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Department of Haematology, Division of Transfusion Medicine, Cambridge Blood Centre, University of Cambridge, Long Road, Cambridge CB2 2PT, UK
Correspondence
Jean-Pierre Allain
jpa1000{at}cam.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). HCV is a single-stranded RNA virus encoding a polypeptide of 
3000 aa that is cleaved into structural and non-structural (NS) viral proteins. Due to genetic variability caused by the non-proofreading NS5B RNA-dependent RNA polymerase, HCV sequences have been classified into six major genotypes and several subtypes (Simmonds et al., 2005). The viral particle consists of an envelope, a nucleocapsid and a highly heterogeneous RNA genome, showing features similar to those of flaviviruses (Kaito et al., 1994; Trestard et al., 1998). The nucleocapsid-forming core protein is encoded by the core gene sequence, which has been shown to produce an additional 17 kDa protein from a translational shift to a +1 open reading frame (ORF), resulting from a ribosomal frameshift between codons 8 and 11 (Lo et al., 1994). This protein has been named alternate reading frame protein (ARFP), or F protein for short (Walewski et al., 2001; Xu et al., 2001). It is highly basic with a pI of 12 and is unstable with a short half-life of approximately 10 min (Roussel et al., 2003). No clear sequence homology to other proteins of known function has been identified (Xu et al., 2001). Unlike the core protein, it is not involved in c-myc activation or in alteration of human telomerase reverse transcriptase (hTERT) and p53 promoter activity (Basu et al., 2004).
Initially, the 10 consecutive adenines at codons 8–11 of HCV genotype 1 isolates were considered essential for the +1 frameshift to take place, at least in vitro. It was later found that these are not critical for the core +1 ORF expression in vivo (Vassilaki & Mavromara, 2003). The core +1 ORF was shown to be expressed efficiently by HCV genotype 1 in vitro using a reticulocyte lysate assay, but not by HCV genotype 1a strain H. Several studies have shown that HCV produces this F protein from the core protein reading frame by more than one type of coding event (Baril & Brakier-Gingras, 2005; Boulant et al., 2003; Choi et al., 2003; Varaklioti et al., 2002; Vassilaki & Mavromara, 2003). HCV could mediate not only a –2/+1 frameshift but also a –1/+2 frameshift by a triple decoding function of the HCV ribosomal frameshift signal both in vitro and in vivo. It has also been reported that a +1 frameshift at codon 42 and a –1 frameshift at codon 144 lead to an F protein containing the first 42 aa of the core protein, followed by 101 aa encoded by the +1 ORF and then aa 144 to the end of the core protein in the genotype 1b sequence (Boulant et al., 2003). Detection of antibodies to the F protein in the sera of HCV-infected patients using purified recombinant F protein or peptides has provided indirect evidence of its existence in HCV infections at different stages (Komurian-Pradel et al., 2004; Varaklioti et al., 2002; Walewski et al., 2001). The cellular response to the F protein has been described in HCV-seropositive patients receiving antiviral treatment using peptides based on a genotype 1b strain (Bain et al., 2004). To date, all studies regarding the F protein have been based on the expression of recombinant proteins or on monoclonal antibodies derived from genotype 1a or 1b sequences, but not from other genotypes. Here, we present data on the humoral response and antibody titres of HCV chronically infected blood donors or donors who had recovered from infection, against a recombinant F protein derived from a genotype 2 sequence, which was compared with anti-core and anti-HCV reactivity and levels.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In addition, 31 plasma samples from Ghanaian blood donors who were negative for anti-HCV antibody were included as a control group. This study was approved by the ethics committee of the Kwame Nkrumah University School of Medical Sciences (Ghana) and Addenbrooke's Hospital Trust (Cambridge, UK), and informed consent was obtained from each donor.
Sequencing of the genotype 2 HCV core in Ghanaian donors with chronic infection.
