vector (Topo-TA cloning kit; Invitrogen). Recombinant plasmids were used to transform competent Escherichia coli cells, and transformants were detected according to the protocol of the manufacturer. We selected 20 to 25 positive clones from each patient at random. Plasmid DNA was isolated from 1.5 ml of a 16-h broth culture and purified with a Qiagen plasmid mini kit, according to the protocol of the manufacturer. The presence of the insert was checked by digestion with EcoRI. We sequenced both strands of 120 clones (20 individual clones per patient) with the M13 universal and M13 reverse primers (Eurogentec).
Nucleotide sequence accession numbers. The sequences have been submitted to GenBank under accession numbers AY506805 to AY506844.
RESULTS
Baseline E2-PePHD sequence and its relationship to IFN- response. The pretreatment amino acid sequence of E2-PePHD, derived by direct sequencing by PCR, was highly conserved among the HCV-1b isolates (from the 15 sustained responders and all 10 nonresponders) and was identical to that of the prototype HCV-J strain for all samples studied. A small number of mutations were detected in the regions flanking the PePHD domain (29 amino acids [aa] upstream and 31 aa downstream), but these mutations were not associated with a particular pattern of response to therapy (Fig. 1).
Changes in PePHD sequence during therapy. The amino acid sequence of E2-PePHD before treatment was compared with that after treatment by direct sequencing. The major profiles detected after M6 in nonresponders showed various amino acid substitutions in the clones from four of the nine patients studied: replacement of an S by an A or T at position 2, replacement of a P by an S at position 6, and replacement of a T by an A at position 12. Amino acid changes were also observed in the clones from two responders between the start of treatment and M2: replacement of the S at position 2 by an A, replacement of the P at position 6 by an S, and replacement of the T at position 12 by an A (Fig. 2). For these six selected patients, we carried out pretreatment PePHD quasispecies analysis for 20 clones per patient. All but two clones had a PePHD motif identical to that of the HCV-1b prototype; these mutations (replacement of a P by a T at position 664) found in two clones from a single nonresponder differed from that found in the same patient at M6 (replacement of an S by an A at position 660) (data not shown).
DISCUSSION
The mechanisms underlying HCV resistance to IFN--ribavirin treatment remain elusive. HCV seems to have its own strategy of defense against the host cellular response induced by IFN- (5). The E2 envelope protein appears to play a major role not only as a potential target for the immune response but also because it is thought to interfere with cellular effectors induced by IFN- in infected cells (18).
We investigated the HCV E2-PePHD domain of viral clones from patients infected with HCV-1b and its possible relationship with treatment outcome. The sequences of the clones obtained from the pretreatment samples for 15 sustained responders and 10 nonresponders after combined IFN--ribavirin therapy were all identical to that of the HCV-J prototype, confirming that this domain is highly conserved within a given HCV genotype.
A total of 470 pretreatment HCV-1b PePHD sequences have been published to date (1, 2, 4, 7, 8, 11, 13-17, 20, 21). Although they are rare, mutations have been observed within the PePHD domain when the HCV-J prototype sequence is used as the reference (Fig. 3). Nine amino acid positions (aa 659, 660, 661, 662, 663, 666, 667, 668, and 669) were affected, with a total of 59 substitutions observed; mutations of the second residue of the domain (aa 660) accounted for 30 of these substitutions. The S-to-A substitution at position 660 was the most frequent mutation and was observed in clones from both nonresponders and responders. Overall, 52% of the mutations (31 of 59) were observed in the strains from pretreatment samples from patients who ended up not responding to treatment. Thus, HCV-1b strains associated with resistance to treatment do not systematically bear the PePHD HCV-J prototype sequence (RSELSPLLLSTT), suggesting that pretreatment PePHD sequencing cannot be used for the reliable prediction of treatment outcome.
During therapy, three different mutated PePHD profiles were identified at M2 in responders and M6 in nonresponders. Two profiles (SA at aa 660 and PS at aa 664 plus TA at aa 670) were found in both responders and nonresponders. The third mutated profile (ST at aa 660) was found in one nonresponder. This amino acid change has mostly been reported in the pretreatment PePHD sequences of the clones from patients defined as nonresponders at the end of treatment (Fig. 3). However, we were unable to identify any particular sequence or mutation affecting the PePHD region or its immediate flanking regions that correlated with the antiviral response. A few studies have explored the changes occurring within the PePHD domain under therapeutic pressure. These studies showed that in the nonresponders the minor variants found in the pretreatment samples disappeared rapidly during treatment (2).
Thus, overall, sequencing of the region corresponding to the E2-PePHD domain or its flanking regions, either from pretreatment samples or after several weeks of therapy, was found to be of no value for predicting or monitoring the treatment outcome in patients infected with HCV-1b. It is, however, possible that the specific and highly conserved PePHD sequence observed in this HCV genotype (RSELSPLLLSTT) is associated with an E2-PKR interaction, which contributes to the higher basal levels of resistance in this genotype. In vivo, HCV resistance to IFN--based therapy appears to be multifactorial and is mediated by a combination of HCV-induced changes in the antiviral and immunomodulatory effects of IFN-.
ACKNOWLEDGMENTS
This work was supported by a grant from the Agence Nationale de Recherche sur le SIDA (ANRS grant 2001/016) and a grant from La Ligue contre le Cancer de l'Indre et Loire.
REFERENCES
Berg, T., A. Mas Marques, M. Hohne, B. Wiedenmann, U. Hopf, and E. Schreier. 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 32:1386-1395.
