Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1998-12-09
2001-05-08
Wortman, Donna C. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007400, C530S350000
Reexamination Certificate
active
06228576
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to newly identified polynucleotides and polypeptides, and their production and uses, as well as their variants, agonists and antagonists, and their uses. In particular, in these and in other regards, the invention relates to novel polynucleotides and polypeptides of viruses of the Flaviviridae family, particularly a novel truncate of the Hepatitis C Virus (HCV) NS5B protein, as well as other variants disclosed herein, all hereinafter referred to as “HCV NS5B”.
BACKGROUND OF THE INVENTION
First identified by molecular cloning in 1989 (Choo, et al.,
Science
244: 359-362 (1989)), hepatitis C virus (HCV) is now widely accepted as the most common causative agent of post-transfusion non A, non-B hepatitis (NANBH) (Kuo, et al.,
Science
244:362-364 (1989)). Infection with HCV is a major cause of human liver disease throughout the world with seroprevalence in the general population ranging from 0.3 to 2.2% (van der Poel, et al., HEPATITIS C VIRUS; Amsterdam: Karger; pp. 137-163 (1994)) to as high as ~10-20% in Egypt (Hibbs, et al.,
J. Infect. Dis.
168: 789-790 (1993)). Although the virus is most commonly transmitted via blood, the mode of transmission remains unknown for a large portion of infected individuals (Alter, M. J.,
Infect. Agents Dis.
2: 155-166 (1993)). Over 50% of infections by HCV progress to acute hepatitis associated with viremia and generally elevated serum alanine aminotranferase (ALT) levels (Alter, H. J., CURRENT PROSPECTIVES IN HEPATOLOGY; New York: Plenum; pp. 83-97 (1989)). Over half of the acute cases progress to a chronic infection with roughly 20% developing cirrhosis (Alter, H. J., supra). Chronic infection by HCV has also been linked epidemiologically to the development of hepatocellular carcinoma (HCC), especially in cirrhotic patients (Blum, et al.,
Hepatology
19: 251-258 (1994)).
Since its initial identification, many isolates of HCV have been sequenced, displaying much genetic diversity and leading to the subclassification of this virus into multiple genotypes. See, e.g., Bukh, et al.,
Proc. Natl. Acad. Sci. USA
90:8234-8238 (1993); Bukh, et al.,
Proc. Natl. Acad. Sci. USA
91: 8239-8243 (1994); Dusheiko, et al.,
Hepatology
19: 13-18 (1994)). Due to its genome structure and sequence homology, this virus was assigned as a new genus in the Flaviviridae family, along with the other two genera, flaviviruses (such as yellow fever virus and Dengue virus types 1-4) and pestiviruses (such as bovine viral diarrhea virus). See, e.g., Choo, et al., supra; Miller, et al.,
Proc. Natl. Acad. Sci. USA
87: 2057-2061 (1990)). Like the other members of the Flaviviridae family, HCV is an enveloped virus containing a single-stranded RNA molecule of positive polarity. The HCV genome is approximately 9.6 kilobases (kb) with a long, highly conserved, noncapped 5′ nontranslated region (NTR) of approximately 340 bases which functions as an internal ribosome entry site (IRES) (Wang, et al.,
Curr. Topics Microbiol. Immunol.
203: 99-112 (1995)). This element is followed by a region which encodes a single long open reading frame (ORF) encoding a polypeptide of ~3000 amino acids comprising both the structural and nonstructural viral proteins. This large polypeptide is subsequently processed into the individual structural and nonstructural proteins by a combination of host and virally-encoded proteinases (reviewed in Rice, VIROLOGY; Raven Press: New York, 2nd Ed.; pp. 931-960 (1996)). In their respective order in the polyprotein precursor from amino-terminus to carboxy-terminus, are core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
Inhibition of the biological activity of viral proteins of the Flaviviridae family, particularly HCV NS5B, is potentially of benefit in controlling, reducing and alleviating the diseases caused by infection with these viral organisms. Clearly, there is a need for factors, such as the novel compounds of the invention, that have a present benefit of being useful to screen compounds for antiviral activity. Such factors are also useful in determining their role in pathogenesis of infection, dysfunction and disease. There is also a need for identification and characterization of such factors and their antagonists and agonists which can play a role in preventing, ameliorating or correcting infections, dysfunctions or diseases.
In an attempt to identify the critical domains of the HCV NS5B protein, Lohmann, et al.,
J. Virol.
8416-8428 (1997) truncated both the amino and carboxy termini of the full-length HCV NS5B protein. While Lohmann, et al. discuss various sequence fragments of the HCV NS5B protein, with one exception of a known GenBank sequence, the reference does not specifically disclose either the cDNA or amino acid sequences of the these fragments.
Clearly, there is a need to discover new antiviral compounds that are useful in the cure, treatment, and prevention of virus infection of the Flaviviridae family, particularly HCV. Because the HCV NS5B protein is difficult to express, it is important to discover forms of the protein which may be more soluble for use in a drug-discovery screen than is the full-length HCV NS5B protein. The instant invention is believed to provide for such a need in the antiviral area.
SUMMARY OF THE INVENTION
It is an object of the invention to provide polypeptides that have been identified as novel HCV NS5B polypeptides by homology between the amino acid sequence set forth in Table 2 [SEQ ID NO:2] and other known HCV amino acid sequences, such as those shown in Table 4.
It is a further object of the invention to provide polynucleotides that encode these novel HCV NS5B polypeptides, particularly polynucleotides that encode the polypeptide, herein designated HCV NS5B.
In a particularly preferred embodiment of the invention, there is provided a polynucleotide which comprises a region encoding the HCV NS5B polypeptides set forth in Table 3 [SEQ ID NO:3] and which includes, for example, one or more variants thereof.
In another particularly preferred embodiment of the invention, there is a novel truncation mutant of the HCV NS5B protein comprising the amino acid sequence set forth in Table 3 [SEQ ID NO:4], or one or more variants thereof.
In a further aspect of the invention there are provided isolated nucleic acid molecules encoding HCV NS5B, including mRNAs, cDNAs, genomic DNAs. Further embodiments of the invention include biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
In accordance with another aspect of the invention, there is provided the use of a polynucleotide of the invention for therapeutic or prophylactic purposes, in particular genetic immunization. Among the particularly preferred embodiments of the invention are naturally occurring allelic variants of HCV NS5B polypeptides.
Another aspect of the invention provides novel polypeptides of HCV NS5B, as well as biologically, diagnostically, prophylactically, clinically or therapeutically useful variants thereof, and compositions comprising the same.
Among the particularly preferred embodiments of the invention are variants of HCV NS5B polypeptide encoded by naturally occurring alleles of the HCV NS5B gene.
In a preferred embodiment of the invention, there are provided methods for producing the aforementioned HCV NS5B polypeptides.
In accordance with yet another aspect of the invention, there are provided for inhibitors to such polypeptides, useful as antiviral agents, including, for example, antibodies.
In accordance with certain preferred embodiments of the invention, there are provided products, compositions and methods for assessing HCV NS5B expression, treating disease, for example, viruses linked to the family, particularly HCV; flaviviruses such as yellow fever virus; Dengue virus types 1-4; and pestiviruses, such as bovine viral diarrhea virus and classic swine fever, among others, assaying genetic variation, and administering a HCV NS5B polypeptide or polynucleotide to an organism to
Han William T.
King William T.
Kinzig Charles M.
SmithKline Beecham Corporation
Wortman Donna C.
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