The HCV core gene is more conserved than is necessary to conserve the encoded protein sequence 
, suggesting other functions for the virus RNA. Indeed, a second protein encoded by the core region of the genome has been discovered, designated here as core+1/ARFP (for review, see 
). The role of core+1/ARFP in the virus cycle and in the pathogenesis of HCV infection remains obscure, and this protein is not required either for virus replication or for production of infectious HCVcc (JFH1) in cell culture systems 
. Nevertheless, several studies have provided indirect evidence that core+1/ARFP is effectively produced during the natural HCV infection 
In this study we have investigated the presence of anti-core and anti-core+1/ARFP antibodies in plasma samples from HCV-infected individuals living in Cambodia. Anti-core+1/ARFP antibodies were detected in 8/58 serum samples; 5 individuals were infected with genotype 6 HCV and 3 with genotype 1a. Generally, the levels of anti-core+1/ARFP antibodies (P/N values) were relatively low, and in six such cases anti-core antibody responses were normal. However, 3 patients infected with genotype 1a HCV developed exceptionally strong anti-core+1/ARFP responses and had no detectable antibodies directed towards the N-terminus of core.
Analyses of the HCV RNA from these patients showed that the entire core region was present and was functional, since the cloning and expression of this RNA resulted in the production of full-length core protein in mammalian cells. Also, the 5′UTR of these isolates was intact, excluding the possibility of abnormal expression of the HCV polyprotein (). Furthermore, analyses of the amino-acid sequence of core protein predicted from its gene sequence did not show any specific changes that could explain the unusual serological profile. The deduced core+1/ARFP amino-acid sequence in the +1 frame was extremely conserved between the three patients, but was considerably different from that of the prototype 1a HCV or from the sequence seen in other Cambodian patients infected with genotype 1a HCV.
Nucleotide sequence alignment of the cDNA sequence that corresponds to the 5′UTR region from patient p34, negative for anti-core and positive for anti-core+1/ARFP antibodies.
Interestingly, the absence of “normal” anti-core antibodies in 3 patients correlated with several identical synonymous mutations in the core gene. Most of these (7/10) were positioned in the region aa (99–124), encoding the so called “short form” of core+1/ARFP 
. There is increasing evidence that “silent” mutations that do not alter the amino-acid composition of a gene product can affect transcription, splicing, or mRNA transport. The synonymous mutations do not alter mRNA levels, but they could influence RNA stability and change the rate of protein synthesis and thus cause a protein to fold into a alternative structure 
. Thus, they would have phenotypic effects with clinically important consequences 
Prediction analyses showed that the mutations in the core gene induce conformational changes in the internal loop of SL248 and in the apical loop of SL332 of HCV RNA, compared to HCV-1a RNA from patients with a normal anti-core response. Since SL248 contains codons 85–87 implicated in internal initiation of translation and production of the core+1/ARFP 
, such conformational changes could affect translation initiation within the core-coding region and favour the synthesis of the core+1/ARFP. The analyses of cloned HCV RNA also showed that although the nucleotide sequence spanning codons 85–87 was not altered, nucleotide changes at position 317 (CC to AT) resulted in a conformational change of the RNA structure that shifts AUG-85 from a stem into an internal loop. Such a change may favour the initiation of translation at position 85. Indeed, we detected an increased level of core+1/ARFP-LUC expression in transiently transfected mammalian cells when the sequence contained these mutations ().
Our findings suggest that the absence of detectable anti-core antibodies, and the production of a strong anti-core+1/ARFP response, can be related to specific nucleotide motifs or mutations in the core and ARFP/core+1-coding region. Thus, in patients harbouring RNA with such motifs/mutations, efficient internal translation initiation at AUG-85 probably leads to the production of higher levels of core+1/ARFP. Core+1/ARFP is considered to be a short–lived and very labile protein 
, thus it can also be hypothesized that core+1/ARFP bearing these alterations would be of increased stability in the core-antibody negative patients.
It is noteworthy that, although core+1/ARFP is conserved amongst different HCV genotypes, the length of core+1/ARFP appears to be genotype-specific: by database analyses the longest form of core+1/ARFP has been deduced to be that of the 1a genotype 
. In agreement with this, the three Cambodian patients in question harboured genotype 1a HCV.
In our study, cDNA from these patients induced the synthesis of the full-length 21 kDA core protein upon cloning and expression in mammalian cells (). In addition, different concentrations of HCV core antigen could be detected in plasma samples from each of the three patients. Thus, the HCV variants (p31, p32, p34) identified in this study are not deficient in core protein production, but the patients rather induce the unusual pattern of the immune response. Indeed, the patients with distinctive (and identical) mutations in the core gene sequence seem to produce anti-core antibodies directed to the less immunogenic, C-terminal part of the protein. The question remains as to the reason of production of such unusual anti-core antibodies in the three Cambodian patients. One possibility is that these patients could produce increased levels of so-called “mini-cores”, as shown in studies in vitro 
. Indeed, the synthesis of short forms of core (8–14 kDa) that contain the C-terminal portion of the protein was demonstrated for several infectious HCV strains that replicate in vitro
in hepatoma cell lines 
. These “mini-cores” are synthesized by internal translation initiation from initiator codons in close proximity to AUG-85, the main initiation codon for core+1/ARFP. Although they lack the RNA-binding region and immunodominant core epitopes, the “mini-cores” seem to be important functional components of the HCV protein repertoire, as they might mediate some biological functions of core, such as binding to lipid droplets 
. The viruses that produce mini-cores (genotypes 1 and 2) also express larger core proteins that could play a role in particle formation 
. The core assay involves antibodies that recognise epitopes in both N-and C-terminal parts of core. Thus it can potentially detect both the full-length and mini-core proteins. It is tempting to speculate that « mini-core » and core+1/ARFP may share common regulatory elements for internal translation initiation and therefore there could be a link between “mini-core” and core+1/ARFP production in vivo
In conclusion, we provide further evidence for the synthesis of core+1/ARFP protein during natural HCV infection. We also demonstrate that unusually high levels of anti-ARFP antibodies can be linked to a lack of normal anti-core antibody responses. The synthesis of core+1/ARFP in such patients might be favoured by synonymous mutations in the core coding sequence, situated close to AUG-85, which induce conformational changes in SL248 and SL337. Thus higher levels of core+1/ARFP production may be linked to the properties of the mutated HCV core. Since HCV core protein is thought to play a major role in HCV pathogenesis, it would be worthy to determine whether negative result in anti-core antibody assay (and/or increased levels of anti-core+1/ARFP) could identify patients harbouring mutated core sequences, and whether this unusual serological pattern is associated with a particular pathology during HCV infection.