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Journal of Clinical Microbiology, November 2001, p. 3858-3864, Vol. 39, No. 11
Departments of
Microbiology1 and
Biochemistry,4 Faculty of
Medicine, and Tropical Disease Center,2
Airlangga University, Surabaya, Indonesia, and Department of
Microbiology3 and International
Center for Medical Research,5 Kobe University
Graduate School of Medicine, Kobe, Japan
Received 9 March 2001/Returned for modification 11 June
2001/Accepted 1 August 2001
In the present study, we analyzed the possible relationship
between interferon (IFN) sensitivity-determining region (ISDR) sequence
variation of various hepatitis C virus (HCV) subtypes and serum HCV
titers in Indonesian patients without IFN treatment. The viremia titers
(mean ± standard deviation) of HCV subtype 1b (HCV-1b) isolates
with low (three or fewer) and high (four or more) numbers of ISDR
mutations were 5.4 ± 0.6 and 4.2 ± 0.9 log10
RNA copies/ml, respectively, with the difference between the two
groups being statistically significant (P < 0.01).
Similarly, the viremia titers of HCV-1c isolates with low and high
numbers of ISDR mutations were 5.3 ± 0.6 and <3.0 ± 0.0 log10 RNA copies/ml, respectively, with the difference
between the two groups being statistically significant
(P < 0.01). Also, the virus titers of HCV-2a
isolates with low and high numbers of ISDR mutations were 4.3 ± 0.7 and 3.5 ± 0.4 log10 RNA copies/ml, respectively,
with the difference between the two groups being statistically
significant (P < 0.01). Thus, our results
demonstrated that virus load in Indonesian patients infected with
HCV-1b, HCV-1c, or HCV-2a correlated inversely with the number of
mutations in the ISDR sequence, implying the possibility that the ISDR
sequence plays an important role in determining the levels of HCV viremia.
Hepatitis C virus (HCV) readily
establishes a chronic persistent infection that often results in
chronic hepatitis and more deteriorating disease such as liver
cirrhosis and hepatocellular carcinoma (14). HCV is
phylogenetically classified into at least six clades (formerly called
genotypes), each of which can be further divided into a number of
subtypes (4, 26, 30). We have previously reported the
prevalence of each HCV subtype, including HCV subtype 1c (HCV-1c)
(formerly referred to as HCV-1d), among various clinical populations in
Surabaya, Indonesia (13, 31). HCV-1c has been found almost
exclusively in Indonesia (12, 13, 23) and shown to be
associated with high viral load and poor prognosis (18,
31).
Interferon (IFN) is the most successful therapeutic agent for the
treatment of chronic hepatitis C, although less than half of the
patients treated with IFN show sustained responses with eradication of
the virus. It is now recognized that HCV viral load in the serum and
the HCV genotype and/or quasispecies complexity as well as sequence
diversity of particular regions of the viral genome may predict the
effectiveness of IFN therapy (2, 3, 10, 16, 25, 27). Lower
pretreatment serum HCV RNA levels have been shown to be associated with
a better response to IFN therapy. Patients infected with HCV-1b tend to
exhibit poor IFN responsiveness compared with those infected with
HCV-2a. Enomoto et al. (6, 7) first demonstrated that
amino acid mutations of the nonstructural protein 5A (NS5A) of HCV-1b
in a region between residues 2209 and 2248 were associated with
improved responsiveness to IFN in Japanese patients, and the region has
therefore been designated as the IFN sensitivity-determining region
(ISDR). This observation was subsequently confirmed by other research
groups mostly in Japan (2, 3, 21, 28, 34). However,
several reports from Europe and the United States failed to show the
correlation between ISDR mutations and IFN responsiveness (5, 11,
22, 32), challenging the ISDR hypothesis. The IFN-mediated
antiviral activity is executed in part by the double-stranded
RNA-activated protein kinase (PKR), which has been suggested to form a
complex with NS5A through a region, designated the PKR-binding region, that spans the ISDR and the adjacent 26 residues (9). In
the present study, we have investigated whether the PKR-binding region of HCV-1b, -1c, and -2a plays a role in determining the levels of
viremia in patients without IFN treatment.
Serum samples.
