Pietschmann et al. 10.1073/pnas.0504877103. |
Supporting Table 1
Supporting Text
Table 1. Chimeric HCV genomes
Chimera | Fused genome segments* | GTs | Peak titer (TCID50/ml +/-SD) |
Luc-Con1/C1 | 1-746(Con1)/751-3034(JFH1) | 1b/2a | Not determined |
Luc-Con1/C2 | 1-809(Con1)/814-3034(JFH1) | 1b/2a | Not determined |
Luc-Con1/C3 | 1-842(Con1)/847-3034(JFH1) | 1b/2a | Not determined |
Luc-Con1/C4 | 1-871(Con1)/876-3034(JFH1) | 1b/2a | Not determined |
Luc-Con1/C5 | 1-949(Con1)/954-3034(JFH1) | 1b/2a | Not determined |
Luc-Con1/C6 | 1-1026(Con1)/1031-3034 | 1b/2a | Not determined |
Con1/C3 | 1-842(Con1)/847-3034(JFH1) | 1b/2a | 1,404 +/- 462 |
Con1/C6 | 1-1026(Con1)/1031-3034 | 1b/2a | 5 +/- 2 |
H77/C3 | 1-842(H77)/847-3034(JFH1) | 1a/2a | 187 +/- 80 |
H77/C6 | 1-1026(H77)/1031-3034(JFH1) | 1a/2a | 3 +/- 1 |
J6/C3 | 1-846(J6CF)/847-3034(JFH1) | 2a/2a | 314,731 +/- 111,880 |
J6/C6 | 1-1030(J6CF)/1031-3034(JFH1) | 2a/2a | 93,903 +/- 40,241 |
452/C3 | 1-848(452)/ 847-3034(JFH1) | 3a/2a | 41 +/- 18 |
452/C6 | 1-1032(452/1031-3034(JFH1) | 3a/2a | 102 +/- 35 |
* Amino acid positions of the fused polyprotein fragments; HCV isolates are given in parentheses.
Titers of infectious HCV present in supernatant of transfected cells at the time of maximum virus release; values were determined by TCID50 assay.
The J6/C3 construct represents the most efficient chimera identified in this study and was given the short name Jc1.Supporting Text
Chimeric HCV Genomes.
Construct pFK-Con1/C6 carries a chimeric HCV ORF flanked by the 5' and 3' nontranslated regions (NTRs) of the JFH1 isolate. Codons 1-1026 of the Con1 isolate encompassing the entire Core, E1, E2, p7, and NS2 coding region are fused in-frame to codons 1031-3034 of the JFH1 isolate (GenBank accession no. AB047639). For cloning purposes, a unique FseI site was introduced 33 nt upstream of the NS2/3 cleavage site by silent mutagenesis. To allow simple generation of in vitro transcripts with authentic 3' ends, the 3' NTR of this and all following constructs is flanked by the coding sequence of the genomic ribozyme derived from hepatitis delta virus. Plasmid pFK-J6/C6 encodes a chimera consisting of codons 1-1030 of the J6/CF isolate (1), GenBank accession no. AF177036) and codons 1031-3034 of JFH1, whereas in pFK-J6/C3 codons 1-846 of J6/CF are combined with codons 847-3033 of JFH1. This construct yields the highest virus titers, and the chimeric viruses were designated Jc1. The mapping of the optimal crossover point for Con1/JFH1 chimeras was performed in the context of bicistronic reporter virus genomes consisting of JFH1 nucleotide residues 1-389 encompassing the entire 5' NTR and the coding region of the 16 amino-terminal residues of core, fused in-frame with the coding sequence of firefly luciferase. The second cistron downstream of the reporter is expressed by an internal ribosome entry sitederived from the Encephalomyocarditis virus and encodes different chimeric ORFs, flanked at the 3' end by the JFH1 3' NTR. pFK-Luc-Con1/C1, pFK-Luc-Con1/C2, pFK-Luc-Con1/C3, pFK-Luc-Con1/C4, pFK-Luc-Con1/C5, and pFK-Luc-Con1/C6 contain Con1-codons 1-746, 1-809, 1-842, 1-871, 1-949, and 1-1026 fused with codons 751, 814, 847, 876, 954, and 1031, respectively, up to the end of the JFH1 genome. Plasmids pFK-Con1/C3 and pFK-Con1/C6 encode the respective chimeric genomes without reporter gene. In the cases of pFK-452/C3 and pFK-452/C6, codons 1-848 and 1-1032 of the genotype 3 isolate HCV-452 (see below) are attached to JFH1-codons 847 and 1031, respectively, and pFK-H77/C3 and pFK-H77/C6 harbor H77-codons 1-842 and 1-1026 connected with JFH1 codons 847 and 1031, respectively. Sequence information of the described plasmids is available on request.
Construction of the Genotype 3a Consensus Genome HCV-452.
A specimen of liver tissue was obtained from a patient infected with a GT3 virus. Collection of the sample had been approved by the local ethical committee, and written consent had been obtained from the donor. Total liver RNA was extracted by using TRIZOL BD (Invitrogen). After reverse transcription cDNAs were amplified by PCR using Expand High Fidelity PCR System Polymerase and Expand long template PCR system (Roche Applied Science). The HCV cDNA corresponding to the complete ORF was then amplified in three fragments. For each fragment at least five clones were sequenced and a consensus sequence was established. Using appropriate restriction enzymes and standard protocols, we assembled fragments covering the complete ORF in PGEMT Easy vector (Promega). The 5' UTR was amplified with a 5' RACE kit (Invitrogen), and it was introduced into the vector containing the ORF consensus sequence. The 3' NTR was obtained by using a primer corresponding to the 29 last nt of the HCV genome (2). Because the 3' UTR shows a high degree of variability within the poly(U/C) region, one cDNA with an intermediate length and U/C content was ligated with the partially assembled genome by using standard cloning techniques. The resulting GT3a consensus genome was designated HCV-452. The nucleotide sequence of this genome has been deposited in GenBank and can be retrieved under accession number DQ437509.1. Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. (1999) Virology 262, 250-263.
2. Kolykhalov, A. A., Feinstone, S. M. & Rice, C. M. (1996) J. Virol. 70, 3363-3371.