Viral RNA was extracted from genotype 2 HCV-infected samples using a High Pure Viral RNA kit (Roche Diagnostics). The core sequence was reverse transcribed in a final volume of 20 µl containing 2 µl 10x PCR buffer, 5 mM MgCl2, 1 mM dNTPs, 2 µM Core-2 primer (5'-CGGGAACGTGGAGMACYGC-3'), 20 U RNAsin and 50 U Moloney murine leukemia virus reverse transcriptase with a cycling profile of 25 °C for 5 min, 55 °C for 60 min and 99 °C for 5 min. The cDNA was amplified by nested PCR using the outer primers Core-1 (5'-GCGAAAGGCCTTGTGGTACTG-3'; Ogata et al., 2002) and Core-2, and inner primers Core-3 (5'-ACTGCCTGATAGGGTGCTTG-3'; Ogata et al., 2002) and Core-4 (5'-GAGCARTCATTGGTCACCATGTA-3'). Both PCRs were performed in 20 µl reactions containing 10 µl PCR MasterMix (Promega), 1 µM each primer and 4 µl cDNA with a cycling profile of denaturation at 95 °C for 3 min, followed by 30 cycles of 60 °C for 45 s, 72 °C for 45 s and 94 °C for 45 s, with a final extension at 72 °C for 10 min. The amplicons were sequenced using the inner primers Core-3 and Core-4.
Preparation of HCV F-2 and core recombinant proteins.
The core amplicon of a representative genotype 2 strain (G2-15) was cloned into pCR4-TOPO (Invitrogen) to generate plasmid DNA and the insert was confirmed by sequencing. The putative genotype 2 F sequence (F-2) was generated by introducing a deletion of 1 nt in codon 43 and one insertion at a point corresponding to codon 144 of the core sequence. To achieve this, three amplifications were performed separately in a 50 µl reaction volume containing 5 µl 10x Pfu polymerase buffer, 0.32 mM dNTP, 2.5 U Pfu polymerase and 10 µg plasmid DNA with 0.25 µM each primer according to the following: reaction 1 (nt 1–138): pET-F (5'-CACCATGGCCACAAATCCTAAA-3') and F2 (5'-GCACACCCAACC_GGGGCCC-3'); reaction 2 (nt 118–446): F3 (5'-AGGGGCCCC_GGTTGGGTG-3') and F4 (5'-CTGGCAACGCCACCAACCCGGGGC-3'); reaction 3 (nt 418–573): F5(5'-TTGGCGCCCCGGGTTGGTGGCGTT-3') and pET-R (5'-GGCAGAGACTGGCACAGAAAT-3'). Underscores indicate the position of deletions and underlining the addition of nucleotides introduced into the amplification. The cycling profile for reactions 1 and 3 consisted of denaturation at 95 °C for 45 s, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 20 s. The cycling profile for reaction 2 consisted of 95 °C for 45 s, followed by 30 cycles of 94 °C for 45 s, 60 °C for 45 s and 72 °C for 45 s. A final extension was performed at 72 °C for 10 min in each profile. The amplicons from fragments 1 and 2 were joined using primers pET-F and F4. All three amplicons were finally joined to form the final F-2 sequence of 573 bp using pET-F and pET-R. Both overlapping PCRs were carried out in a 50 µl reaction volume containing 5 µl 10x Pfu polymerase buffer, 0.32 mM dNTP, 2.5 U Pfu polymerase, 0.5 µl each cDNA and 2.5 µM each primer.
The core gene was amplified directly using primers pET-F and pET-R in a 50 µl reaction volume using the conditions and cycling profile described above.
The PCR product of the F-2 or core sequence was inserted into pET101/D-TOPO (Invitrogen), which includes a V5 and 6xHis tag at the C terminus of the insert. The recombinant vector was then expressed in Escherichia coli strain BL21 Star (DE3) according to the manufacturer's instructions. The selected colonies were grown in 2x TY medium (Sigma) containing 50 µg ampicillin ml–1. Four litres of culture were grown from selected colonies at 37 °C overnight, followed by induction with 1 mM IPTG at 25 °C for 6 h. The cultures were centrifuged and the pelleted bacteria were lysed by sonication on ice. The inclusion body layer was collected and washed twice with PBS/1 % Triton X-100/10 mM dithiothreitol. The insoluble pellet was solubilized in 6 M guanidine buffer and purified using Ni-agarose (ProBond; Invitrogen) under denaturing condition. The 6xHis-tagged proteins were eluted using eluting buffer (300 mM imidazole, pH 4.0) containing 1 % Triton X-100. The eluted proteins were refolded by gradual dialysis against 10 mM Tris/HCl (pH 8.0) followed by chromatography with a HiLoad 16/60 Superdex 200 prep-grade column using a perfusion chromatography system (Perseptive Biosystems). The eluted proteins were analysed by 15 % SDS-PAGE under reducing conditions, followed by Coomassie blue staining.
