SEC14L2 enables pan-genotype HCV replication in cell culture.
Since its discovery in 1989, efforts to grow clinical isolates of the hepatitis C virus (HCV) in cell culture have met with limited success. Only the JFH-1 isolate has the capacity to replicate efficiently in cultured hepatoma cells without cell culture-adaptive mutations. We hypothesized that cultured cells lack one or more factors required for the replication of clinical isolates. To identify the missing factors, we transduced Huh-7.5 human hepatoma cells with a pooled lentivirus-based human complementary DNA (cDNA) library, transfected the cells with HCV subgenomic replicons lacking adaptive mutations, and selected for stable replicon colonies. This led to the identification of a single cDNA, SEC14L2, that enabled RNA replication of diverse HCV genotypes in several hepatoma cell lines. This effect was dose-dependent, and required the continuous presence of SEC14L2. Full-length HCV genomes also replicated and produced low levels of infectious virus. Remarkably, SEC14L2-expressing Huh-7.5 cells also supported HCV replication following inoculation with patient sera. Mechanistic studies suggest that SEC14L2 promotes HCV infection by enhancing vitamin E-mediated protection against lipid peroxidation. This provides a foundation for development of in vitro replication systems for all HCV isolates, creating a useful platform to dissect the mechanisms by which cell culture-adaptive mutations act.
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10065, USA.
Mohsan
M
Saeed
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10065, USA.
Ursula
U
Andreo
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10065, USA.
Hyo-Young
HY
Chung
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10065, USA.
Christine
C
Espiritu
Division of Liver Diseases, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.
Andrea D
AD
Branch
Department of Pathology, Icahn School of Medicine at Mount Sinai, New York, New York 10029, USA.
Jose M
JM
Silva
Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York 10065, USA.
Charles M
CM
Rice
D000975:Antioxidants / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism* / Q000821:virology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D015723:Gene Library; D016679:Genome, Viral / Q000235:genetics; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000254:growth & development* / Q000502:physiology; D058605:Host-Derived Cellular Factors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D015227:Lipid Peroxidation; D008074:Lipoproteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012093:Replicon / Q000235:genetics; D044967:Serum / Q000821:virology; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014161:Transduction, Genetic; D014779:Virus Replication / Q000235:genetics; D014810:Vitamin E / Q000378:metabolism
D000975:Antioxidants; D002352:Carrier Proteins; D058605:Host-Derived Cellular Factors; D008074:Lipoproteins; D012367:RNA, Viral; C432422:SEC14L2 protein, human; D015534:Trans-Activators; D014810:Vitamin E