Title:  Production of replicative hepatitis C virus

United States Patent:  6,828,139

Issued:  December 7, 2004

Inventors:  Schmidt; Emmett Vance (Andover, MA); Chung; Raymond Taeyong (Boston, MA)

Assignee:  The General Hospital Corporation (Boston, MA)

Appl. No.:  205962

Filed:  July 26, 2002

Abstract

A nucleic acid having a first nucleotide sequence encoding an infectious hepatitis C virus, a second nucleotide sequence encoding a ribozyme, and an inducible promoter operably linked to the first and second nucleotide sequences, the ribozyme being configured to remove a 3' sequence unnecessary for replication of the infectious hepatitis C virus from a transcript initiated by the inducible, is described. A cell containing the nucleic acid and methods of using the cell are also described.

Description of the Invention

FIELD OF THE INVENTION

The invention relates to virology and antiviral drug screening.

BACKGROUND OF THE INVENTION

The development of anti-viral strategies against hepatitis C virus (HCV) infection has been hindered by the lack of an ideal animal model, or even a cell culture system, for HCV replication. One characteristic of an ideal HCV animal model or cell culture system would be the ability to induce expression of an infectious HCV in a cell in vitro or in vivo.

The HCV genome consists of a positive strand RNA that encodes a single precursor viral protein that is cleaved by cellular and viral proteases to generate viral structural and non-structural proteins, respectively. Non-translated regions 5' and 3' (5'NTR and 3'NTR) to the open reading frame encoding the precursor protein are also involved in viral replication. For a review of HCV, see Houghton, "Chapter 32, Hepatitis C Viruses," in: Fields Virology, 3rd ed., Fields et al. eds., pp 1035-1058, 1996, Lippincott-Raven Publishers, Philadelphia, Pa.

Infection with HCV is one of the leading causes of chronic liver disease throughout the world. Chronic infection nearly always ensues after acute exposure to HCV, and chronically infected individuals develop cirrhosis and hepatocellular carcinoma at a dramatically elevated rate compared with the rate of disease in an uninfected population.

The development of more effective treatments has been limited by the lack of an effective tissue culture or small animal model of infection. HCV replication systems based on a self-replicating HCV RNA replicon is dependent only on expression of the nonstructural viral proteins. Therefore, such systems do not recapitulate all steps of the HCV virus life cycle, some of which may be effective targets for antiviral intervention. Cell lines stably transfected with the HCV precursor are not capable of HCV RNA replication. Consequently, such cell lines cannot be used to screen for antiviral drugs that block viral RNA replication. RNA transcripts from an infectious HCV cDNA clone can replicate in chimpanzees, but any model that requires the use of large, expensive primates is impractical.

SUMMARY OF THE INVENTION

To address the historical and intractable limitations in the above HCV replication models, the invention provides an inducible system for producing infectious or replicative HCV, as described in the Example below, thereby fulfilling a long-felt need for an ideal HCV replication system.

The invention is based on the development and implementation of an inducible cell-based system for producing replicative HCV. Because the HCV produced by this system is replicative and has the full complement of genetic material found in HCV (e.g., naturally occurring HCV), all aspects of the HCV life cycle can be examined, e.g., in screening assays for candidate antiviral compounds. The inducibility of the system allows the propagation of cells or animals containing HCV genetic material without the damaging effects of HCV replication. Thus, viral replication is induced only when desired, for example, in a particular step that requires infectious HCV replication in an assay.

Accordingly, the invention features a nucleic acid having a first nucleotide sequence encoding an infectious hepatitis C virus, a second nucleotide sequence encoding a ribozyme (e.g., a hepatitis D virus ribozyme), an inducible promoter (e.g., a T7 promoter) operably linked to the first and second nucleotide sequences, and optionally a transcription termination signal (e.g., a T7 transcription termination signal) operably linked to the first and second nucleotide sequences, the ribozyme being configured to remove a 3' sequence unnecessary for replication of the infectious hepatitis C virus from a transcript initiated by the inducible promoter and optionally terminated by the transcription termination signal. The invention also includes cells that harbor a nucleic acid of the invention.

