Differential sensitivity of hepatitis C virus quasispecies to interferon-α therapy

Cop)'right© Journalof Hepatology 1994

Journal o.f Hepatoh~gy 1994: 21:88'4-886 Prhated 0~ Denmark. All rights reserved Munksgaard. Copenhagen

Journal of Hepatology

ISSN 0168-8278

Rapid Publication

Differential sensitivity of hepatitis C virus quasispecies to interferon-a therapy Masashi Mizokami, Johnson Y. N. Lau I, K a o r u Suzuki, Tatsunori N a k a n o and Takashi G o j o b o r i 2 Second Department o f Medicine, Nagoya City University, Nagoya, Japan: ~Section oJ" Hepatobiliary Diseases, Departmetlt o/" Medicine, University o f Florida, GahTesville, Florida, USA; attd 2DNA Research Center, National htstitute o / Genetics, Mishh~a, Japan

The quasispecies nature of hepatitis C virus was investigated in a patient with chronic hepatitis C virus infection who underwent interferon-a therapy. The hepatitis C virus E2/NS1 region was amplified and cloned, and multiple clones were sequenced before and after interferon-or therapy. The hepatitis C virus quasispecies can be grouped into three groups by phylogenetic tree analysis. Quasispecies from all the groups were present before interferon-or therapy. However, only group 3 remained after interferon-a therapy. In addition, only group 3 hepatitis C virus quasispecies were present during the early biochemical relapse. These data indicate that various groups of hepatitis C virus quasispecies may have different sensitivity to interferon-a. © Journal of Hepatology. Key words: Hepatitis C virus; Interferon-a; Molecular evolutionary analysis; Quasispecies; Selection

Most RNA viruses, including hepatitis C virus (HCV), exist as a heterogeneous mixture of closely related mutant genomes, resulting from their high error rates in RNA replication, and are referred to as quasispecies. Charles Darwin's hypothesis of natural selection predicts the selection of the most stringent quasispecies (I). With reference to HCV infection, these factors may include viral replication efficacy, immune recognition by the host, immunomodulation of the host, therapeutic interventions, and coinfection with other viruses (viral-viral interaction). We report here that interferon-a (IFN) therapy is one external factor that selects HCV quasispecies.

Case Report, Methods and Results A 42-year-old Japanese male who had chronic hepatitis C, with liver biopsy showing chronic persistent hepatitis, was treated with natural IFN for 8 weeks. He had a history of acute non-A, non-B hepatitis acquired by blood transfusion 25 years previously. He showed biochemical

response to IFN, but relapsed shortly after IFN was discontinued. Genotyping of the HCV by the method of Okamoto et al. showed the same HCV genotype lb before and after IFN therapy [(2), naming of HCV according to Simmonds et al. (3)]. Five time points were studied (A: 1 year before IFN, B: just before IFN, C: just after IFN, D: when he had biochemical relapse, E: 1 year after IFN; Fig. IA). The HCV E2/NSI region was amplified by reverse transcription-polymerase chain reaction (RT-PCR), cloned and sequenced by the dideoxynucleotide chain termination method using a 373A DNA sequence (Applied Biosystems, Foster City, CA) (4). A total of 106 clones were sequenced (timepoint A: II, B: 47, C: 12, D: 24, E: 12, clones were named by the time point followed by the clone number). A phylogenetic tree was then constructed by conventional evolutionary analyses based on the estimation of nucleotide substitutions per site for the genomic region analyzed by the 6-parameter method and construction of the phylogenetic tree by the neighbor-joining method,

Correspondenceto: Johnson Y. N. Lau, M.D., Section of Hepatobiliary Diseases, Universityof Florida, PO Box 100214JHMHC, Gainesville, FL

32610, USA.

