Enhanced replication of HCV RNA

Details Enhanced replication of HCV RNA Enhanced replication of HCV RNA

2001-08-03

2003-12-09

Housel, James (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S004000, C435S007210, C435S325000, C435S235100, C435S238000, C435S366000, C435S370000

Reexamination Certificate

active

06660471

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the generation of cellular environments that are favorable for the replication of sub-genomic hepatitis C virus (HCV) replicons. The present invention also relates to use of cells presenting such replicon replication-favorable environments to screen for compounds useful for the treatment of HCV infection and related diseases.
BACKGROUND OF THE INVENTION
Because of the large number of HCV infected individuals worldwide, therapeutic drugs are critically needed. Currently, it is not possible to efficiently propagate HCV in culture, or to generate actively infected non-primate animal models of the disease.
The efforts to develop therapeutic drugs against HCV would be aided by the establishment of cell lines that carry replicating HCV RNA or synthesize de novo HCV virus. In particular, a reproducible system which accommodates replication of sub-genomic RNA would facilitate drug screening. However, no such system currently exists that is both robust and amenable to drug screening applications. Recently, Lohmann et al. (1999, Science, 285:110-113) described neomycin-selectable, HCV sub-genomic replicons, which were used to establish a few stable cell clones producing autonomously replicating sub-genomic HCV RNAs, in Huh-7 cells. Lohmann et al. report that efficiency of recovery, however, was low, and speculate that recovery was due to particular host cell conditions or factors present in only a few cells.
The present invention is directed to a cell-based system to establish the replication of sub-genomic viral replicons, such as that of HCV, with high efficiency.
SUMMARY OF THE INVENTION
The present invention is directed to methods of generating cells, with a disabled host anti-viral response factor, that stably replicate sub-genomic virus replicons. In one aspect, the methods comprise disabling PKR activity in a cell prior to or concurrent with introducing a sub-genomic virus replicon into the cell. Preferably, the sub-genomic virus replicon is an HCV sub-genomic replicon.
In a further aspect, the present invention is directed to cells that are generated according to the aforementioned methods. These cells have disabled PKR activity and stably replicate sub-genomic virus RNA.
In yet another aspect, the present invention is directed to methods of screening for compounds that modulate viral RNA replication.
These and other aspects of the invention are described more fully below.
All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.
DETAILED DESCRIPTION OF THE INVENTION
HCV is an enveloped, positive-strand (messenger sense) RNA virus belonging to the family Flaviviridae. The HCV genome is a single-stranded RNA of about 9.5 kb in length. Like other single-stranded RNA viruses, HCV is believed to replicate its genomic RNA via a double-stranded RNA (ds-RNA) intermediate.
The HCV genome codes for a polyprotein that is subsequently spliced and processed into the structural proteins C (core) and E1, and E2 (both envelope proteins) and the non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B) of the virus. The positions of the various proteins produced from the polyprotein, numbered relative to HCV-1 (see, Choo et al. (1991)
Proc. Natl. Acad. Sci. USA
88:2451-2455) is shown in Table 1.
TABLE 1
Domain
Approximate Boundaries*
C (core)
  1-191
E1
 192-383
E2
 384-746
P7
 747-809
NS2
 810-1026
NS3
1027-1657
NS4a
1658-1711
NS4b
1712-1972
NS5a
1973-2420
NS5b
2421-3011
*Numbered relative to HCV-1. See, Choo et al. (1991) Proc. Natl. Acad. Sci. USA 88:2451-2455.
It is well documented that virus infection triggers an interferon response in the host, including the induction of the expression of the cellular ds-RNA-dependent protein kinase (PKR). PKR becomes activated, through autophosphorylation, upon binding to viral ds-RNA. Activated PKR phosphorylates the eukaryotic translation initiation factor 2 (eIF2&agr;), leading to a dramatic reduction in both cellular and viral protein synthesis. This, among other interferon-induced effects, results in apoptosis of infected cells.
The difficulty in obtaining HCV replicon-transfected cell clones may be related to the induction of the activation of PKR by the viral ds-RNA in the cells. Therefore, blocking PKR activity would be expected to facilitate the establishment of clones of cells that are capable of stably replicating HCV viral replicons.
Cells deficient in PKR activity have been described for the generation of viral vaccines and persistent viral infections (U.S. Pat. No. 5,840,565; Yeung et al., 1999, Proc. Natl. Acad. Sci. USA, 96:11860-11865). In these systems, persistent infections are established when cell cultures are infected with virus particles, i.e., complete viral genomes, for the generation of progeny virus. These systems have not been applied to the generation of stable cell lines capable of replicating sub-genomic viral replicons, nor have they been applied to HCV.
The invention provides, inter alia, methods to prepare cells, having disabled PKR activity, for the replication of sub-genomic viral replicons, preferably HCV sub-genomic replicons. The invention also provides methods of using these cells to screen for drugs that modulate viral replication.
The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Maniatis et al., eds., Molecular Cloning: A Laboratory Manual (2
nd
ed.) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Glover, ed., DNA Cloning: A Practical Approach, Vols. I & II; Colowick & Kaplan, eds., Methods in Enzymology, Academic Press; Weir & Blackwell, eds., Handbook of Experimental Immunology, Vols. I-IV Blackwell Scientific Pubs. (1986); Fields, Knipe, & Howley, eds., Fields Virology, 3
rd
Edition, Vols. I &II, Lippincott Williams & Wilkins Publishers (1996); Coligan et al., eds., Current Protocols in Immunology, John Wiley & Sons, New York, N.Y. (2000).
Various definitions are made throughout this document. Most words have the meaning that would be attributed to those words by one skilled in the art. Words specifically defined either below or elsewhere in this document have the meaning provided in the context of the present invention as a whole and as typically understood by those skilled in the art.
As used herein, the term “replicon” refers to a viral nucleic acid that is capable of directing the generation of copies of itself. As used herein, the term “replicon” includes RNA as well as DNA. For example, double-stranded DNA versions of HCV genomes can be used to generate a single-stranded RNA transcript that constitutes an HCV replicon. Generally, a viral replicon contains the complete genome of the virus. “Sub-genomic replicon,” as used herein, refers to a viral nucleic acid that contains something less than the full complement of genes and other features of the viral genome, yet is still capable of directing the generation of copies of itself. For example, the sub-genomic replicons of HCV described below contain most of the genes for the non-structural proteins of the virus, but are missing most of the genes coding for the structural proteins. Sub-genomic replicons are capable of directing the expression of all of the viral genes necessary for the replication of the viral sub-genome (replication of the sub-genomic replicon), without the production of viral particles.
An HCV sub-genomic replicon, may be derived from any of the various HCV strains and isolates, such as, but not limited to, any of the isolates from strains 1, 2, 3, 4, 5 or 6 of HCV. Moreover, the various genes included in the sub-genomic replicon can be derived from different strains. The complete genotypes of many of these strains are known. See, e.g., U.S. Pat. No. 6,150,087 and GenBank Accession Nos. AJ238800 and AJ238799, International Publication No

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