Full-length open reading frame of a recombinant hepatitis C virus strain from St Petersburg: proposed mechanism for its formation

Abstract

The full-length ORFs for the hepatitis C virus recombinant RF1_2k/1b (N687) and the non-recombinant 1b strain N589 were sequenced. A single recombination point was found and the sizes of the genes (C, E1, E2, p7, NS2, NS3, NS4 and NS5) were according to the parental subtypes. The PKR-eIF2a phosphorylation site homology domain sequence of the E2 protein was identical to those of genotype 2 strains, while the IFN-a-sensitivity-determining region of the NS5A protein was identical to those of interferon-resistant 1b strains. For the parental strains, two hairpin structures, HS1 and HS2, were predicted for the plus-strand up-and downstream of the crossover site, which were not present in the recombinant strain. HS2 shared similarity with the motif1 hairpin of turnip crinkle virus RNA that binds to the RNA-dependent RNA polymerase and facilitates 39-terminal extension during recombination. This study suggests that RF1_2k/1b has emerged by homologous recombination during minus-strand synthesis via template switching because of constraints imposed by the HS1 hairpin of the 39-parental genome.Hepatitis C virus (HCV) is an RNA virus belonging to the family Flaviviridae. It has a single-stranded plus-sense genome of approximately 9?6 kb with a single open reading frame (ORF) and three structural (C, E1 and E2) and seven non-structural genes (p7, NS2, NS3, NS4A, NS4b, NS5A and NS5B) (de Francesco, 1999). HCV exhibits a high genetic variability, enabling its classification into four hierarchical strata: clades, genotypes, subtypes and isolates (Robertson et al., 1998;Simmonds, 1999). A high mutation rate is considered to be a major factor behind its genetic diversity (Simmonds, 1999(Simmonds, , 2001Farci & Purcell, 2000). Recombination provides another mechanism for creating genetic variation and is known to be widespread among nonsegmented RNA viruses, including other members of Flaviviridae (Lai, 1992;Figlerowicz et al., 1997;Worobey et al., 1999;Guillot et al., 2000;Twiddy & Holmes, 2003). Some viruses, such as coronaviruses, bromoviruses and carmoviruses, for example turnip crinkle virus (TCV), may undergo both homologous and non-homologous recombination, while some non-segmented plus-strand RNA viruses, such as picornaviruses, may only undergo homologous recombination (Lai, 1992). Recombination was not recognized as a likely mechanism for creating diversity of HCV until our recent finding of a spontaneous HCV recombinant in St Petersburg, RF1_2k/1b (Kalinina et al., 2002).The virus genotype is one factor influencing the outcome of interferon (IFN) therapy (Zein, 2000). Infections with genotype 1 are more resistant to IFN therapy than infections with genotypes 2 and 3 (Taylor et al., 2000). Two domains, the PePHD (PKR-eIF2a phosphorylation site homology domain) in the E2 protein and the ISDR (IFNa-sensitivity-determining region) in the NS5A protein, are claimed to mediate IFN resistance through inhibition of the PKR (an IFN-a-induced, double-stranded RNA-activated protein kinase), although the importance of the ISDR is s...

Efficient Rescue of Hepatitis C Virus RNA Replication bytrans-Complementation with Nonstructural Protein 5A

“…Surprisingly, in no case was RNA recombination observed, although naturally occurring recombinant HCV isolates have been described (10,11,27). Very recently, a model was proposed in which homologous recombination occurs during minus-strand RNA synthesis by template switching of the polymerase that is facilitated by conserved hairpin structures (26). For members of the Picornaviridae family, recombination is a well-known phenomenon.…”

Hepatitis C virus genetic variability and evolution

“…An apparent hotspot has been identified between amino acids 1022 and 1042 (corresponding to the vicinity of the NS2/NS3 junction). Considering how short this region seems to be, and despite the existence of only a few reports inquiring into RNA secondary structures involved in the recombination process [130,139] , all seems to indicate that a copy choice mechanism might be responsible for the generation of these recombinant forms [49] . With respects to intragenotypic recombination, nine recombinant forms have been described.…”

Complete genomic sequences for hepatitis C virus subtypes 4b, 4c, 4d, 4g, 4k, 4l, 4m, 4n, 4o, 4p, 4q, 4r and 4t

“…Recombinants of different HCV genotypes and subtypes have been described in patients from Russia (2k/1b) (Kalinina et al, 2002(Kalinina et al, , 2004, Peru (1a/1b) (Colina et al, 2004), France (2k/5) and Vietnam (2i/6p) (Noppornpanth et al, 2006). In order to exclude such a possibility, pair-wise nucleotide similarity curves were plotted along HCV genomes.…”

“…Bootstrap resampling was performed using 500 neighbour-joining replicates. To exclude recent virus recombination events (Kalinina et al, 2002(Kalinina et al, , 2004Colina et al, 2004;Legrand-Abravanel et al, 2007;Noppornpanth et al, 2006), the RDP3 software was run with default settings modified as follows: (i) window size was 40 nt; (ii) the option of linear sequences was chosen; (iii) six different methods (RDP, GENECONV, MaxChi, Bootscan, Chimaera and SiScan) were run simultaneously against the sequences listed below; and (iv) only the events detected by more than two of the above methods were considered positive. For pair-wise comparison and detection of HCV recombination, the following 39 complete HCV genomic sequences (subtypes) were co-analysed: GenBank accession no.…”