Sera were obtained from the Red Cross Blood
Transfusion Center, Surabaya, Indonesia, and from patients with chronic
liver disease at Dr. Soetomo Hospital, Faculty of Medicine, Airlangga University, Surabaya, Indonesia. They were tested for anti-HCV antibodies by enzyme-linked immunosorbent assay (UBI HCV EIA [United Biologicals, Inc., New York, N.Y.]; Ortho HCV Ab ELISA Test II [Ortho
Diagnostics, Inc., Tokyo, Japan]) and for hepatitis B surface antigen
(subtypes ad and ay) by using AUSAB EIA (Abbott Laboratories, Diagnostics Division). Sera that were positive for anti-HCV antibodies and negative for hepatitis B surface antigen were used for further analysis. A total of 57 HCV isolates obtained from 57 individuals (23 isolates of HCV-1b, 15 isolates of HCV-1c, and 19 isolates of HCV-2a)
were analyzed. Table 1 summarizes the
number, sex, and age of the subjects, and mean HCV viremia titers for
each HCV subtype with low (three or fewer) and high (four or more) numbers of mutations in the ISDR (see below). The grouping of HCV
isolates on the basis of low (three or fewer) and high (four or more)
numbers of ISDR mutations has been reported (2, 3, 6, 7).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3858-3864.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Correlation between Mutations in the Interferon
Sensitivity-Determining Region of NS5A Protein and Viral Load of
Hepatitis C Virus Subtypes 1b, 1c, and 2a
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Comparison between the number of ISDR mutations and serum
HCV RNA titers for HCV subtypes 1b, 1c, and 2a
HCV subtype analysis. RNA was extracted from the anti-HCV antibody-positive sera (60 µl each) using Trizol LS (Life Technologies, Gaithersburg, Md.) and reverse transcribed into cDNA using Rous-associated virus type 2 reverse transcriptase (Takara Shuzo, Co., Ltd., Kyoto, Japan) and a primer specific for a portion of the NS5B region of the HCV genome (167R), as described previously (13, 18, 31). The resultant cDNA was then amplified by nested PCR using Tth DNA polymerase (Toyobo Co., Ltd., Osaka, Japan) and appropriate sets of primers. Anti-HCV-negative sera or saline served as a negative control in the reverse transcription (RT)-PCR analysis to monitor the possible cross-contamination between the samples. Also, other standard precautions were taken to minimize possible cross-contamination. The amplified fragments were sequenced by using the Taq DiDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer) and an ABI 373A DNA sequencer (Applied Biosystems, Inc.). Based on the sequence similarity to the reported sequences, each HCV isolate was assigned an HCV subtype.
Measurement of HCV viral load. Levels of HCV viral load were assessed using a commercially available kit (Amplicor HCV Monitor Test, version 1.0; Roche Diagnostic Systems, Inc., Branchburg, N.J.) according to the manufacturer's instructions. The lowest detectable titer with this kit was 3.0 log10 RNA copies/ml. A viral load of 4.1 log10 RNA copies/ml or higher was regarded as a high virus titer, and that of 4.0 log10 RNA copies/ml or lower was regarded as a low titer, according to the titers for the high and low controls included in the kit.
Analysis of NS5A sequences.
RNA extracted from the sera (60 µl each) was reverse transcribed into cDNA by using Rous-associated
virus type 2 reverse transcriptase (Takara Shuzo) and an appropriate
primer (Fig. 1). The resultant cDNA was subjected to the first-round PCR over 35 cycles, with each cycle consisting of 1 min at 94°C, 1 min at 50°C, and 2 min at
72°C, followed by the second-round PCR under the same conditions described above. The primers used to analyze the entire NS5A region of
HCV-1b, -1c, and -2a were selected on the basis of the sequences that
had been reported to be conserved among HCV-1b and -1c or -2a. The
sequences and positions of the primers used are shown in Fig. 1. The
PCR products were electrophoresed in an agarose gel containing ethidium
bromide and were visualized by UV illumination. Nucleotide sequences of
the amplified fragments were determined with the Big Dye Deoxy
Terminator Cycle Sequencing kit (Perkin-Elmer) and ABI 377 or ABI 310 DNA sequencer (Applied Biosystems, Inc.), and the amino acid sequences
were deduced.
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Statistical analysis. The data obtained were statistically analyzed by one-way analysis of variance (ANOVA) and Student's t test. When appropriate, the nonparametric Mann-Whitney test was also used. A P value of <0.05 was considered significant.