A method of producing infectious HCV is also included in the invention. A cell containing the nucleic acid of the invention (e.g., as an episome or an integrated cassette) can then be used to generate infectious HCV by inducing the inducible promoter of the nucleic acid. For example, if the promoter is a T7 bacteriophage promoter, HCV is produced by expressing a T7 RNA polymerase in the cell. The T7 RNA polymerase in turn can be expressed by infecting the cell with a viral vector (e.g., a vaccinia vector) encoding the T7 RNA polymerase. Alternatively, the cell can contain an episomal plasmid or genomic transgene (e.g., delivered by a retrovirus) that expresses T7 RNA polymerase. Regardless of the vectors used to express T7 RNA polymerase, the expression of the polymerase can itself be regulated, depending on the genetic elements operably linked to the sequence encoding the polymerase.

The invention further includes a screening method for identifying a compound (e.g., a polypeptide, small molecule, or nucleic acid, such as an antisense nucleic acid or ribozyme) that inhibits replication of an HCV. The method includes (1) providing a test cell containing a nucleic acid of the invention, (2) inducing the inducible promoter of the nucleic acid, (3) contacting the test cell with a candidate compound, and (3) detecting a decrease in the amount of infectious hepatitis C virus produced by the test cell compared to the amount of the infectious hepatitis C virus produced by a control cell. The detecting step can include measuring (e.g., by PCR) the amount of negative strand hepatitis C viral RNA in the cell or the amount of positive strand hepatitis C viral RNA in the cell or in cell-free virions produced by the cell.

Since it is possible that the candidate compound inhibits HCV replication by inhibiting a viral or cellular protease responsible for cleaving the HCV precursor protein, the screening method can further include determining whether a hepatitis C virus structural or non-structural protein is cleaved from a hepatitis C virus precursor protein in the cell after the contacting step, e.g., by protein gel electrophoresis.

As used herein, "inhibits" or "inhibition" means any measurable decrease (e.g., 10%, 20%, 50%, 90%, or 100%) in an activity of interest.

As used herein, an "infectious hepatitis C virus" means an HCV that is capable of propagation in a population of cells in vivo or in vitro. Therefore, an infectious hepatitis C virus minimally contains (1) a sequence encoding a precursor protein and (2) 5' and 3' non-translated flanking sequences sufficient to support virus replication (i.e., each step of the virus life cycle) in a cell population.

By one genetic element being "operably linked" to another is meant that a genetic element (either in a plus strand, minus strand, or double stranded form) is structurally configured to operate or affect another genetic element. For example, a promoter operably linked to a sequence encoding a polypeptide means that the promoter initiates transcription of a nucleic acid encoding the polypeptide, and a transcription termination signal operably linked to the sequence encoding the polypeptide means that the transcription termination signal terminates transcription of a nucleic acid encoding the polypeptide.

The nucleic acids and methods of the invention provide a HCV replication system amenable to comprehensive, yet relatively inexpensive (as compared to infection of a primate), antiviral drug screening methods. Because the HCV replication system performs all steps of the virus life cycle, candidate antiviral compounds can be screened for activity against any vital viral or cellular drug target involved in virus replication. In addition, the HCV replication system is inducible, thereby allowing cells to vigorously replicate in the absence of HCV until virus replication becomes necessary for the particular step of a screening assay performed. Thus, the nucleic acids and methods of the invention remove a substantial obstacle in anti-HCV drug development.

DETAILED DESCRIPTION OF THE INVENTION

The nucleic acid, such as an expression plasmid, of the invention can be constructed using standard methods and reagents in the art of molecular biology. For example, inducible promoters, such as the T7 promoter system (Aoki et al., Virology 250:140-150, 1998; WO 98/39031) can be used to provide controlled expression of the infectious HCV clone. Alternatively, a tetracycline-inducible promoter system can be used (Moradpour et al., Hepatology 28:192-201, 1998). The inducible promoter is then operably linked to an infectious clone of an HCV, such as the one described in Yanagi et al., Proc. Natl. Acad. Sci. USA 94:8738-8743, 1997; or Kolykhalov et al., Science 277:570-574, 1997. At the 3' end of the HCV clone, a ribozyme (e.g., a hepatitis D virus ribozyme) and optionally a transcription termination signal (e.g., a T7 transcription termination signal) is attached. Ideally, a cis-acting ribozyme, such as a HDV ribozyme, that cleaves at the boundary with the 3' terminus of the HCV RNA is suitable for this purpose. After the HCV clone is transcribed, the ribozyme serves to remove itself and other 3' sequences which may hinder virus replication.

