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Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Standard

Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7. / Galli, Andrea; Scheel, Troels K H; Prentoe, Jannick C; Mikkelsen, Lotte S; Gottwein, Judith M; Bukh, Jens.

I: The Journal of general virology, Bind 94, Nr. Pt 10, 10.2013, s. 2221-35.

Publikation: Bidrag til tidsskriftTidsskriftartikelForskningfagfællebedømt

Harvard

Galli, A, Scheel, TKH, Prentoe, JC, Mikkelsen, LS, Gottwein, JM & Bukh, J 2013, 'Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7', The Journal of general virology, bind 94, nr. Pt 10, s. 2221-35. https://doi.org/10.1099/vir.0.053868-0

APA

Galli, A., Scheel, T. K. H., Prentoe, J. C., Mikkelsen, L. S., Gottwein, J. M., & Bukh, J. (2013). Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7. The Journal of general virology, 94(Pt 10), 2221-35. https://doi.org/10.1099/vir.0.053868-0

Vancouver

Galli A, Scheel TKH, Prentoe JC, Mikkelsen LS, Gottwein JM, Bukh J. Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7. The Journal of general virology. 2013 okt;94(Pt 10):2221-35. https://doi.org/10.1099/vir.0.053868-0

Author

Galli, Andrea ; Scheel, Troels K H ; Prentoe, Jannick C ; Mikkelsen, Lotte S ; Gottwein, Judith M ; Bukh, Jens. / Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7. I: The Journal of general virology. 2013 ; Bind 94, Nr. Pt 10. s. 2221-35.

Bibtex

@article{7739650a1ac748268526094bbec719ca,
title = "Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7",
abstract = "Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1-7 core-NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.",
keywords = "Carcinoma, Hepatocellular, Cell Culture Techniques, Cell Line, Tumor, Gene Expression Regulation, Viral, Genotype, Hepacivirus, Humans, Viral Core Proteins, Viral Nonstructural Proteins, Virus Assembly, Virus Cultivation, Virus Replication",
author = "Andrea Galli and Scheel, {Troels K H} and Prentoe, {Jannick C} and Mikkelsen, {Lotte S} and Gottwein, {Judith M} and Jens Bukh",
year = "2013",
month = oct,
doi = "10.1099/vir.0.053868-0",
language = "English",
volume = "94",
pages = "2221--35",
journal = "Journal of General Virology",
issn = "0022-1317",
publisher = "Society for General Microbiology",
number = "Pt 10",

}

RIS

TY - JOUR

T1 - Analysis of hepatitis C virus core/NS5A protein co-localization using novel cell culture systems expressing core-NS2 and NS5A of genotypes 1-7

AU - Galli, Andrea

AU - Scheel, Troels K H

AU - Prentoe, Jannick C

AU - Mikkelsen, Lotte S

AU - Gottwein, Judith M

AU - Bukh, Jens

PY - 2013/10

Y1 - 2013/10

N2 - Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1-7 core-NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.

AB - Hepatitis C virus (HCV) is an important human pathogen infecting hepatocytes. With the advent of infectious cell culture systems, the HCV particle assembly and release processes are finally being uncovered. The HCV core and NS5A proteins co-localize on cytoplasmic lipid droplets (cLDs) or on the endoplasmic reticulum (ER) at different stages of particle assembly. Current knowledge on assembly and release is primarily based on studies in genotype 2a cell culture systems; however, given the high genetic heterogeneity of HCV, variations might exist among genotypes. Here, we developed novel HCV strain JFH1-based recombinants expressing core-NS2 and NS5A from genotypes 1-7, and analysed core and NS5A co-localization in infected cells. Huh7.5 cells were transfected with RNA of core-NS2/NS5A recombinants and putative adaptive mutations were analysed by reverse genetics. Adapted core-NS2/NS5A recombinants produced infectivity titres of 10(2.5)-10(4.5) f.f.u. ml(-1). Co-localization analysis demonstrated that the core and NS5A proteins from all genotypes co-localized extensively, and there was no significant difference in protein co-localization among genotypes. In addition, we found that the core and NS5A proteins were highly associated with cLDs at 12 h post-infection but became mostly ER associated at later stages. Finally, we found that different genotypes showed varying levels of core/cLD co-localization, with a possible effect on viral assembly/release. In summary, we developed a panel of HCV genotype 1-7 core-NS2/NS5A recombinants producing infectious virus, and an immunostaining protocol detecting the core and NS5A proteins from seven different genotypes. These systems will allow, for the first time, investigation of core/NS5A interactions during assembly and release of HCV particles of all major genotypes.

KW - Carcinoma, Hepatocellular

KW - Cell Culture Techniques

KW - Cell Line, Tumor

KW - Gene Expression Regulation, Viral

KW - Genotype

KW - Hepacivirus

KW - Humans

KW - Viral Core Proteins

KW - Viral Nonstructural Proteins

KW - Virus Assembly

KW - Virus Cultivation

KW - Virus Replication

U2 - 10.1099/vir.0.053868-0

DO - 10.1099/vir.0.053868-0

M3 - Journal article

C2 - 23907394

VL - 94

SP - 2221

EP - 2235

JO - Journal of General Virology

JF - Journal of General Virology

SN - 0022-1317

IS - Pt 10

ER -

ID: 122662765