Expression of hepatitis C virus proteins interferes with the antiviral action of interferon independently of PKR-mediated control of protein synthesis.

Hepatitis C virus (HCV) of genotype 1 is the most resistant to interferon (IFN) therapy. Here, we have analyzed the response to IFN of the human cell line UHCV-11 engineered to inducibly express the entire HCV genotype 1a polyprotein. IFN-treated, induced UHCV cells were found to better support the growth of encephalomyocarditis virus (EMCV) than IFN-treated, uninduced cells. This showed that expression of the HCV proteins allowed the development of a partial resistance to the antiviral action of IFN. The nonstructural 5A (NS5A) protein of HCV has been reported to inhibit PKR, an IFN-induced kinase involved in the antiviral action of IFN, at the level of control of protein synthesis through the phosphorylation of the initiation factor eIF2alpha (M. Gale, Jr., C. M. Blakely, B. Kwieciszewski, S. L. Tan, M. Dossett, N. M. Tang, M. J. Korth, S. J. Polyak, D. R. Gretch, and M. G. Katze, Mol. Cell. Biol. 18:5208-5218, 1998). Accordingly, cell lines inducibly expressing NS5A were found to rescue EMCV growth (S. J. Polyak, D. M. Paschal, S. McArdle, M. J. Gale, Jr., D. Moradpour, and D. R. Gretch, Hepatology 29:1262-1271, 1999). In the present study we analyzed whether the resistance of UHCV-11 cells to IFN could also be attributed to inhibition of PKR. Confocal laser scanning microscopy showed no colocalization of PKR, which is diffuse throughout the cytoplasm, and the induced HCV proteins, which localize around the nucleus within the endoplasmic reticulum. The effect of expression of HCV proteins on PKR activity was assayed in a reporter assay and by direct analysis of the in vivo phosphorylation of eIF2alpha after treatment of cells with poly(I)-poly(C). We found that neither PKR activity nor eIF2alpha phosphorylation was affected by coexpression of the HCV proteins. In conclusion, expression of HCV proteins in their biological context interferes with the development of the antiviral action of IFN. Although the possibility that some inhibition of PKR (by either NS5A or another viral protein) occurs at a very localized level cannot be excluded, the resistance to IFN, resulting from the expression of the HCV proteins, cannot be explained solely by inhibition of the negative control of translation by PKR.

Sequence analysis of the NS5A protein of European hepatitis C virus 1b isolates and relation to interferon sensitivity.

Japanese studies have defined the discrete 2209-2248 amino acid region of the non-structural 5A protein (NS5A(2209-2248)) of hepatitis C virus genotype 1b (HCV 1b) isolates as the interferon sensitivity determining region (ISDR). European studies did not confirm these results since most of the ISDR sequences harboured an intermediate profile. Recently, a direct interaction between the NS5A protein, involving the ISDR, and the interferon-induced protein kinase (PKR) has been reported and presented as a possible explanation of HCV interferon resistance. In the present study, the entire NS5A amino acid sequence from 11 resistant and eight sensitive strains from European HCV 1b isolates was inferred from direct sequencing. The previously described important amino acid stretches and positions in NS5A were compared between the resistant and sensitive groups. Although some variations were observed, no clear differences could be directly correlated with the interferon sensitivity. However, sensitive strains were different, owing to more amino acid changes when compared to a consensus sequence from all strains. The carboxy-terminal region and especially the previously reported NS5A/V3 region showed most of the variations. Moreover, the conformational analysis of NS5A by secondary structure prediction allowed the differentiation of most sensitive strains from resistant ones. It was concluded that other regions different from ISDR were involved in resistance to interferon maybe via the interaction between NS5A and PKR.

Amplification and detection of the terminal 3' non-coding region of hepatitis C virus isolates.