Identification by mass spectrometry.
The purified F-2 and core proteins were prepared by reducing 15 % SDS-PAGE with Coomassie blue staining and sent to the Department of Biochemistry (University of Cambridge, UK) for matrix-assisted laser desorption/ionization fingerprinting and peptide fragmentation analysis by tandem mass spectrometry (MS/MS).
Western blot analysis.
The eluted proteins were separated by 15 % SDS-PAGE and transferred to nitrocellulose membrane at 20 V for 1 h. The membrane was treated with blocking buffer (Sigma) at 37 °C for 1 h and incubated with alkaline phosphatase-conjugated anti-V5 monoclonal antibody (Invitrogen) at 37 °C for another 1 h. The membrane was washed six times with PBS/0.05 % Tween 20 and incubated with an appropriate amount of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium substrate (Sigma) until the bands corresponding to the appropriate proteins became visible.
ELISA and titration of anti-F-2, anti-core and HCV antibodies.
Microplates (96-well) were coated with 100 µl purified F-2 or core protein (1 µg ml–1) in carbonate coating buffer (0.15 M sodium carbonate, 0.435 M sodium bicarbonate, 0.03 M sodium azide, pH 9.6) at 4 °C overnight. Plates were washed six times with PBS/0.5 % Tween 20 and blocked with 100 µl PBS/0.05 % Tween 20/4 % BSA per well at 37 °C for 1 h. Plates were then washed six times with PBS/0.5 % Tween 20. Each plasma sample was serially diluted threefold starting from 1 : 100 to give dilutions of 1 : 300, 1 : 900, 1 : 2700, 1 : 8100, 1 : 24 300 and 1 : 72 900 in PBS/0.05 % Tween 20/2 % BSA. Each dilution (100 µl) was added to the well and incubated at 37 °C for 1 h. Four HCV-negative plasma samples were diluted 1 : 100 and added as negative controls. After washing with PBS/0.5 % Tween 20, 100 µl 1 : 50 000-diluted horseradish peroxidase-conjugated anti-human whole IgG monoclonal antibody (Sigma) was added per well and incubated at 37 °C for 1 h. Plates were washed with PBS/0.5 % Tween 20 and 100 µl 3,3',5,5'-tetramethylbenzidine substrate was added per well, followed by incubation at room temperature in darkness for 20 min. The reaction was stopped by the addition of 100 µl 2 M sulfuric acid and the absorbance of each well was read at 450 nm. The cut-off was determined as the mean absorbance value of three anti-HCV and HCV RNA-negative controls plus 3 SD. The reactivity of each sample was expressed as a sample to cut-off ratio (S : CO) of the absorbance values obtained at a 1 : 100 dilution. The antibody titre was determined as the last dilution with an absorbance value above the cut-off. To determine the overall HCV antibody titre in plasma samples, samples were tested using an HCV 3.0 ELISA (Ortho Diagnostic Systems) with some modifications. The samples were serially diluted as 1 : 10, 1 : 100, 1 : 300, 1 : 900, 1 : 2700, 1 : 8100, 1 : 24 300 and 1 : 72 900 in the diluent provided, and 200 µl each dilution was added to a well and tested for anti-HCV antibodies according to the manufacturer's instructions.
Anti-F-2 ELISA after adsorption with core protein.
