Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2002-01-31
2004-03-02
Housel, James (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007910, C435S007210, C435S008000, C435S465000, C435S235100, C435S325000, C435S069200, C536S023100, C536S024500
Reexamination Certificate
active
06699657
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed toward the pharmaceutical and molecular biology arts, more particularly this invention is an in vitro system for the replication of the viral genomes of viruses that depend upon the enzyme RNA-dependent RNA polymerase (RDRP) for replication. The method of the invention provides an efficient means for measuring genomic replication in RDRP viruses, and also for the rapid screening of compounds for their ability to inhibit genomic replication of RDRP viruses, including the Hepatitis C virus (HCV).
BACKGROUND OF THE INVENTION
It is known that viral genomes can be made of DNA or RNA and can be double-stranded or single-stranded. Typically, viral genomes encode viral coat proteins that serve to package the genome after replication, and also nonstructural proteins that facilitate enzymatic replication of the viral genome in conjunction with cellular enzymes. In the case of some viruses having a single-stranded RNA genome, one of the nonstructural proteins encoded by the viral genome is RNA-dependent RNA polymerase (RDRP), which is needed by the virus to replicate its genomic sequence. The viral enzyme RNA-dependent RNA polymerase is also called RNA replicase.
The viral family Flaviviridae is one such type of virus which is dependent upon its own RNA-dependent RNA polymerase in order to replicate. Flaviviridae is a family of viruses having a single-stranded RNA genome in the (+) orientation. The term “(+) orientation” is a convention used to designate single-stranded nucleic acid molecules which exist in the coding or sense orientation when read from the 5′ to 3′ direction. The Flaviviridae family comprises the flaviviruses, the animal pathogenic pestiviruses, the recently characterized GB viruses (GBV-A, GBV-B and GBV-C/hepatitis G), and most importantly from a human disease perspective, the genus Hepacivirus or Hepatitis C virus (HCV). The RNA genome of these viruses typically includes a single long open reading frame encoding a polyprotein that is proteolyically cleaved into a set of distinct structural and nonstructural protein products. Translation of the open reading frame of the genome is directed via a 5′ untranslated region (UTR) which functions as an internal ribosomal entry site (IRES). The 3′ end of the genome in these viruses comprises a highly conserved UTR region of variable length which is thought to be essential for replication.
The most well-known member of the Flaviviridae family of viruses is the Hepatitis C virus (“HCV”), which is a parenterally transmitted, hepatotropic virus that in primates causes acute and chronic hepatitis, as well as hepatocellular carcinoma. Approximately 2% of the world's human population is thought to be afflicted with HCV infections. No vaccine for HCV is currently available, and present treatment is generally limited to interferon monotherapy, or the combination of alpha-interferon with the nucleoside analog ribavirin. (1)(2)(3)(4)(5).
HCV is a positive-stranded RNA virus having a genome 9.6 kb long comprised of a single, uninterrupted open reading frame encoding a polyprotein of about 3000-3011 amino acids. The HCV polyprotein is a precursor to the individual HCV proteins necessary for replication, packaging and infectivity. The structural region of the polyprotein precursor (including the C, E1, E2 and p7 proteins) is processed by host cell signal peptidases. The nonstructural region of the precursor (including the NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins) is processed between NS2 and NS3 by NS2-3 protease, while processing in the NS3-NS5B region of the polyprotein is accomplished by NS3 protease activity. (6)(7)(8)(9)(10).
The mode of replication of the HCV virus is still speculative and current understanding is based upon analogy with other of the flavi-and pestiviruses. It is believed that HCV replication begins by viral penetration of the host cell and liberation of the viral genomic (+)single-stranded RNA from the virus particle into the cytoplasm of the cell. The viral RNA is translated by cellular enzymes, and the encoded viral polyprotein is processed into several distinct functional viral proteins including RNA-dependent RNA polymerase protein (RDRP). RDRP then proceeds to synthesize (−)stranded RNA intermediates (from template viral genomes) which in turn serve as templates for synthesis of new (+)stranded RNA molecules. These (+)stranded viral RNA molecules can then be used for further viral polyprotein expression, for synthesis of new (−)stranded RNA molecules, or for packaging into progeny virions which can then be released from the infected cell to spread the HCV infection. (1).
Presently, there are no efficient systems for in vitro monitoring of the replication of the RDRP viruses of the Flaviviridae family. As a result, there is a lack of means for studying the mechanism of replication of these (+) stranded RNA viruses, or for determining the ability of a compound or condition to inhibit such replication. While cell-based systems for HCV replication have been described (11), these systems rely on protocols and endpoints that are not easily formatted into platforms for screening large numbers of compounds for anti-viral activity (12). The present invention provides a solution to these problems by providing a system for the efficient in vitro manipulation and monitoring of the replication of RDRP viruses. The system of the invention can be assembled so as to provide a convenient platform for screening inhibitors to RDRP viral replication. The method of the invention also provides a means to design therapies for the in vivo treatment of cells that are infected with RDRP viruses.
SUMMARY OF THE INVENTION
The present invention provides an efficient in vitro method for measuring the replication of the genome of viruses that are dependent upon RNA-dependent RNA polymerase for replication (these types of viruses are herein referred to as “RDRP viruses”). The method comprises the steps of culturing virally-compatible eukaryotic cells, which have been transfected with the cDNA of the genome of the RDRP virus, and transfecting these cultured cells with a construct of the invention, which construct comprises the cDNA, in antisense orientation, of a reporter gene sequence. The reporter gene cDNA sequence of the construct is operably linked on its 5′ end with the cDNA of the untranslated region (hereinafter “UTR”) in antisense orientation of the native 3′ end of said RDRP virus, and is operably linked on its 3′ end with the cDNA of the UTR in antisense orientation of the native 5′ end of said RDRP virus. Thus, the construct will be comprised of the cDNA, in antisense orientation, of a reporter gene flanked by the 3′ and 5′ UTRs of the native RDRP viral genome. Transfected cells containing the construct of the invention are cultured for a sufficient period of time under conditions which are permissive for replication of said RDRP virus, and the cells are analyzed for the presence of the protein encoded by the reporter gene. If the cDNA of the RDRP viral genome has been replicated and processed within the cultured cell, viral RDRP enzyme will have been synthesized, thereby enabling polymerization of the construct and synthesis of the protein encoded by the reporter gene. Thus, detection of the reporter protein in the cells provides a means to monitor and measure the genomic replication of said RDRP virus.
In another aspect, the invention provides an efficient in vitro method for identifying compounds or conditions which inhibit the genomic replication of viruses that are dependent for replication on RNA-dependent RNA polymerase (an RDRP virus). The method comprises the steps of culturing virally-compatible eukaryotic cells, which have been transfected with the cDNA of all or a portion of the genomic sequence of the RDRP virus, and transfecting these cultured cells with a construct of the invention, which comprises the cDNA in antisense orientation of a reporter gene sequence.
Jeffries Matthew W.
King Robert W.
Pasquinelli Claudio
Bristol--Myers Squibb Company
Housel James
Lamerdin John A.
Li Bao Qun
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