Nucleotide sequence accession numbers. The nucleotide sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases with the accession numbers AB056520 through AB056569.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Levels of viremia in patients infected with HCV-1b, -1c, or -2a. Levels of viremia of HCV-1b isolates ranged from 3.6 to 6.3 (5.3 ± 0.8) log10 RNA copies/ml (values are presented throughout as means ± standard deviations [SD]). The titers of HCV-1c isolates ranged from <3.0 to 6.0 (5.0 ± 1.0) log10 RNA copies/ml. The titers of HCV-2a isolates ranged from <3.0 to 5.8 (3.9 ± 0.7) log10 RNA copies/ml. The apparent difference in the viremia titers between HCV-2a and the other subtypes is likely due to the fact that the Amplicor HCV Monitor Test, version 1.0, underestimates the viral load for HCV genotypes other than genotype 1 (19).
Correlation between HCV viremia levels and the number of ISDR
mutations in HCV-1b, -1c, and -2a.
We determined deduced amino
acid sequences of the entire NS5A protein of representative isolates
with high and low virus titers for HCV-1b, -1c, and -2a. When compared
with each of the reference strains
HCV-1bJ (15) for
HCV-1b, HC-G9 (23) for HCV-1c, and HC-J6 (24)
for HCV-2athe HCV isolates tested possessed 15 to 30 amino acid
substitutions in NS5A excluding the PKR-binding region; however, those
substitutions outside the PKR-binding region did not appear to
correlate with serum HCV RNA titers (data not shown).
4.1
(5.4 ± 0.6) log10 RNA copies/ml. On the other hand, two of three isolates with high numbers of ISDR mutations showed viremia levels of <4.0 log10 RNA
copies/ml, and the mean titer ± SD of this group was 4.2 ± 0.9 log10 RNA copies/ml. The difference in the
mean virus titers between the two groups was statistically significant
(P < 0.01).
|
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, P < 0.01 by one-way
ANOVA and P < 0.05 by Mann-Whitney test.
4.2 (5.3 ± 0.6) log10
RNA copies/ml. On the other hand, both of the two isolates with high
numbers of ISDR mutations showed viremia levels of <3.0
log10 RNA copies/ml, with the mean titer being at
its highest 3.0 log10 RNA copies/ml. The
difference in the mean virus titers between the two groups was
statistically significant (P < 0.01).
When HCV-1b and HCV-1c isolates were combined, the viremia titer for
those with low numbers of ISDR mutations (three or fewer) was 5.4 ± 0.6 log10 RNA copies/ml, whereas that for
those with high numbers of ISDR mutations (four or more) was 3.7 ± 0.9 log10 RNA copies/ml, with the difference
between the two groups being statistically significant
(P < 0.01).
The sequences of HCV-2a isolates were compared with that of HC-J6,
which had been used as a reference strain in a previous work
(20). We noticed a tendency, similarly to the cases with HCV-1b and HCV-1c isolates, for serum HCV titers to be correlated inversely with the number of ISDR mutations (Fig. 2C). Again, HCV-2a
isolates were divided into the two groups of low (three or fewer) and
high (four or more) numbers of ISDR mutations, and serum HCV titers
were plotted (Fig. 3C and Table 1). The virus titers (mean of HCV-2a
isolates with low [three or fewer] and high [four or more] numbers
of ISDR mutations) were 4.3 ± 0.7 and 3.5 ± 0.4 log10 RNA copies/ml, respectively, with the
difference between the two groups being statistically significant
(P < 0.01).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We previously observed that serum HCV RNA titers varied considerably with different isolates of the same HCV subtype in Indonesia, such as HCV-1b, -1c, and -2a (18). In the present study, we analyzed amino acid sequences of NS5A of those HCV isolates in order to see whether or not there is any correlation between serum HCV titers and NS5A sequences, especially the sequence of the ISDR and PKR-binding region. Our results clearly demonstrated that with all subtypes tested, i.e., HCV-1b, -1c, and -2a, the high number of ISDR mutations was associated with low HCV viral load in patients without IFN treatment. (Fig. 3). Pretreatment viral load was reported to correlate with IFN responsiveness of HCV-infected patients (2, 3). It is reasonable, therefore, to assume that ISDR sequence analysis can predict IFN responsiveness of HCV-infected Indonesian patients, as has been reported with Japanese patients (2, 3, 6, 7, 16).