Microplates (96-well) coated with 100 µl purified F-2 (1 µg ml–1) were prepared as above. To determine the appropriate quantity of purified core protein required to adsorb the majority of anti-core antibody present in the plasma samples, 100 µl 1 : 100-diluted plasma samples from three donors with chronic infection were pre-incubated with 0, 1, 5, 10 and 20 µg purified core protein ml–1 before transfer to the wells. After the optimal blocking concentration has been determined, plasma samples from eight donors with chronic infection of mixed genotypes (C1–C8) and eight donors who had recovered from infection (R1–R8) were tested for specific anti-F-2 reactivity following pre-adsorption to block anti-core antibody.
ELISA against synthetic peptide of the F-2 protein.
Based on the theoretical hydrophobicity and antigenic index, a peptide (LASPGESQDTPGPC) corresponding to aa 72–85 of the F-2 protein was synthesized (Proimmune) and tested against antibodies in donor plasma samples. The peptide ELISA was similar to the previous assay using plates coated with F-2 or core protein, but with some modifications. The 96-well microplates were coated with 100 µl peptide (1 µg ml–1) at 4 °C overnight. Plates were washed five times with PBS/0.1 % Tween 20 and then blocked with 200 µl PBS/0.05 % Tween 20/4 % BSA per well at 37 °C for 1 h, followed by five washes with 200 µl PBS/0.1 % Tween 20 per well. A volume of 100 µl 1 : 100-diluted plasma samples from all donors was added to each well in duplicate and tested for anti-IgG as described above.
Statistical analysis.
All statistical analyses performed in this study were carried out using SPSS or Prism. The results were considered statistically significant when a two-tailed analysed P value was equal to or less than 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The partial nucleotide core sequences obtained from these 24 donors were analysed against reference sequences of both genotype 1 and 2 subtypes. It has been reported that the +1 frameshift for HCV genotype 1a occurs between nt 364 and 373, where 10 consecutive adenines are observed only in some HCV genotype 1 strains, but not in other genotype 1 subtypes. None of the genotype 2 references or the Ghanaian samples contained similar consecutive adenines, although a cluster of adenines was present in the region. Similar to other genotype 1 subtypes, all genotype 2 sequences contained a guanine at position 367 and a cytosine at position 373 instead of an adenine. In contrast, the ribosomal frameshift in genotype 1b was shown to take place at nucleotides corresponding to codon 42 and to return to the normal ORF at codon 144. To examine whether genotype 2 HCV has a similar sequence pattern at these positions, nt 451–480 of reference and Ghanaian samples were aligned as shown in Table 1. This region is relatively conserved in both genotype 1 and genotype 2 reference sequences except for a few nucleotides between nt 467 and 471. A thymine at position 467 was only observed in genotype subtype 1a, whilst subtypes 1b and 1c and genotype 2 possessed a cytosine in that position. This is the position (codon 44) where the frameshift was observed in genotype 1b. Furthermore, a guanine at nt 470 was present in both genotype 1b and genotype 2 sequences, whilst a guanine at nt 471 was unique to genotype 1c. Out of 24 Ghanaian genotype 2 HCV sequences, 11 had C467 and G470 whilst others had one of either C468 or A471. The exact elements in terms of sequences or secondary structure required for the frameshift initiation in different genotypes was not clearly defined. Based on the nucleotide homology observed at the site where the frameshift in genotype 1b takes place, it is possible that some genotype 2 strains may adopt the same mechanism for frameshift initiation. To examine further the influence of frameshifting on HCV genotype 2 strains, the partially +1 ORF translated sequences of the core from the Ghanaian donors were deduced. A stop codon appeared at codon 126 in genotype 2a. Other sequences of Ghanaian samples terminated at various positions including codons 141, 144, 147, 150, 153, 154 and 162.
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). In addition, the core with the +0 ORF from the same strain was expressed and purified using the same system. A recombinant F protein (F-2) with theoretical molecular mass of 23 287 Da and the corresponding core protein of 24 267 Da were obtained as shown by Coomassie blue staining (Fig. 1b). The identity of the two proteins was determined by detecting the V5 tag using anti-V5 antibody by Western blotting (Fig. 1c). Because the two proteins had very similar molecular masses on the gel, the single bands corresponding to the F-2 and core protein were each excised from the stained gels and tested by MS/MS analysis. Trypsin-digested oligopeptides from both proteins were ionized and detected according to molecular mass as shown in the mass spectra in Fig. 2. The recovered peptides represented by high signal intensity (major peaks) at particular mass-to-charge ratios were compared with the putative tryptic peptides from both proteins and the matched peptides are listed in Table 2. Given that the translated sequences of both proteins were available, the different peptide fragmentation profiles confirmed the corresponding purified bands as the F-2 and core proteins, respectively.
