Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.

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...

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Autores principales:	Troels K H Scheel, Andrea Galli, Yi-Ping Li, Lotte S Mikkelsen, Judith M Gottwein, Jens Bukh
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/62ceabffd31041f089cd5936d2865f60
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spelling	oai:doaj.org-article:62ceabffd31041f089cd5936d2865f602021-11-18T06:05:52ZProductive homologous and non-homologous recombination of hepatitis C virus in cell culture.1553-73661553-737410.1371/journal.ppat.1003228https://doaj.org/article/62ceabffd31041f089cd5936d2865f602013-03-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/23555245/pdf/?tool=EBIhttps://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374Genetic 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.Troels K H ScheelAndrea GalliYi-Ping LiLotte S MikkelsenJudith M GottweinJens BukhPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 9, Iss 3, p e1003228 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Troels K H Scheel Andrea Galli Yi-Ping Li Lotte S Mikkelsen Judith M Gottwein Jens Bukh Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.
description	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.
format	article
author	Troels K H Scheel Andrea Galli Yi-Ping Li Lotte S Mikkelsen Judith M Gottwein Jens Bukh
author_facet	Troels K H Scheel Andrea Galli Yi-Ping Li Lotte S Mikkelsen Judith M Gottwein Jens Bukh
author_sort	Troels K H Scheel
title	Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.
title_short	Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.
title_full	Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.
title_fullStr	Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.
title_full_unstemmed	Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.
title_sort	productive homologous and non-homologous recombination of hepatitis c virus in cell culture.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/62ceabffd31041f089cd5936d2865f60
work_keys_str_mv	AT troelskhscheel productivehomologousandnonhomologousrecombinationofhepatitiscvirusincellculture AT andreagalli productivehomologousandnonhomologousrecombinationofhepatitiscvirusincellculture AT yipingli productivehomologousandnonhomologousrecombinationofhepatitiscvirusincellculture AT lottesmikkelsen productivehomologousandnonhomologousrecombinationofhepatitiscvirusincellculture AT judithmgottwein productivehomologousandnonhomologousrecombinationofhepatitiscvirusincellculture AT jensbukh productivehomologousandnonhomologousrecombinationofhepatitiscvirusincellculture
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Productive homologous and non-homologous recombination of hepatitis C virus in cell culture.

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