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using the system ODEN, version 1.1, of the National Institute of Genetics (Mishima, Japan, Fig. I B) (5). The clones can be grouped together into three groups, based on their evolutionary distance. Before IFN therapy (timepoint A and B), clones from groups 1-3 were obtained. However, all the clones obtained after IFN therapy (timepoint C, D, and E) belonged to group 3, indicating that group 1 and 2 HCV quasispecies were eliminated by IFN therapy and the biochemical relapse was related to group 3 HCV quasispecies which persisted for at least l year after IFN. The elimination of some of the HCV quasispecies was also reflected by the calculated nucleotide diversity at various timepoints (6). Timepoints A and B had nucleotide diversity values of 0.0404 and 0.0405, whereas timepoints C, D and E had lower nucleotide diversity values: 0.0228, 0.0298 and 0.0188, respectively. When the encoded amino acids in these sequences were

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compared (from amino acid position 369-508), there was only a single encoded amino acid residue (serine) that was conserved for all group I and group II sequences, but a different residue, threonine, was conserved in all the group III sequences (Fig. 2). Interestingly, this encoded amino acid was within the HVRl region.

Discussion

These data show that two groups of HCV quasispecies were eliminated during IFN therapy. In addition, HCV relapse was only associated with one group of HCV quasispecies, indicating that most, if not all, of the quasispecies of group 1 and 2 were eliminated. These observations suggest that the various HCV quasispecies may have different sensitivity to IFN and, therefore, IFN therapy represents an external selection pressure for HCV quasispecies. These findings have two important implications. First, some HCV quasispecies may be more sensitive than others. This raises the possibility that some quasispecies

886 may contain IFN-sensitive elements or that the resistant quasispecies contain IFN-resistant elements. If these elements can be located, they may serve as predictors o f subsequent response to I F N therapy. Secondly, HCV may behave like bacteria and malignant tumors in that therapy may lead to the dominance of resistant quasispecies, similar to the selection of antibiotic-resistant bacteria or chemotherapy-refractory t u m o r clones. If this hypothesis is true, then this virus has to be treated with multiple antiviral agents, as in the treatment o f mycobacterium tuberculosis and malignancies. In an attempt to identify the difference in amino acid sequence that may account for this different sensitivity, we identified one amino acid position that was conserved in both group I and II sequences and was also conserved but different in all the group III sequences, and this encoded amino acid was within the HVR1 region. Although this finding is interesting, no extrapolation is warranted without further confirmation with other patients.

Acknowledgements M M was supported by grants from the Japanese Ministry of Welfare and Viral Hepatitis Research Foundation

M. MIZOKAMI et al. o f Japan. J Y N L was supported in part by the D S R - D - I 5, D S R - R D A - I - 1 5 grants from the Division of Sponsored Research of the University of Florida, Gainesville, Florida, USA; the American Liver Association Hans Popper Liver Scholar Award; and the G l a x o Institute o f Digestive Health Clinical Investigator Award.

References 1. Darwin C. On the Origin of Species. London; Murray, 1859. 2. Okamoto H, Sugiyama Y, Okada S, Kuari K, Akahane Y, Sugai Y. et al. Typing hepatitis C virus by polymerase chain reaction with type-specific primers: application to clinical surveys and tracing infectious sources. J Gen Virol 1992; 73: 673-9. 3. Simmonds E Alberti A, Alter HJ, Bonino E Bradley DW, Brechot C, et al. A proposed system for the nomenclature for genotypes of hepatitis C virus. Hepatology 1994; 19: 1321-4. 4. Okada S, Akahane Y, Suzuki H, Okamoto H, Mishiro S, et al. The degree of variability in the amino terminal region of the E2/ NSI protein of hepatitis C virus correlates with responsiveness to interferon therapy in viremic patients. Hepatology 1992; 16: 619-24. 5. Gojobori T, Ishii K, Nei M. Estimation of average number of nucleotide substitutions when the rate of the substitution varies with nucleotide. J Mol Evol 1982; 18: 414-23. 6. Nei M. Molecular Evolutionary Genetics. Ch. 10. DNA polymorphism within and between populations. New York; Columbia University Press, 1987: 254-86.