Identification of Hepatitis C Virus (HCV) Subtype 1b Strains That Are Highly, or Only Weakly, Associated with Hepatocellular Carcinoma on the Basis of the Secondary Structure of an Amino-Terminal Portion of the HCV NS3 Protein

Serum samples.Sera were collected from patients with and without HCC in Hyogo and Yamagata Prefectures, Japan. The diagnosis of HCC was made on the basis of clinical and histopathological criteria. The sera were tested for anti-HCV antibodies and HBsAg by using commercial kits (Ortho HCV Ab ELISA Test III [Ortho Diagnostics, Tokyo, Japan] and AUSAB EIA [Abbott Diagnostics], respectively). Anti-HCV antibody-positive sera were tested for HCV RNA by reverse transcription (RT)-PCR, and the HCV subtypes were determined as reported previously (5, 21, 30). A total of 167 serum samples positive for HCV-1b RNA and negative for HBsAg were further analyzed, as described below. The age and sexes of the patients are summarized in Table 1.

NS3 analysis.RNA was extracted from 50 μl of serum with the RNeasy Mini kit (Qiagen). To amplify a portion of the HCV genome encoding an amino-terminal region of NS3, a one-step RT-PCR was performed in a tube by the Superscript One-Step RT-PCR with Platinum Taq (GIBCO BRL) and an outer set of primers, primer NS3-F1 (sense primer; 5′-ACACCGCGGCGTGTGGGGACAT-3′; nucleotides 3295 to 3316) and primer NS3-AS2 (antisense primer; 5′-GCTCTTGCCGCTGCCAGTGGGA-3′; nucleotides 4040 to 4019). PCR was initially performed at 45°C for 30 min for RT and then at 94°C for 2 min, followed by the first-round PCR over 40 cycles, with each cycle consisting of 1 min each at 94, 55, and 72°C. The second-round PCR was performed with Pfu DNA polymerase (Promega) and an inner set of primers, primer NS3-F3 (sense primer; 5′-CAGGGGTGGCGGCTCCTT-3′; nucleotides 3390 to 3407) and primer NS3-AS1 (antisense primer; 5′-GCCACTTGGAATGTTTGCGGTA-3′; nucleotides 4006 to 3985). The second-round PCR was performed for 35 cycles, with each cycle consisting of 1 min at 94°C, 1.5 min at 55°C, and 3 min at 72°C. This method allowed us to amplify the corresponding portion of the HCV genome from more than 90% of HCV-1b RNA-positive samples. The amplified fragments were purified with the QIAquick PCR Purification kit (Qiagen) and directly sequenced, without being subcloned, in both directions with the dRhodamine Terminator Cycle Sequencing Ready Reaction kit and an ABI 377 sequencer (PE Applied Biosystems). The secondary structure of the amino-terminal portion of NS3 was predicted by computer-assisted Robson analysis (9) with GENETYX-MAC software (version 10.1; Software Development Co., Ltd., Tokyo, Japan) with α-helix and extended decision constants of 0.

Statistical analysis.The data obtained were statistically analyzed by the χ² test for independence with a two-by-two contingency table and Student's t test. A P value of <0.05 was considered significant.

Nucleotide sequence accession numbers.The nucleotide sequence data reported in this paper appear in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession numbers AB072043 through AB072113 , AB089512 through AB089583 , and AB100806 through AB100829 .

Classification of HCV-1b strains on the basis of the secondary structure of an amino-terminal portion of NS3 and their possible association with HCC.On the basis of the secondary structure of the amino-terminal 120 residues of NS3, HCV-1b isolates were classified into group A, group B, and an indeterminate group, each of which was further divided into a number of subgroups (Fig. 1). The criteria for the group classification, which should match the reported one (25), are as follows. In group A isolates, the carboxy-terminal portion of 20 residues (aa 1125 to 1146) goes leftward relative to a domain composed of the remaining amino-terminal portion. In group B isolates, on the other hand, the carboxy-terminal portion of 20 residues goes rightward relative to the amino-terminal domain. It should be noted that the turn structures at about positions 1048 and 1125 were conserved among all 167 isolates tested (data not shown). Isolates of group A and group B were further classified into subgroups, such as subgroups A1-1, A1-2, A2-1, A2-2, B1-1, B1-2, B2-1, and B2-2, on the basis of the positions of the additional turn structures. For example, subgroups A1-1 and B1-1 have a turn structure at about position 1083, but not at about position 1075. On the other hand, subgroups A1-2 and B1-2 have a turn structure at about position 1075 but not at about position 1083. Subgroups A2-1 and B2-1 have two turn structures at about positions 1075 and 1083, while subgroups A2-2 and B2-2 do not have any turn structure between positions 1048 and 1125. Eighteen (10.8%) of 167 isolates had an additional turn structure near the amino terminus at about positions 1033 to 1037, with the partial secondary structure thereafter resembling that of one of the subgroups mentioned above. Those isolates were classified into an indeterminate group and were further divided into a number of subgroups, such as subgroups C-1 (six isolates), C-2 (five isolates), and C-3 (four isolates). Three isolates of the indeterminate group differed from each other and were tentatively classified into a subgroup referred to as C-etc. (Table 2).