A reverse transcription polymerase chain reaction (RT-PCR) assay was set up to amplify, from chronically infected patients, the recently discovered hepatitis C virus (HCV) 3'non-coding region (3'NCR). A panel of 149 samples was tested by RT-PCR for the 3'NCR. Two detection methods of amplified products were evaluated: ethidium bromide staining on 3% agarose gel electrophoresis and DNA enzyme immunoassay ("DEIA"). Results were compared with those obtained by amplification of the 5' non-coding region (5'NCR), i.e. the "Amplicor" HCV RNA qualitative assay. Genotype distribution of the 86 Amplicor-positive samples was subtype 1a: n = 15 (17.4%); subtype 1b: n = 32 (37.2%); subtype 2a/2c: n = 7 (8.1%); type 3: n = 25 (29%); type 4: n = 2 (2.3%); type 5: n = 1 (1.2%); not determined: n = 4 (2.3%). Sixty-three sera were HCV RNA-Amplicor-negative, 32 of which were from HCV-seronegative patients and 31 from HCV-seropositive patients. All seronegative samples were negative by both PCR methods. None of the Amplicor-negative samples from seropositive patients were positive by the 3'NCR assay. Forty-seven (54.7%) and 83 (96.5%) of the 86 Amplicor-HCV-RNA-positive samples were positive after ethidium bromide staining and by the 3'NCR assay using DEIA, respectively. The limit of detection by end-point dilution was lower with Amplicor. No difference between genotypes was detected for the 3'NCR RT-PCR, and a high degree of concordance was obtained between the Amplicor and the 3'NCR DEIA results (97.4%). Nevertheless, further studies are needed before the 3'NCR RT-PCR assay could be used instead of the 5'NCR RT-PCR for diagnostic purposes.

Pattern of HCV antibodies with special reference to NS5A reactivity in HCV-infected patients: relation to viral genotype, cryoglobulinemia and response to interferon.

BACKGROUND/AIMS: We aimed to compare the anti-hepatitis C virus reactivity in confirmatory assays (RIBA 3.0 Ortho Diagnostic and INNO-LIA HCV Ab III Innogenetics) among patients infected with different hepatitis C virus genotypes, with or without cryoglobulinemia, and in patients treated with interferon. METHODS: One hundred and three patients followed in our hepatogastroenterology unit were included in the study and compared to 320 consecutive patients tested using RIBA 3.0. Seventy-nine of the 103 patients were treated with interferon. Long-term responders to interferon were defined as having normal alanine aminotransferase levels and being HCV RNA negative 6 months after the end of treatment. Initial responders were defined as having normal alanine aminotransferase levels at the end of interferon therapy but abnormal alanine aminotransferase levels and/or detectable HCV RNA during the following 6 months. Non-responders were defined as still having elevated alanine aminotransferase during and after interferon. Serological tests (RIBA and INNO-LIA) were performed according to the manufacturers' instructions. HCV RNA was detected by nested polymerase chain reaction. Hepatitis C virus genotype was determined by using a Line Probe Assay (Innogenetics). RESULTS: There was no significant difference in the pattern of hepatitis C virus reactivity according to the hepatitis C virus genotype or presence of cryoglobulinemia. Twenty-three patients were classified as non-responders, 35 as initial responders, 21 as long-term responders. NS5 reactivity was significantly different (p<0.01) between these three groups: 34% of non-responders (8/23) had RIBA 3.0 NS5 reactivity and 13% (3/23) were reactive in the INNO-LIA III. Almost all long-term responders (95%) had NS5 reactivity by both RIBA 3.0 and INNO-LIA III. CONCLUSION: We conclude that patients who respond to interferon have stronger reactivity against NS5 antigens than non-responders. Molecular changes in the NS5A region may be responsible for such differences, as recently suggested.

Mutations of hepatitis C virus 1b NS5A 2209-2248 amino acid sequence do not predict the response to recombinant interferon-alfa therapy in French patients.

BACKGROUND/AIMS: Studies of HCV quasispecies during interferon treatment have shown the selection of resistant clones. Enomoto et al. have defined the interferon sensitivity-determining region in an amino acid stretch of the HCV-1b NS5A region. Patients with a mutant strain before treatment were complete responders, whereas those with wild-type HCV-J strain were resistant to interferon. The same region was studied in HCV isolates of French patients. METHODS: Forty-three HCV-1b chronically infected patients, consisting of 26 non-responders and 17 complete responders to interferon-alfa treatment (3 MUI tiw for 6 months), were included retrospectively. We directly sequenced the NS5A(2209-2248) HCV region of these patients before treatment. The viral load could be obtained from six complete responders and 15 non-responders. RESULTS: We detected wild-type and intermediate strains, but only two mutant strains were present. One of them was found in a non-responder. In three complete responders, we found a wild-type strain. The distribution of the various strains was rather different from that found in Japan. Before treatment, the viral load was lower in complete responders (p=0.01). CONCLUSIONS: Only two mutant strains were detected in our study. This could partially explain the low response rate to interferon treatment of French HCV-1b-infected patients, although the dose regimen was lower than in Japanese studies. Also, wild-type strains were found in some complete responders, and no correlation was determined between the mutation number in the NS5A(2209-2248) region and response to alfa interferon therapy. This may be related to epidemiological differences between HCV-1b strains present in France and those in Japan. Searching for the mutant NS5A pattern before treatment does not appear to be useful in French patients as it is too uncommon.

