Flavivirus-based system for production of hepatitis C virus (HCV)
Inventors
Saunier, Bertrand • Triyatni, Miriam • Berger, Edward A.
Assignees
Institut National de la Sante et de la Recherche Medicale INSERM • US Department of Health and Human Services
Publication Number
US-9052321-B2
Publication Date
2015-06-09
Expiration Date
2029-09-28
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Abstract
Provided herein is a mammalian cell transformed to contain a plasmid encoding a T7 or SP6 promoter operably linked to one or more HCV genes, a subgenomic replicon from a flavivirus and a cytoplasmic T7 and SP6 RNA amplification system. Also provided herein are isolated replication-competent HCV particles produced by the method comprising the steps of providing a transformed mammalian cell according to the first embodiment, culturing the cell, and recovering the replication-competent HCV particles from the cell culture. Provided herein are isolated HCV structural proteins produced by the method comprising the steps of providing a transformed mammalian cell according to the first embodiment, culturing the cell, and recovering the HCV structural proteins from the cell culture. Further provided herein is a system for assaying HCV entry into a cell comprising a first plasmid encoding a T7 or SP6 promoter operably linked to an HCV polynucleotide comprising at least the 5′-UTR to NS2 operably linked to an EMCV IRES in frame with an SP6 or T7 polymerase gene, respectively, a first host cell line expressing a replicon from a flavivirus and comprising a cytoplasmic T7 and SP6 RNA amplification system, a second plasmid encoding a reporter gene operably linked to both T7 and SP6 promoters in tandem, and a second host cell line comprising a cytoplasmic T7 polymerase or SP6 polymerase RNA amplification system.
Core Innovation
Hepatitis C virus (HCV) infects millions worldwide and current treatments are not fully effective, particularly against prevalent subtypes. The rapid replication and mutation rate of HCV complicate antiviral therapy and vaccine development. There remains a need for improved systems to produce HCV particles across multiple genotypes and to specifically study viral entry without interference from replication.
Provided herein is a mammalian cell transformed to contain a plasmid encoding a T7 or SP6 promoter operably linked to one or more HCV genes, a subgenomic replicon from a flavivirus, and a cytoplasmic T7 and SP6 RNA amplification system. This system enables the production of isolated replication-competent HCV particles and structural proteins by culturing transformed mammalian cells and recovering these products from culture.
Further, a system for assaying HCV entry into a cell is described, comprising a first plasmid encoding a T7 or SP6 promoter operably linked to an HCV polynucleotide (from the 5′-UTR to NS2) linked to an EMCV IRES in frame with an SP6 or T7 polymerase gene; a first host cell expressing a flavivirus subgenomic replicon and the cytoplasmic amplification system; a second plasmid encoding a reporter gene linked to both T7 and SP6 promoters; and a second host cell containing a cytoplasmic polymerase RNA amplification system. Methods employing these systems enable assays of HCV particle entry, identification of cellular proteins necessary for entry, identification of HCV glycoproteins essential for entry, and screening of compounds blocking entry or genome uncoating.
Claims Coverage
The patent contains multiple independent claims covering recombinant mammalian cells, production methods, and assay systems related to HCV production and entry.
Recombinant mammalian cell containing specific genetic elements
A mammalian cell transformed with a plasmid encoding a T7 or SP6 promoter operably linked to one or more HCV genes; a subgenomic flavivirus replicon; and a cytoplasmic T7 and SP6 RNA amplification system, wherein the HCV genes encode structural proteins or replication-competent HCV particles, and the flavivirus is West Nile or dengue 2 virus.
Construction of the cytoplasmic RNA amplification system
The cytoplasmic T7 and SP6 polymerase RNA amplification system comprises a plasmid encoding a T7 polymerase promoter operably linked to an EMCV IRES in frame with the SP6 polymerase open reading frame, and a plasmid encoding an SP6 polymerase promoter operably linked to an EMCV IRES in frame with the T7 polymerase open reading frame.
Methods of producing HCV structural proteins or replication-competent particles
Methods comprising providing the recombinant mammalian cell of the first inventive feature, culturing the cell, and recovering HCV structural proteins or replication-competent HCV particles, optionally produced without HCV virus replication and purified by sucrose gradient or filtration.
The independent claims collectively cover recombinant mammalian cells transformed with specific genetic components for HCV production, the design of the RNA amplification system enabling efficient transcription, and methods for producing HCV structural proteins and replication-competent particles from these cells.
Stated Advantages
Enables production of HCV particles across multiple genotypes without requiring viral replication.
Allows uncoupling of viral entry from replication facilitating specific study of envelope interactions and internalization.
Provides a system to identify cellular proteins and compounds affecting HCV entry, assembly, maturation, egress, or genome packaging.
Supports production of infectious wild-type HCV particles without adaptive mutations.
Documented Applications
Producing replication-competent HCV particles and isolated HCV structural proteins from transformed mammalian cells.
Assaying HCV particle entry into target cells using dual polymerase reporter systems to measure reporter gene expression proportional to viral entry.
Identifying cellular proteins necessary for HCV particle entry by siRNA knockdown, gene overexpression, or lentiviral transduction in target cells.
Identifying HCV glycoproteins necessary for viral entry by antibody blocking prior to infection.
Screening test compounds that block HCV particle entry or viral genome uncoating in cell-based assays.
Identifying cellular factors involved in release of infectious HCV particles by comparing productive and non-productive clones using subtractive hybridization.
Identifying test compounds inhibiting HCV particle assembly, maturation, egress, or genome packaging by assessing reductions in viral entry.
Studying viral spreading and dissemination in organs, tissues, or animals using HCV genomes tagged with tetracysteine.
Assessing contributions of HCV structural or non-structural proteins to viral production, assembly, and egress using bicistronic genomes and trans-complementation.
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