Chayama, K., F. Suzuki, A. Tsubota, M. Kobayashi, Y. Arase, S. Saitoh, Y. Suzuki, N. Murashima, K. Ikeda, N. Takahashi, M. Kinoshita, and H. Kumada. 2000. Association of amino acid sequence in the PKR-eIF2 phosphorylation homology domain and response to interferon therapy. Hepatology 32:1138-1144.
Deuffic, S., L. Buffat, T. Poynard, and A. J. Valleron. 1999. Modeling the hepatitis C virus epidemic in France. Hepatology 29:1596-1601.
Gerotto, M., F. Dal Pero, P. Pontisso, F. Noventa, A. Gatta, and A. Alberti. 2000. Two PKR inhibitor HCV proteins correlate with early but not sustained response to interferon. Gastroenterology 119:1649-1655.
He, Y., and M. G. Katze. 2002. To interfere and to anti-interfere: the interplay between hepatitis C virus and interferon. Viral Immunol. 15:95-119.
Hu, K. Q., J. M. Vierling, and A. G. Redeker. 2001. Viral, host and interferon-related factors modulating the effect of interferon therapy for hepatitis C virus infection. J. Viral Hepat. 8:1-18.
Hung, C. H., C. M. Lee, S. N. Lu, J. F. Lee, J. H. Wang, H. D. Tung, T. M. Chen, T. H. Hu, W. J. Chen, and C. S. Changchien. 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. 10:87-94.
Lo, S., and H. H. Lin. 2001. Variations within hepatitis C virus E2 protein and response to interferon treatment. Virus Res. 75:107-112.
Lunel, F., P. Veillon, I. Fouchard-Hubert, V. Loustaud-Ratti, A. Abergel, C. Silvain, H. Rifflet, A. Blanchi, X. Causse, Y. Bacq, and C. Payan. 2003. Antiviral effect of ribavirin in early non-responders to interferon monotherapy assessed by kinetics of hepatitis C virus RNA and hepatitis C virus core antigen. J. Hepatol. 39:826-833.
Martinot-Peignoux, M., P. Marcellin, M. Pouteau, C. Castelnau, N. Boyer, M. Poliquin, C. Degott, I. Descombes, V. Le Breton, V. Milotova, et al. 1995. Pretreatment serum hepatitis C virus RNA levels and hepatitis C virus genotype are the main and independent prognostic factors of sustained response to interferon alfa therapy in chronic hepatitis C. Hepatology 22:1050-1056.
Polyak, S. J., J. B. Nousbaum, A. M. Larson, S. Cotler, R. L. Carithers, Jr., and D. R. Gretch. 2000. The protein kinase-interacting domain in the hepatitis C virus envelope glycoprotein-2 gene is highly conserved in genotype 1-infected patients treated with interferon. J. Infect. Dis. 182:397-404.
Poynard, T., P. Marcellin, S. S. Lee, C. Niederau, G. S. Minuk, G. Ideo, V. Bain, J. Heathcote, S. Zeuzem, C. Trepo, J. Albrecht, et al. 1998. Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. Lancet 352:1426-1432.
Puig-Basagoiti, F., J. C. Saiz, X. Forns, S. Ampurdanes, M. Gimenez-Barcons, S. Franco, A. Sanchez-Fueyo, J. Costa, J. M. Sanchez-Tapias, and J. Rodes. 2001. Influence of the genetic heterogeneity of the ISDR and PePHD regions of hepatitis C virus on the response to interferon therapy in chronic hepatitis C. J. Med. Virol. 65:35-44.
Quer, J., P. Murillo, M. Martell, J. Gomez, J. I. Esteban, R. Esteban, and J. Guardia. 2004. Subtype mutations in the envelope 2 region including phosphorylation homology domain of hepatitis C virus do not predict effectiveness of antiviral therapy. J. Viral Hepat. 11:45-54.
Saito, T., T. Ito, H. Ishiko, M. Yonaha, K. Morikawa, A. Miyokawa, and K. Mitamura. 2003. Sequence analysis of PePHD within HCV E2 region and correlation with resistance of interferon therapy in Japanese patients infected with HCV genotypes 2a and 2b. Am. J. Gastroenterol. 98:1377-1383.
Sarrazin, C., M. Bruckner, E. Herrmann, B. Ruster, K. Bruch, W. K. Roth, and S. Zeuzem. 2001. Quasispecies heterogeneity of the carboxy-terminal part of the E2 gene including the PePHD and sensitivity of hepatitis C virus 1b isolates to antiviral therapy. Virology 289:150-163.
Sarrazin, C., I. Kornetzky, B. Ruster, J. H. Lee, B. Kronenberger, K. Bruch, W. K. Roth, and S. Zeuzem. 2000. Mutations within the E2 and NS5A protein in patients infected with hepatitis C virus type 3a and correlation with treatment response. Hepatology 31:1360-1370.
Taylor, D. R. 2000. Hepatitis C virus: evasion of the interferon-induced antiviral response. J. Mol. Med. 78:182-190.
Taylor, D. R., S. T. Shi, P. R. Romano, G. N. Barber, and M. M. Lai. 1999. Inhibition of the interferon-inducible protein kinase PKR by HCV E2 protein. Science 285:107-110.
Watanabe, H., K. Nagayama, N. Enomoto, J. Itakura, Y. Tanabe, C. Sato, N. Izumi, and M. Watanabe. 2003. Amino acid substitutions in PKR-eIF2 phosphorylation homology domain (PePHD) of hepatitis C virus E2 protein in genotype 2a/2b and 1b in Japan and interferon efficacy. Hepatol. Res. 26:268-274.
Yang, S. S., M. Y. Lai, D. S. Chen, G. H. Chen, and J. H. Kao. 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. 23:426-433.