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FIG. 1.

Schematic representation of secondary structure of the amino-terminal 120 residues of NS3 of HCV-1b isolates obtained from patients with and without HCC. (A) Representative forms of group A, which can be further classified into subgroups A1-1, A1-2, A2-1, and A2-2, based on the number and positions of turn structures; (B) representative forms of group B, which can be further classified into subgroups B1-1, B1-2, B2-1, and B2-2; (C) representative forms of an indeterminate group, which can be further classified into subgroups C-1, C-2, C-3, C-etc. The looped, zigzag, straight, and bent lines represent α-helix, β-sheet, coil, and turn structures, respectively. The numbers along the secondary structure indicate amino acid positions.

HCV-1b isolates of subgroup B1-1 were found in 53 (59.6%) of 89 patients with HCC and in 19 (24.4%) of 78 patients without HCC, with the difference in the prevalence between the two patient groups being statistically significant (P < 0.00001) (Table 2). Thus, subgroup B1-1 was highly associated with HCC. Although the number of isolates was small, subgroup B2-1 was also highly associated with HCC, with all five isolates being found in patients with HCC (P < 0.05). Isolates of subgroup A1-1, which was weakly associated with HCC, were found in 6 (6.7%) of 89 patients with HCC and in 25 (32.1%) of 78 patients without HCC, with the difference between the two patient groups being statistically significant (P < 0.0001). The other subgroups, such as A1-2, A2-1, and B1-2 and the indeterminate ones, C-1 to C-3, were moderately associated with HCC; their distribution patterns among patients with HCC did not significantly differ from those among patients without HCC.

Sequence alignment of the amino-terminal 120 residues of NS3 of HCV-1b strains.The amino acid sequences of the isolates were aligned with each other, and a consensus sequence was determined on the basis of the alignment result (Fig. 2). The consensus sequence differed from the sequence of a standard strain, strain HCV-J (GenBank accession number D90208 ) (15), by five residues at positions 1056 (Asp to Glu), 1062 (Leu to Val), 1112 (Pro to Gln), 1120 (Met to Leu), and 1140 (Val to Ile). Subgroup B1-1 had the consensus sequence, whereas subgroup A1-1 had the HCV-J sequence. Sequences of representative isolates of each of the HCV subgroups were aligned with the consensus sequence (Fig. 2). Tyr-1082 was associated, in general, with the presence of a turn structure at about position 1083, as observed in subgroups A1-1, A2-1, B1-1, and B2-1, with some exceptions. On the other hand, Phe-1082 was associated with the absence of the turn structure at about position 1083 and generation of a turn structure at about position 1075, as observed in subgroups A1-2 and B1-2. Also, a mutation(s) upstream of position 1082 appeared to affect the turn structures at about positions 1075 and 1083. It was difficult to predict the presence or absence of a turn structure at about position 1103 simply by comparing amino acid sequences. Isolates of the indeterminate subgroups, C-1, C-2, C-3, and C-etc., had a unique mutation(s) near the amino terminus, such as Leu to Phe at position 1040 (13 isolates) and Ser to Cys or Thr at position 1033 (3 isolates), that created an additional turn structure.

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FIG. 2.

Sequence alignment of the amino-terminal 120 residues of NS3 of HCV-1b isolates obtained from patients with and without HCC. Representative sequences of each of the subgroups are aligned with a consensus sequence, shown on the top. Dashes indicate residues identical to the residue in the consensus sequence. The numbers along the consensus sequence indicate amino acid positions.

At the primary structure level, we did not find any particular residue that is unique to and yet common among the majority of isolates from patients with HCC (data not shown). It should be noted, however, that isolates of subgroup B2-1, which are highly associated with HCC but small in number (Table 2), have more mutations or a unique mutation from a hydrophobic residue (Val) to a hydrophilic residue (Thr) at about positions 1055 to 1080 (Fig. 2).

The average number of mutations in the 120-residue sequence differed by subgroup. Subgroup A1-1 had significantly more mutations than B1-1 but significantly fewer mutations than A1-2, B1-2, and B2-1 (Table 3). When isolates from patients with HCC and those without HCC were compared, there was no significant difference in the average number of mutations between them (2.9 ± 1.8 and 3.2 ± 1.5, respectively).