Expression of alternate reading frame protein (F1) of hepatitis C virus in Escherichia coli and detection of antibodies for F1 in Indian patients.

Apart from the core (21kD), a novel hepatitis C virus (HCV) frame shift protein (F1) is synthesized from the initiation codon of the polyprotein sequence followed by ribosomal frame shift into the -2/+1 reading frame. To date, no information is available on F1 protein of Indian isolates, and hence detection of antibodies for F1 protein in Indian patients assumes great relevance. Specific primers have been designed to amplify sequence coding for 120aa of truncated F1 (tF1). The amplified tF1 has been cloned in bacterial expression vector, pET21b for expression in Escherichia coli. Partially purified expressed protein has been subjected to western blot analysis using patients' sera. Three HCV positive sera employed in western analysis showed positive signals to tF1, while sera from uninfected individuals failed to give any signals. Further, results of western blots, carried out with patients sera titrated with purified core protein, confirmed the presence of antibodies specific to F1. The positive signal observed for F1 in western analysis with HCV infected sera suggests that F1 protein is synthesized in the natural course of HCV infection in Indian patients as well. Presence of antibodies against F1 protein of subtype 1c has been demonstrated, for the first time, in Indian patients.

Introduction

Hepatitis C virus (HCV) is the major causative agent of post-transfusion and parenterally transmitted, non-A, non-B hepatitis throughout the world (Alter and Seeff, 2000). HCV is an enveloped RNA virus that is classified in the family Flaviviridae (Robertson et al., 1998). HCV has high genomic variability and at least six different genotypes and an increasing number of subtypes have been reported (Simmonds, 1999). The genome of HCV comprises a single stranded positive-sense RNA of ∼9.6 kb in length and contains a single open reading frame (ORF) that encodes for a non-functional polyprotein of about 3000 aminoacids (Grakoui et al., 1993). This polyprotein is cleaved co- and post-translationally by cellular and viral proteases to yield 10 different functional proteins. Structural proteins are the major components of the mature virion, which are coded by the 5′ quarter of the ORF and arranged as C–E1–E2 and p7, while the non-structural proteins are coded by the 3′ three-quarters of the ORF in the order NS2, NS3, NS4A, NS4B, NS5A and NS5B (Barbara and Contreras, 1991); these proteins are involved in polyprotein processing and replicative functions of the virus (Suzuki et al., 1999, Suzuki et al., 2007). Translation of the HCV polyprotein sequence was reported to be regulated by a cap-independent mechanism that requires most of the 5′-non-coding region and the first nine codons of the polyprotein-coding sequence to serve as the internal ribosomal entry sequence (IRES) (Rijnbrand and Lemon, 2000). Initial expression studies indicated that, besides the core protein (21 kD), another protein (17 kD) also expressed from the same core protein coding sequence both in vitro and in mammalian cells and it was thought to be a truncated core protein (Lo et al., 1994, Lo et al., 1995, Basu et al., 2004). Recent observation, in members of family Flaviviridae, revealed that a novel translation mechanism of a ribosomal frame shift exists within the capsid-encoding region, which results in a frame shift protein (Varaklioti et al., 2002, Walewski et al., 2001, Xu et al., 2001). The frame shift protein was named as F1 or ARFP (alternative ribosomal frame shift protein) based on translation initiated at a non-AUG codons in a −2/+1 reading frame relative to the polyprotein of HCV (Baril and Brakier-Gingras, 2005). Similar synthesis of capsid protein via frame shift was also observed in various other viruses such as SARS-CoV (Baranov et al., 2005).

It was first demonstrated that the 17 kD protein was synthesized by ribosomal frame shift and was mostly derived from the coding sequence that overlaps the HCV core protein reading frame (Xu et al., 2001, Choi et al., 2003). The expressed F1 protein was localized in the cytoplasm of HepG2 cells, with a notable perinuclear localization (Roussel et al., 2003), and was found to be associated with the endoplasmic reticulum (Xu et al., 2003). This subcellular localization of HCV F1 protein is similar to that of the HCV core and NS5A proteins, raising the hypothesis that the F1 protein may participate in HCV morphogenesis or replication (Xu et al., 2003). In addition, sera from patients who were positive for HCV genotype 1a or 1b were shown to react differently to synthetic peptides of F1 (Boulant et al., 2003). The present study mainly deals with the cloning and expression of F1 coding sequence of the HCV Indian isolate belonging to genotype 1c. Further, antibodies against F1 protein have been detected in Indian patients. The recombinant plasmid containing HCV core coding sequence, belonging to genotype 1c (GenBank Acc. No. AY051292), was used as template for amplification of truncated F1 (tF1) coding sequence employing F1F 5′ATT CAT ATG GCA CGA ATC CTA AAC C 3′ and F1R 5′ ATT AAG CTT ACC CAA ATT GCG TGA CCT GC 3′ as forward and reverse primers, respectively. The PCR amplification was performed using conditions of 94 °C/30 s, 52 °C/45 s, 72 °C/1 min for 35 cycles and a final extension of 72 °C/5 min. PCR product was gel purified and subjected to restriction digestion using NdeI and HindIII and subsequently cloned at same sites of pET21b. The pET21b-tF1 was subjected to automated DNA sequencing. E. coli BL21(DE3) competent cells were transformed with pET21b-tF1 to carryout the expression studies. Western blot analysis was carried out using three positive (anti-HCV and HCV RNA positive) and three negative sera (anti-HCV and HCV RNA negative). Westerns were also performed employing patients’ sera titrated with purified core protein. The deduced aminoacid sequences in +1 reading frame of the standard reference set representing various genotypes were utilized in multiple alignment of F1 sequences. The phylogenetic tree was generated based on alignment using clustalW program (http://swift.embl-heildelberg.de).