Direct blotting electrophoresis for sequencing and genotyping hepatitis C virus.

Many methods have been used to differentiate the hepatitis C virus (HCV) genotypes based on, for example, type specific primers, probes and restriction fragment length polymorphism. However, determination of the nucleotide sequence remains the reference. Therefore, a simple non-radioactive cycle sequencing technique was developed for clinical tests. PCR-amplified products of the 5' non-coding region (from position -274 to -31) were sequenced using a 5' digoxygenin-labeled primer. After denaturation, the samples were loaded on a direct blotting electrophoresis system (GATC 1500). Sequencing products were blotted onto a nylon membrane during the electrophoresis. The DNA fragments were then UV-cross-linked, incubated with phosphatase-labeled anti-digoxygenin antibody and stained with a precipitating substrate. Reading the sequence of six samples were possible within 2 days. In 41 different samples, five different genotypes were found by sequence analysis from position -245 to -69, of which 17 were type 1a, 7 type 1b, 5 type 2a, 8 type 3a, 3 type 4 and 1 type 5. These results agreed with those obtained by reverse hybridization assay. Direct blotting electrophoresis offered a good non-radioactive method of performing clinical sequencing on a medium scale, with a minimum of investment.

Comparison of hepatitis C virus serotyping and genotyping in French patients.

BACKGROUND: Hepatitis C virus (HCV) serotyping has been proposed as an alternative assay to determine the respective genotype as it is more rapid, simple and less expensive than polymerase chain reaction (PCR) based typing methods. OBJECTIVES: A serotyping assay was compared with a genotyping assay to determine the infecting hepatitis C virus type in chronically HCV infected patients eligible for interferon therapy. STUDY DESIGN: An enzyme immunoassay (HC01, Murex) was tested to identify HCV types 1, 2 and 3 specific antibodies in 134 PCR-positive sera from chronically infected patients which had been previously genotyped by a reverse hybridization assay (INNO-LiPA HCV I, Innogenetics). Respectively nine and seven sera were from HIV-seropositive and hemodialysis patients. Unreactive sera and those with discrepant results were retested by a new version (HC02) extended to types 4, 5 and 6. RESULTS: The distribution frequency of HCV genotypes was subtype 1a, 16.4%; subtype 1b, 46.3%; subtype 2a, 7.5%; subtype 3a, 20.9%; type 4, 4.5%; type 5, 0.7%; and co-infections, 3.7%. Among all the patients, 95% were of type 1, 2 or 3. The antibody reactivities of hemodialysis (1/7; P < 0.05) and HIV-seropositive patients (4/9; P = 0.06) were lower than for the patients seen at the hepatology unit (87/118). For these latter patients, the serotyping assay was interpretable in 71% and concordant in 64% of the samples with the genotyping assay. Out of the 84 samples with interpretable results, 75 sera were correctly serotyped (89% specificity). The two mixed results obtained by serotyping did not correspond to genotype co-infections (n = 3) and reciprocally. Six discrepancies were ruled out by the new assay, but the 2 untypeable sera remained unsolved, and four out of six sera with genotype 4 were serotyped as type 5. CONCLUSIONS: Serotyping could be an attractive approach if the reactivity was improved and the subtyping possible.

[Research of type 1, 2 and 3 hepatitis C virus specific antibodies in relation to the viral genotype in chronically infected patients]. / Recherche des anticorps spécifiques des types 1, 2 et 3 du virus de l'hépatite C en relation avec le génotype viral chez des patients chroniquement infectés.

The determination of the viral genotype in chronically HCV infected patients is used as an epidemiologic tool, a predictive marker of the evolution of the disease and especially of the response to the interferon alfa therapy. Its determination needs an amplification of the selected genomic region and remains expensive. Thus an indirect determination of the genotype has been proposed using the characterization of type-specific antibodies. In 101 chronically HCV infected patients, a serologic method specific for HCV type 1, 2 and 3 has been evaluated in relation to the genotyping one. The test was interpretable in 64 sera and in agreement with the genotyping method in 92% of the samples. The reactivity of the test was lower in hemodialysis and IVDUs patients (p < 0.02). The mixed results obtained by the serologic typing method were not in agreement with the results of the genotyping coinfections. We could not differentiate the possible past infections from the cross-reactivities of the test. The serotyping methods was simple and rapid, allowing generally a deduction of the viral genotype. It could be an attractive approach if the reactivity was improved and the subtyping was possible.