Productive homologous and non-homologous recombination of hepatitis C virus in cell culture
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Productive homologous and non-homologous recombination of hepatitis C virus in cell culture. / Scheel, Troels K H; Galli, Andrea; Li, Yi-Ping; Mikkelsen, Lotte S; Gottwein, Judith M; Bukh, Jens.
In: P L o S Pathogens, Vol. 9, No. 3, e1003228, 03.2013, p. 1-12.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - Productive homologous and non-homologous recombination of hepatitis C virus in cell culture
AU - Scheel, Troels K H
AU - Galli, Andrea
AU - Li, Yi-Ping
AU - Mikkelsen, Lotte S
AU - Gottwein, Judith M
AU - Bukh, Jens
PY - 2013/3
Y1 - 2013/3
N2 - Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13-36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5' end to the NS2-NS3 region followed by JFH1 sequence from Core to the 3' end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients.
AB - Genetic recombination is an important mechanism for increasing diversity of RNA viruses, and constitutes a viral escape mechanism to host immune responses and to treatment with antiviral compounds. Although rare, epidemiologically important hepatitis C virus (HCV) recombinants have been reported. In addition, recombination is an important regulatory mechanism of cytopathogenicity for the related pestiviruses. Here we describe recombination of HCV RNA in cell culture leading to production of infectious virus. Initially, hepatoma cells were co-transfected with a replicating JFH1ΔE1E2 genome (genotype 2a) lacking functional envelope genes and strain J6 (2a), which has functional envelope genes but does not replicate in culture. After an initial decrease in the number of HCV positive cells, infection spread after 13-36 days. Sequencing of recovered viruses revealed non-homologous recombinants with J6 sequence from the 5' end to the NS2-NS3 region followed by JFH1 sequence from Core to the 3' end. These recombinants carried duplicated sequence of up to 2400 nucleotides. HCV replication was not required for recombination, as recombinants were observed in most experiments even when two replication incompetent genomes were co-transfected. Reverse genetic studies verified the viability of representative recombinants. After serial passage, subsequent recombination events reducing or eliminating the duplicated region were observed for some but not all recombinants. Furthermore, we found that inter-genotypic recombination could occur, but at a lower frequency than intra-genotypic recombination. Productive recombination of attenuated HCV genomes depended on expression of all HCV proteins and tolerated duplicated sequence. In general, no strong site specificity was observed. Non-homologous recombination was observed in most cases, while few homologous events were identified. A better understanding of HCV recombination could help identification of natural recombinants and thereby lead to improved therapy. Our findings suggest mechanisms for occurrence of recombinants observed in patients.
KW - Base Sequence
KW - Carcinoma, Hepatocellular
KW - Cell Line, Tumor
KW - Genome, Viral
KW - Hepacivirus
KW - Hepatocytes
KW - Homologous Recombination
KW - Humans
KW - Molecular Sequence Data
KW - RNA, Viral
KW - Sequence Analysis, RNA
KW - Transfection
KW - Virus Replication
U2 - 10.1371/journal.ppat.1003228
DO - 10.1371/journal.ppat.1003228
M3 - Journal article
C2 - 23555245
VL - 9
SP - 1
EP - 12
JO - P L o S Pathogens
JF - P L o S Pathogens
SN - 1553-7366
IS - 3
M1 - e1003228
ER -
ID: 122663240