Analyses of F1 coding −2/+1 reading frame indicated a protein product of 142aa in the Indian isolate AY051292 belonging to genotype 1c. Different genotypes of HCV were reported to code for varied lengths of F1—the genotype 1a encoded 162aa, 1b coded for 144aa and 2a coded for 126aa (Kolykhalov et al., 1997, Lohmann et al., 1999, Yanagi et al., 1999, Xu et al., 2001). PCR amplification product of ∼370 bp region coding for truncated F1 (tF1) was cloned at NdeI and HindIII sites of pET21b. The clone pET21b-tF1 released a fragment of ∼370 bp upon double digestion with NdeI and HindIII.

The clone having the tF1 insert when subjected to sequencing revealed the presence of F1 coding sequence (Fig. 1 ). The deduced aa of tF1 sequence subjected to BLAST search exhibited domain based identity of 74–77% with POCO45, a core frame shift product of HCV isolate H belonging to type 1a. Sequence alignment of deduced aa of F1 belonging to different genotypes displayed substantial diversity in F1 sequences. Despite these variations, presence of various conserved aa clusters in F1 indicates the conserved nature of its secondary structure among isolates. Phylogenetic analysis of F1 showed close clustering of sequences belonging to various subtypes of specific genotype (Fig. 2 ) implicating that F1 sequences are genotype specific. Motif search analysis of F1 revealed the presence of Caesin Kinase 2-phosphorylation site; Protein Kinase C-phosphorylation site and LDL class B (LDLRB) receptor binding site. The function of F1 protein in the life cycle of HCV remains unknown (Baril and Brakier-Gingras, 2005). Presence of a binding site for LDLRB indicates the possibility of interaction of F1 with lipids in the natural course of infection. The protein expressed upon IPTG induction yielded ∼17 kD band which was absent in un-induced samples (Fig. 3A). The expressed protein tF1 was in the insoluble fraction as inclusion bodies. Inclusion bodies were purified by washing the pellet after lysis using 0.1% Triton-X100 and subsequently the pellet was dissolved in phosphate buffer (pH 8.0) containing 0.5% sodium laural sarcosine (SLS). The partially purified tF1 was employed in western blot analyses. Three HCV positive sera employed in western analysis showed the presence of antibodies to tF1, while sera from uninfected individuals failed to give any signals (Fig. 3B). Similar results were observed with patients’ sera titrated with purified core protein (Fig. 3C). Purified core protein was electro-transferred on to nitrocellulose membrane from SDS-PAGE. Several strips of the membrane containing purified core protein were used to titrate out anti-core antibodies in three different positive sera. Finally a western blot without a signal for purified core protein, when titrated sera were used, confirmed the absence of anti-core antibodies. The positive signal observed for F1 in western blot analysis with HCV infected sera and its absence with uninfected sera suggests that F1 protein is plausibly synthesized in the natural course of HCV infection in Indian patients as well.

Fig. 1

Nucleotide sequence of clone pET21b-tF1 with deduced aminoacids. The deduced aminoacid sequence of F1 is represented in red and the sequence in black indicate the aminoacids derived from pET21B plasmid. The sequence in bold represents the sequence of cloning sites.

Fig. 2

Phylogenetic analysis based on deduced aminoacid sequences of F1 of various genotypes of HCV. The phylogenetic analysis was done using ClustalW and the tree was constructed using TreeView program. Scale bar shows number of nucleotide substitutions per site.

Fig. 3

Expression and western blot analysis of tF1 protein of HCV. (A) Expression of tF1. M, protein marker. Lane 1, pET21b-tF1 before IPTG induction. Lane 2, pET21b-tF1 after IPTG induction showing ∼17 kD induced protein. (B) Western blot analysis of tF1. 1P, 2P, 3P: Three different positive sera were used as primary antibody. 1N, 2N, 3N: Three different negative sera were used as negative controls. (C) Western blot analysis of tF1 using patients’ sera titrated with purified C120. 1P, 2P, 3P: Three different positive sera used as primary antibody after titration with purified core protein. N represents negative sera and P for positive sera.

An overview of the results amply indicates that F1 protein is also synthesized in the natural course of HCV in Indian patients. Phylogenetic analysis of F1 of various HCV isolates revealed that aminoacid sequences of F1 are genotype specific. Establishment of the presence of antibodies to F1, in this investigation, emphasizes the need for further studies dealing with the role of F1 in HCV pathogenesis.