Characterization of hepatitis C virus deletion mutants circulating in chronically infected patients

Abstract

Hepatitis C virus (HCV) has a linear positive-stranded RNA genome of approximately 9,600 nucleotides in length and displays a high level of sequence diversity caused by high mutation rates and recombination. However, when we performed long distance reverse transcription-PCRs on HCV RNA isolated from serum of chronic HCV patients, not only full-length HCV genomes but also HCV RNAs which varied in size from 7,600 to 8,346 nucleotides and contained large in-frame deletions between E1 and NS2 were amplified. Carefully designed control experiments indicated that these deletion mutants are a bona fide natural RNA species, most likely packaged in virions. Moreover, deletion mutants were detected in sera of patients infected with different HCV genotypes. We observed that 7/37 (18.9%) of genotype 1, 5/43 (11.6%) of genotype 3, and 4/13 (30.7%) of genotype 6 samples contained HCV deletion mutant genomes. These observations further exemplify HCV's huge genetic diversity and warrant studies to explore their biological relevance.

FIG. 1.

Identification of an HCV genotype 6h virus with a deletion in the E1-NS2 region in a patient (D54) doubly infected with genotypes 2i and 6h. (A) RT-PCR analysis (nucleotides 66 to 3636) of HCV RNA from sample D54 (lane 1) and sample D42 (lane 2) and the subsequent Southern blot analysis are shown in the left and right panels, respectively. Amplicons were separated in 0.8% agarose gels along with a marker (M) for which the molecular masses are given on the left side (in kilobase pairs). The deletion mutant and wild-type HCV genome are indicated on the right side. (B) The upper panel depicts the genome organization of HCV, with boxes indicating the coding regions for the core protein (C), envelope 1 and 2 (E1 and E2) proteins, p7, and nonstructural proteins 1 to 5 (NS1 to NS5). Also indicated are the 5′ and 3′ UTRs and the nucleotide and amino acid numbers, according to the numbering system for the prototype strain HCV-H (accession number M67463). In the lower panel, black lines indicate the D54 full-length (genotype 2i, DQ155561) and deletion (genotype 6h) sequences. The shaded box displays the position of the in-frame deletion region (Δ674).

FIG. 2.

HCV deletion mutants occur in the serum of patients chronically infected with HCV genotypes 1, 3, or 6. (A and B) RT-PCR products obtained from HCV genotype 1 (G1; P21), genotype 3 (G3; R30), and genotype 6 (G6; D33 and D88) serum samples, using degenerate primers for amplification of HCV nucleotides 66 to 3636 (A) and 1992 to 3636 (B), were separated on 0.8% agarose gels. The molecular mass marker (M) is indicated on the left side in kilobase pairs. (C) Schematic representation of complete and deletion genomes determined for the different HCV genotypes. The upper panel depicts the genome organization of HCV as in Fig. 1. Forward and reverse primers to detect complete and deletion HCV genomes are presented. In the lower panel, black lines indicate the full-length and deletion sequences. The shaded boxes display the positions of the in-frame deletion region.

FIG. 3.

Consensus sequence alignments of different HCV full-length complete genomes (com) and deletion mutants (del). Nucleotide differences between complete and deletion mutant sequences are shown in bold. Residues identical to the major sequence are indicated by a dash, the deletion region is indicated by dots, and slashes indicate discontinuous sequences of the deletion regions. The nucleotide positions are presented above the sequences (numbering system for the prototype strain HCV-H), and sample names are indicated in the left-hand column. Complement nucleotide codes: R = T or C; Y = G or A.

FIG. 4.

Experimental confirmation of HCV E1-E2 deletion mutants. (A to C) RT-PCR (left panel) and Southern blot analysis (right panel) of HCV genotype 1 (P21), genotype 3 (R30), and genotype 6 (D33 and D88), using sequence-specific primers to amplify the regions 5′UTR to NS3 (nucleotides 17 to 3277) (A), 5′UTR to core (nucleotides 17 to 750) (B), and NS3 to NS5 (nucleotides 4039 to 8625) (C). Amplicons were separated in 0.8% agarose gels along with a marker (M) for which the molecular masses are given on the left side in kilobase pairs. (D) Gel electrophoresis of RT-PCR products amplified from RNA of samples P21, R30, D33, and D88 with primer 3636 and a junction site primer overlapping the deleted region that was specifically designed for each sample (Table 1). The molecular mass marker (M) is indicated on the left side (in kilobase pairs).

FIG. 5.

HCV deletion mutants are bona fide RNA species. (A) Comparison of RT-PCR amplification of HCV nucleotides 66 to 3636 (left panel) and Southern blot analysis (right panel) of HCV genotype 1 (P21), genotype 3 (R30), and genotype 6 (D33 and D88) serum samples, using Expand reverse transcriptase (lanes 1, 3, 5, and 7) and Transcriptor reverse transcriptase (lanes 2, 4, 6, and 8). (B) RT-PCR analysis of in vitro-transcribed full-length HCV RNA (left panel) and deletion mutant RNA (right panel) of sample P21 (genotype 1), using primer 3636 and either primer 66 or a junction site primer overlapping the deleted region (DelFP). Reactions were performed in the presence (+) or absence (−) of T7 RNA polymerase. As controls, a cDNA clone containing either full-length or deletion mutant HCV (C) or RNA isolated from the P21 serum sample (P) was used. The molecular mass marker (M) is shown in kilobase pairs on the left side. The amplicons representing deletion forms (Del) are indicated with an arrow.

FIG. 6.

The HCV RNA genotype 1 (P21) deletion mutant circulating in plasma is protected from RNase A digestion. The P21 serum sample was treated with or without 0.1% Triton X-100 and 1 μg RNase A before RNA extraction. RT-PCR amplifications of nucleotides 66 to 3636 (deletion mutant; left panel) or 1992 to 3636 (full-length; middle panel) or of the deletion mutant with a junction site primer overlapping the deleted region (DelFP) and primer 3636 (deletion mutant; right panel) are shown. The molecular mass marker (M) is indicated on the left side (in kilobase pairs).