Depending on the inducible promoter used to drive transcription of the infectious HCV clone, the method for inducing HCV production will vary. For example, if a T7 promoter is used, then transcription is induced by expressing a T7 RNA polymerase in a cell harboring the nucleic acid (e.g., as a result of transfection, viral vector delivery, or genomic DNA integration). The polymerase can be expressed using a viral vector, such as the vaccinia vector described in the Example below, or the adenovirus vector described in Aoki et al., supra. Alternatively, a T7 polymerase expression vector under the control of a mammalian promoter can be introduced into the cell to induce HCV production. In addition, the cell can already contain a stably integrated expression cassette that is induced to express T7 polymerase, thereby producing replicative HCV.

To allow propagation of HCV, induced cells are optionally mixed with a population of cells that do not produce HCV but are permissive for HCV infection. A mixed or unmixed population can then be used in screening assays for candidate antiviral compounds. Depending on the nature of a candidate compound, contacting the population of cells with the compound can involve a variety of standard techniques. If the candidate compound is a small molecule drug that can pass through lipid membranes, all that is needed for the contacting step is the addition of the compound to an aqueous mixture containing the cells. However, if the compound is a protein or nucleic acid intended to elicit its antiviral effect inside a cell, the contacting step may require other techniques. A protein compound can be delivered into the cell by encapsulation in liposomes or fusion to a viral protein that is delivered inside a cell by viral infection. A nucleic acid can be delivered into the cell using a viral vector or a transfection protocol (e.g., electroporation). Methods of introducing proteins and nucleic acids into a cell are well known in the art of molecular biology.

After the contacting step, the amount of infectious hepatitis C virus produced in the presence of the compound is measured and compared to a control amount of infectious hepatitis C virus. The control amount is generally an amount observed in a similar population of cells grown in the absence of the candidate compound.

The amount of HCV can be measured using any suitable direct or indirect method. Several suitable methods are known in the art. For example, the amount of positive strand viral RNA in cells and in virions can be measured using quantitative PCR. The amount of minus strand RNA can also be measured using PCR. In addition, viral proteins, rather than viral nucleic acids, can be measured, e.g., in the supernatant of an HCV-infected cell culture, using an enzyme linked immunosorbent assay (ELISA). Such assays typically utilize monoclonal or polyclonal antibodies to capture viral antigens in a sample. The captured antigens are then detected using labeled antibodies that specifically bind to the captured antigen at an epitope different from the one to which the capture antibody binds. HCV enzyme-linked immunoassays are available from Abbott Laboratories. In addition, any decrease in the level of cleavage of the viral precursor protein caused by the presence of the candidate compound provides information regarding the mode of action of the compound. For example, decreased cleavage could lead to asking whether the compound binds and inhibits a viral or cellular protease. Thus, an examination of the level of precursor protein cleavage can be a valuable step in the screening methods of the invention.

The methods of the invention can be performed repetitively and in parallel to screen libraries of compounds (e.g., a small molecule library, a peptide library, or a single chain antibody library) for candidate antiviral drugs. For example, cells having a nucleic acid of the invention can be cultured in the wells of a 96-well microtiter plate. Before or after induction of HCV replication, each member of the compound library is deposited into one well, and the level of HCV replication determined in the well. Thus, automation of the methods of the invention are especially amenable to high throughput screening of chemical libraries.

This invention can also be used to identify key cellular proteins involved in HCV RNA replication by identifying replication complexes, and to identify host cellular genes that are induced by expression of replicative HCV.

Claim 1 of 7 Claims

What is claimed is:

1. A method of inducibly producing infectious hepatitis C virus, the method comprising

providing a cell containing a nucleic acid comprising a first nucleotide sequence encoding an infectious hepatitis C virus, a second nucleotide sequence encoding a ribozyme, and an inducible promoter operably linked to the first and second nucleotide sequences, the ribozyme being configured to remove a 3'sequence unnecessary for replication of the infectious hepatitis C virus from a transcript initiated by the inducible promoter; and

inducing the inducible promoter, thereby producing the infectious hepatitis C virus.

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