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. There was a significant difference in reactivity expressed as the S : CO ratio between donors with chronic infection and those who had recovered or controls (P<0.001). Although the recovered group seemed to have a slightly higher median than the controls, the difference was not significant. To examine the cross-reactivity of antibodies from individuals infected with other HCV genotypes, F-2 ELISA reactivity was tested in samples from donors chronically infected with HCV genotypes 1, 2 or 3. No significant difference in reactivity was found among genotypes (Fig. 3b). In parallel, the immunoreactivity against the core protein was examined in all three groups. Similar to the above results, a significant difference in reactivity was found between individuals with chronic and recovered infections or the control group (P<0.001), whilst no significant difference was found between recovered individuals and the control group (Fig. 3c). By comparing the anti-F-2 and anti-core results for each individual of a given genotype, HCV-infected genotype 1 and 2 donors both showed significant Pearson correlation coefficients between these two antibody reactivities (P<0.01). The correlation was less significant in patients infected with HCV genotype 3 (P<0.05).
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shows that individuals with chronic infection had significantly higher F-2 antibody titres than those who recovered from infection (P<0.0001): 72/82 chronically infected donors (87.8 %) had anti-F-2 antibody titres ranging from a 1 : 900 to 1 : 24 300 dilution, whilst 31/35 (88.6 %) of recovered donors had F-2 antibody titres ranging from 1 : 100 to 1 : 900. Likewise, a significant difference was found between the anti-core titres of the two groups (P<0.0001) (Fig. 4b): 77/82 chronically infected donors (93.9 %) had anti-core titres ranging from a 1 : 900 to 1 : 24 300 dilution, whilst 26/35 recovered individuals (74.3 %) had titres ranging from 1 : 100 to 1 : 300. Furthermore, the anti-HCV titre was examined in both groups using a commercial anti-HCV kit that detects anti-core, anti-NS3 and anti-NS5. Fig. 4(c) shows that the chronic infection group had a significantly higher anti-HCV titre than the recovered group (P<0.0001): 67/81 chronically infected donors (82.7 %) had anti-HCV titres between 1 : 900 and 1 : 8100 and 30/35 recovered donors (85.71 %) had HCV antibody titres of 1 : 100. These data demonstrated that the relatively higher anti-F-2 and anti-core reactivity in the donors with chronic HCV infection correlated well with anti-HCV level. To investigate further the relationship between anti-F-2, anti-core and anti-HCV titres in chronically infected donors, the correlation between titre values of all three antibodies in each individual was ranked and analysed by calculating Pearson correlation coefficients. Both genotype 1 and genotype 2 HCV-infected individuals showed significant correlation between anti-F-2 and anti-core titres (P<0.001) compared with genotype 3-infected individuals (P=0.155). No significant correlation between anti-F-2 and anti-HCV titres was obtained for all genotypes, but a significant correlation was obtained between anti-core and anti-HCV titres in genotype 2-infected donors (P<0.05).
