Functional DNA clone for hepatitis C virus (HCV) and uses thereof
AssigneeWashington University
InventorsCharles M. Rice, Alexander A. Kolykhalov
Life of this patent
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Abstract
The present invention relates to the determination of an authentic HCV genome RNA sequences, to construction of infectious HCV DNA clones, and to use of the clones, or their derivatives, in therapeutic, vaccine, and diagnostic applications. The invention is also directed to HCV vectors, e.g., for gene therapy of gene vaccines.
Claims68
A genetically engineered hepatitis C virus (HCV) nucleic acid clone which comprises from 5′ to 3′ on the positive-sense acid a functional 5′ non-translated region (NTR) comprising an extreme 5′-terminal conserved sequence, an open reading frame (ORF) encoding at least a portion of an HCV polyprotein whose cleavage products form functional components of HCV virus particles and RNA replication machinery, and a 3′ non-translated region (NTR) comprising an extreme 3′-terminal conserved sequence, or a derivative thereof selected from the group consisting of adapted virus, line-attenuated virus, replication-competent non-infectious virus, and defective virus.
A plasmid clone harboring a full-length HCV cDNA which can be transcribed to produce infectious HCV RNA transcripts as deposited with the American Type Culture Collection and assigned accession no. 97879, having a sequence as depicted in SEQ ID NO:5, or a derivative thereof selected from the group consisting of
- a) a derivative wherein a 5′-terminal sequence is homologous or complementary to an RNA sequence selected from the group consisting of GCCAGCC, GGCCAGCC, UGCCAGCC, AGCCAGCC, AAGCCAGCC,k GAGCCAGCC, GUGCCAGCC, and GCGCCAGCC, wherein the sequence GCCAGCC is the 5′-terminus of SEQ ID NO:3;
- and
- b) a derivative wherein a 3′-NTR comprises a short poly-pyrimide region.
A plasmid clone harboring a full-length HCV cDNA which can be transcribed to produce infectious HCV RNA transcripts as deposited with the American Type Culture Collection and assigned accession no. 97879, having a sequence as depicted in SEQ ID NO:5, or a derivative thereof selected from the group consisting of
- a) a derivative produced by substitution of homologous regions from other HCV isolates or genotypes;
- b) a derivative produced by mutagenesis;
- c) a derivative selected from the group consisting of adapted, live-attenuated, replication competent non-infectious, and defective variants;
- d) a derivative comprising a heterologous gene operatively associated with an expression control sequence;
- e) a derivative consisting of a functional fragment of any of the abovementioned derivatives.
A method for identifying a cell line that is permissive for infection with HCV, comprising contacting a cell line in tissue culture with an infectious amount of the HCV RNA of claiim 15, and detecting replication of HCV in cells of the cell line.
A method for identifying a cell line that is permissive for infection with HCV, comprising contacting a cell line in tissue culture with an infectious amount of an infectious HCV RNA of claiim 19 under conditions that select for cells that express the heterologous expression control sequence.
A host cell line transfected, transformed, or transduced with the HCV DNA of claiim 16.
32 An in vitro cell line infected with HCV, wherein the HCV has a genomic RNA sequence corresponding to the HCV nucleic acid of claim 1, 3, or 5.
The cell line of claim 32 which is a hepatocyte cell line.
A method for producing HCV virus particles comprising:
- a) culturing the cell line of claim 32 under conditions that permit HCV replication and virus particle formation;
- and
- b) isolating HCV virus particles from the cell line culture.
A method for screening for agents capable of modulating HCV replication comprising:
- a) contacting the cell line of claim 32 with a candidate agent;
- and
- b) testing for increase or decrease in a level of HCV infection or activity compared to a level of HCV infection or activity in a control cell line or in the cell line prior to administration of the candidate agent;
- wherein a decrease in the level of HCV infection or activity compared to the level of HCV infection or activity in a control cell line or in the cell line prior to administration of the candidate agent is indicative of the ability of the agent to inhibit HCV infection or activity.
A method for preparing an HCV nucleic acid comprising joining from 5′ to 3′ on the positive-sense DNA a functional 5′ non-translated region (NTR) comprising an extreme 5′-terminal conserved sequence, a polyprotein coding region encoding HCV proteins that provide for expression of functional HCV proteins, and a 3′ non-translated region (NTR) comprising an extreme 3′-terminal conserved sequence.
An in vitro method for detecting the presence of HCV in a biological sample from a subject comprising:
- a) contacting a cell line permissive for productive HCV infection with a biological sample, wherein the cell line has been modified to contain a transgene that express a reporter gene product expressed under control of a trans-acting factor produced by HCV;
- and
- b) detecting expression of the reporter gene product,
- wherein detection of expression of the reporter gene product is indicative of the presence of HCV in the biological sample from the subject.
An in vitro method for detecting the presence of HCV in a biological sample from a subject comprising:
- a) contacting a cell line permissive for productive HCV infection with a biological sample, wherein the cell line has been modified to contain a defective virus transgene, which defective virus transgene will express a reporter gene product at high levels under control of a trans-acting factor produced by HCV;
- and
- b) detecting expression of the reporter gene product,
- wherein detection of expression of the reporter gene product is indicative of the presence of HCV in the biological sample from the subject.
A test kit for HCV comprising authentic HCV virsu components.
A diagnostic test kit for HCV comprising components derived from an authentic HCV virus.
Dependencies are inferred from the claim text.