Despite the consistent observations by Japanese research groups that the number of amino acid substitutions in ISDR correlates well with sensitivity to IFN therapy in patients infected with HCV-1b or HCV-2a, conflicting observations were reported in that there was no significant correlation between ISDR mutations and IFN responsiveness in patients infected with HCV-1b in Europe and the United States (5, 11, 22, 32). Recently, Nakano et al. (21) pointed out sequence variation among HCV-1b isolates, based on which they divided the HCV-1b isolates into three groups, J, NJ, and W. The same authors concluded that correlation between ISDR mutations and IFN responsiveness was observed only with the J group, the group representing approximately 40% of HCV-1b isolates in Japan but rarely found in Europe and the United States. According to our sequence data published previously (12) (DDBJ accession no. D13729 to D13731, D13734, and D13735), two (40%) of five Indonesian HCV-1b isolates could be classified into the J group, with the remaining three (60%) being classified into an as-yet-undefined fourth group. This observation may also support the hypothesis that the ISDR sequence can be used to predict IFN responsiveness of some, if not all, Indonesian patients infected with HCV.
It is noteworthy that an HCV-1b isolate (isolate 378 [Fig. 2A]) had
five mutations in the ISDR and a total of 11 mutations in the
PKR-binding region and yet showed a high virus titer. It was recently
reported that envelope glycoprotein E2 of HCV contains a sequence
identical with phosphorylation sites of PKR and eIF-2
and that E2
inhibited the kinase activity of PKR, which might be one of the
mechanisms counteracting antiviral activity of IFN (33).
It is possible that the sequence, designated the PKR-eIF-2 phosphorylation homology domain (PePHD), of this particular HCV-1b isolate (isolate 378) is involved in maintaining the high level of
viremia while apparently lacking the IFN-inhibiting function of the
ISDR. On the other hand, we found a few isolates of HCV-2a with low
virus titers, which had low numbers of ISDR mutations. It would be
interesting to see whether or not those isolates had high numbers of
mutations in PePHD of E2, as reported recently (29).
Another possibility should also be taken into consideration, i.e., that
there exists another viral mechanism regulating IFN sensitivity and
viral load, such as quasispecies complexity of the HCV genome
(10, 25) and an as-yet-undetermined viral factor(s) (21, 34). Further study is needed to elucidate the issue.
Previous study suggested a possible correlation between the presence of the arginine residue at position 2218 and sensitivity to IFN therapy in patients infected with HCV-1b (8). In the present study, however, no significant correlation was observed for HCV-1b or HCV-1c (Fig. 2). Consistent with our observation, the lack of such a correlation was reported by other investigators (22).
It was recently demonstrated that, upon adaptation to the Huh-7 human hepatoma cell line, NS5A underwent mutations at positions 2163, 2177, 2189, 2196, 2197, 2199, and 2204, which were clustered in a defined region just upstream of the ISDR, suggesting that those mutations were responsible for the higher degrees of HCV RNA replication in Huh-7 cells (1, 17). In the present study, however, irrespective of HCV viremia titers, all of those residues as well as the serine residue at position 2201 were completely conserved among the HCV isolates of each subtype tested (data not shown). It should also be noted that all but one (position 2163) are completely conserved even across different subtypes, including subtypes other than HCV-1b, -1c, and -2a. In this connection, Pawlotsky et al. (25) reported that serine residues at positions 2197, 2201, and 2204 showed remarkable conservation, suggesting the importance of NS5A phosphorylation at those residues. Taken together, these results imply the possibility that the defined region of NS5A just upstream of the ISDR, with certain serine residues being phosphorylated, plays a crucial role in HCV replication and that the viral adaptation mechanism differs in continuously growing Huh-7 cells and nongrowing, mature hepatocytes in the human liver.
In conclusion, we have demonstrated that virus load in patients infected with HCV-1b, HCV-1c, or HCV-2a correlates inversely with the number of mutations in the ISDR sequence of NS5A of HCV in patients in Indonesia. Our results imply the possibility that the ISDR sequence plays an important role in determining the levels of HCV viremia, through differentially inhibiting antiviral activity of endogenous IFN and/or through another mechanism(s) regulating HCV replication.
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ACKNOWLEDGMENTS |
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We are grateful to Budi Arifah, Red Cross Blood Transfusion Center, Surabaya, Indonesia, for providing serum samples obtained from blood donors.
This work was carried out during the large-scale cooperative study between Southeast Asian countries and Japan conducted by the Japan Society for the Promotion of Science. This work was also supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture, Japan, and a research grant from Mitsui Life Social Welfare Foundation.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan. Phone: 81-78-382-5500. Fax: 81-78-382-5519. E-mail: hotta{at}kobe-u.ac.jp.
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References
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Journal of Clinical Microbiology, November 2001, p. 3858-3864, Vol. 39, No. 11
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.11.3858-3864.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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