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shows that the anti-F-2 response could be reduced substantially in donor samples with chronic infection by the absorption of anti-core antibody at a concentration of 1 µg core protein ml–1. No clear influence on the reactivity in plasma samples from recovered donors (IV–VI) was observed. Pre-incubation with >5 µg core protein ml–1 did not markedly modify the S : CO ratio compared with unblocked samples. The anti-core response was also tested after core protein absorption at various concentrations (Fig. 5b), showing a similar trend to anti-F-2 in Fig. 5(a), but with a greater S : CO reduction at 1 µg ml–1. With this concentration of core protein, all donor samples with chronic infection (I–III) showed a >50 % decrease in anti-core reactivity. It thus appeared that the anti-F-2 response observed previously was partially attributable to anti-core antibody, but substantial anti-F-2 reactivity remained. A concentration of 1 µg core protein ml–1 was determined as the appropriate concentration for testing samples with a wide range of anti-F-2 reactivity. To illustrate further the anti-F-2 reactivity in donors with chronic infection and recovery from HCV infection, samples from 16 donors with chronic infection (C1–C8) or who had recovered from infection (R1–R8) were tested for anti-F-2 and anti-core antibody after incubation with 1 µg core protein ml–1 (Fig. 5c). At this core protein concentration, some anti-core reactivity remained detectable after core protein absorption but at lower levels than anti-F-2 reactivity in both chronic and recovered infections. The F-2 reactivity of core-adsorbed plasmas remained higher in donors with chronic compared with recovered infection (P<0.01).
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). The level of reactivity to the F2 peptide was considerably lower than to the F-2 protein in both infected donor groups (Fig. 6b). However, using 31 Ghanaian anti-HCV-negative donor samples as controls, 19/82 donors (23.17 %) with chronic infections and 4/35 recovered donors (11.43 %) were truly reactive to the peptide (S : CO>1). The difference in reactivity was significantly higher in donors with chronic infection than in recovered donors (P<0.05).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Both references and most of our genotype 2 HCV strains shared the same nucleotides at the frameshift site with genotype 1b, but not with the well-studied genotype 1a. Therefore, a recombinant putative genotype 2 F protein based on this frameshift sequence feature along with core protein from the same strain were produced to assess their respective antigenicity. There is no direct evidence that the frameshift artificially introduced here exists in vivo. Attempts to express the F protein based on the frameshift sites observed in genotype 1a or in the common region shared by all genotype 2 F proteins have been undertaken. However, none of them was successful in our laboratory. The recombinant F protein constructed here contains sufficient amino acids and antigenic determinants in common with the natural F protein to be immunologically similar. It has been reported that the prevalence of anti-F antibodies in patients chronically infected with HCV ranges between 41.6 and 89 % using F protein generated from the +1 ORF of the genotype 1a core sequence (Cohen et al., 2007; Komurian-Pradel et al., 2004; Troesch et al., 2005). Sera from genotype 1a infections exhibit the lowest prevalence of reactivity. The frequency of reactivity is much less when using a truncated form of the protein (25 %). Four out of ten treated patients with cleared viraemia displayed an anti-F response, although the number of patients studied was small. From these studies, the prevalence of anti-core antibody ranges from 95 to 100 % in chronically infected patients when using whole core protein as antigen. However, none of these studies examined thoroughly the anti-F response in both chronically infected and recovered individuals, either because of lack of samples or because of an inadequate number of recovered patients.
The present study demonstrates the significantly higher level of anti-F antibodies in chronically infected patients compared with recovered individuals, irrespective of genotype. Both groups were previously confirmed to be seropositive by ELISA and Western blotting using three recombinant HCV proteins (Chuang et al., 2007). Most importantly, the reactivity pattern paralleled anti-core antibody in terms of both the S : CO ratio and antibody titres (Figs 3 and 4). It has been shown that the expression level of the F protein is considerably lower than that of the core protein in vivo (Fiorucci et al., 2007). Accordingly, one would anticipate a lower antibody level against the F protein than the core protein. However, this phenomenon was not observed using genotype 2 recombinant protein. The first 42 aa shared by both proteins might partially explain this result, as this region contains a major antigenic domain (Jackson et al., 1997; Park et al., 1999; Pereboeva et al., 1998). If the high anti-F-2 reactivity observed in donors with chronic infection was a result of anti-core antibodies targeting the shared antigenic domain, the blocking of anti-core antibodies by the addition of excess core protein