SEC14L2 enables pan-genotype HCV replication in cell culture

Nature. 2015 Aug 27;524(7566):471-5. doi: 10.1038/nature14899. Epub 2015 Aug 12.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antioxidants / metabolism
  • Carcinoma, Hepatocellular / genetics
  • Carcinoma, Hepatocellular / metabolism*
  • Carcinoma, Hepatocellular / virology*
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Culture Techniques*
  • Cell Line, Tumor
  • Cells, Cultured
  • Gene Library
  • Genome, Viral / genetics
  • Genotype*
  • Hepacivirus / genetics*
  • Hepacivirus / growth & development*
  • Hepacivirus / physiology
  • Host-Derived Cellular Factors / genetics
  • Host-Derived Cellular Factors / metabolism*
  • Humans
  • Lentivirus / genetics
  • Lipid Peroxidation
  • Lipoproteins / genetics
  • Lipoproteins / metabolism*
  • Mutation / genetics
  • RNA, Viral / biosynthesis
  • RNA, Viral / genetics
  • Replicon / genetics
  • Serum / virology
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*
  • Transduction, Genetic
  • Virus Replication* / genetics
  • Vitamin E / metabolism

Substances

  • Antioxidants
  • Carrier Proteins
  • Host-Derived Cellular Factors
  • Lipoproteins
  • RNA, Viral
  • SEC14L2 protein, human
  • Trans-Activators
  • Vitamin E