in plasma samples would abolish the anti-F-2 response. However, the anti-F-2 reactivity remained detectable after sample pre-incubation with recombinant core protein (Fig. 5c). Various levels of anti-core reactivity remained detectable but were considerably lower than anti-F-2, suggesting that the blocking effect of the core protein was more effective against anti-core antibodies than against anti-F-2 antibodies, although this is not completely convincing. Studying the reactivity of samples from HCV-infected donors with a peptide unique to F-2 (Fig. 6b) provided additional evidence that there was specific anti-F-2 reactivity at a higher level and frequency in donors with chronic infection. It has also been shown that 42 % of the reactivity was observed when using a peptide of aa 43–141 of the HCV genotype 1b +1 ORF (Komurian-Pradel et al., 2004). These data strongly suggested that the observed high reactivity against F-2 protein in plasma samples from HCV-infected individuals is unlikely to be accounted for totally by the antigenic domains shared with the core protein. By using whole protein instead of a truncated form or linear peptides, antigenic determinants, including conformational epitopes, may mimic more closely those involved in the natural humoral response if the double frameshift is responsible for F protein expression during HCV infection of particular genotypes such as 1b or 2. Furthermore, the antibody titre against each recombinant protein and the commercial test demonstrated that the level of anti-F-2 antibodies correlated with anti-core and anti-HCV antibody levels. The anti-core titre of donors (Fig. 4b) in this study confirmed the data reported by Nikolaeva et al. (2002), who showed a range of 1 : 800–1 : 40 000 of anti-core IgG titres in chronic HCV and an antibody titre value of 1 : 5–1 : 200 in a recovered group. Because of the lack of variation of high anti-HCV reactivity in donors chronically infected with different genotypes using the HCV 3.0 ELISA, it was unlikely that a reliable correlation between anti-F and anti-HCV could be found in terms of the S : CO ratio, even though a 4–4.5-fold greater seroreactivity of genotype 1 samples compared with genotype 2 or 3 was reported using a commercial assay (Dhaliwal et al., 1996). However, the higher correlation between anti-F and anti-core antibody levels in genotypes 1 and 2 than in genotype 3 suggest that the F protein produced along with core protein during natural HCV infection is cross-reactive but potentially genotype-specific. The same correlation was observed in antibody titres. These data demonstrated that the genotype-specific reactivity of the F protein could be demonstrated by methods other than sequence variation and the direct measurement of S : CO ratio in the sera of HCV-infected patients, at least between genotypes 1/2 and 3.
It is known that the F protein of HCV genotype 1a can be detected in serum samples of a few patients infected with different genotypes using either whole or a truncated protein (Komurian-Pradel et al., 2004). The monoclonal antibody generated from a genotype 1a F protein was able specifically to detect a protein of 17 kDa in cells transfected with genotype 1b core protein-coding sequence (Fiorucci et al., 2007). In this study, we did not find a significant difference in antibody reactivity against F-2 protein among genotypes 1, 2 and 3, which accounted for the majority of our samples. This might be due to the functional constraints of the F protein in natural infection, even though its function remains unclear. To date, no homologous function of the F protein has been identified except for an interaction with prefoldin (PFD) 2, a subunit of the PFD complex, resulting in some disturbance of the tubulin cytoskeleton organization (Tsao et al., 2006). It has also been suggested that the F protein could serve as a modulator to prevent cytolysis of the host cells. However, no clinical evidence supporting this hypothesis was available. Also, no physical interaction with any other HCV proteins has been found, even though the F protein displays a subcellular localization similar to the core and NS5A in the endoplasmic reticulum membrane of infected Huh7 cells (Xu et al., 2003). It has been reported that genotype 2 and 3 strains terminate at a more variable position than genotype 1 in the +1 ORF sequence of the core, and the pattern of divergence in the encoded F protein was suggested to be unaffected by the functional constraint (Cristina et al., 2005).
Although the ribosomal frameshift of HCV seems to take place at different locations in different genotypes, at least in subtypes 1a and 1b, the cross-genotype reactivity indicates that the common central domain of the F protein is conserved and recognized by antibody in infected individuals in a conformational manner rather than as linear sequences that are constrained by sequence diversity. Depending on the expression construct, viral strain and experimental design, the genotype specificity of the F protein might be further revealed in relation to other host or viral proteins.
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ACKNOWLEDGEMENTS |
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Received 21 December 2007;
accepted 18 April 2008.
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