Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1999-03-31
2001-02-27
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120
Reexamination Certificate
active
06194141
ABSTRACT:
BACKGROUND OF THE INVENTION
Single stranded RNA viruses are unique in having an RNA template which directs the biosynthesis of RNA. Upon infection of a cell, the genome of positive strand RNA viruses directs the synthesis of viral proteins which are required for the replication of the RNA. During the replication process, a negative strand RNA molecule is first produced followed by a subsequent transcription of the negative strand to produce new copies of the positive strand RNA. Richards, O. C. et al., (1990)
Current Topics in Microbiol. and Immun
. 161:89-119.
Despite the fact that RNA synthesis has been studied for the past thirty years or so in single stranded RNA viruses such as poliovirus, the biochemistry of single stranded RNA virus replication has not been clearly elucidated. Richards O. C. et al., 1990. Complicating the replication process is the finding that two groups of animal viruses (picornavirus and calicivirus) and five groups of plant viruses (comovirus, luteovirus, nepovirus, potyvirus, and sobemovirus) have been shown to contain a genome-linked viral protein (VPg) attached to the 5′-end of the single-stranded genome RNA. Salas, M. (1991)
Annu. Rev. Biochem
. 60: 39-71. See Table 3, taken from Salas, M., (1991). For example, poliovirus and encephalomyocarditis virus (EMCV), although polyadenylated at their 3′ ends, are not conventionally capped at their 5′ ends. Instead of having a 7-methyl guanosine triphosphate group, the RNAs are covalently linked to a VPg. Poliovirus-specific mRNA, however, isolated from infected cells lack VPg. HeLa cell extracts and rabbit reticulocyte lysates contain an enzyme activity that cleaves VPg from the 5′ terminus of picornavirus RNA in vitro. It is thought that this activity modifies all newly synthesized viral RNAs destined to become mRNAs in vivo. Jang, S. K., et al., (1988)
J. Virol
. 62(8):2636-2643.
Picornaviridae are a large family of non-enveloped, positive-sense RNA animal viruses, comprised of six genera (Enterovirus, Parechovirus, Rhinovirus, Hepatovirus, Cardiovirus, and Aphthovirus) that contain numerous viral species (more than 200) known to cause important diseases of humans and animals, including type A viral hepatitis, aseptic meningitis, chronic heart disease and the common cold. Table 2. VPg proteins in picornaviruses are between 20 to 26 amino acids in length. Salas, 1991. In poliovirus, VPg is a protein of only 22 amino acids (Kitamura, N., et al. (1981)
Nature
291, 547-553.) whose single tyrosine residue provides the link to the 5′-terminal uridylylic acid of the genome. Rothberg, P. G., et al. (1978)
Proc. Natl. Acad. Sci. USA
75, 4868-4872. Ambros, V., et al. (1978)
J. Biol. Chem
253, 5263-5266. Biochemical and genetic data have implicated protein(s) mapping to the 3D region of the viral polyprotein, in the formation of the O
4
-(5′-uridylyl)tyrosine bond. (Takegami, T., et al. (1983)
Proc. Natl. Acad. Sci. USA
80, 7447-7451; Takeda, N., et al. (1986)
J. Virol
. 60, 43-53; Toyoda, H., et al. (1987)
J. Virol
. 61, 2816-2822.
The precise role of VPg linked to the 5′-end of single stranded RNA viruses has not been clearly understood heretofore. In poliovirus, for example, the finding of VPg covalently linked to the 5′-end of poliovirus RNA, to nascent strands of poliovirus replicative intermediates, and to the poly(U) of minus strands, suggests that VPg serves as a primer for both the plus and minus strands of poliovirus RNA. Other findings suggest that a VPg precursor or a uridylylated form of VPg serves as a primer. Still another model suggests that a hairpin formed at the 3′-end of poliovirus RNA acts as a primer for minus-strand synthesis by the RNA polymerase. Salas, M. (1991).
In accordance with the present invention, it has been surprisingly found that VPg, 3D
pol
, poly(A) and UTP form a complex that facilitates transfer of UMP to the hydroxyl of Y3 of VPg. Transcription of a poly(A) template is then initiated at the 3′ hydroxyl group of UMP (
FIG. 7
, arrow). Knowledge of the biochemistry of picornavirus replication allows for intervention with various compositions in order to inhibit such replication. Inhibition of replication is especially valuable in preventing and ameliorating the progression of disease caused by picornaviruses.
SUMMARY OF THE INVENTION
The present invention provides methods of inhibiting picornavirus genome replication. In particular, methods for interfering with VPg-nucleotidylylation and elongation are provided.
In one embodiment of the invention, an effective amount of VPg, VPg analog, VPg homolog or a biologically active fragment thereof is administered to a subject in order to inhibit picornavirus genome replication. In this embodiment, the VPg, VPg analog, VPg homolog or biologically active fragment thereof competitively binds to the 3D
pol
enzyme, poly(A) and UTP and prevents the picornavirus native VPg from binding to the 3D
pol
enzyme, polyA and UTP.
In another embodiment of the invention, an oligonucleotide constituting adenylate residues (AMP) is administered to a subject in order to inhibit picornavirus replication. The oligonucleotide competes with the poly(A) tail of the picornavirus in complexing with the VPg, UTP and 3D
pol
. Since the poly(A) tail of the picornavirus has to compete with the polyA oligonucleotide, replication of the virus is inhibited. Picornaviruses generally have polyA tails of about 70 adenylate residues. The oligonucleotides for use in inhibiting the replication of picornavirus may be anywhere from about five residues to about seventy residues.
In yet another embodiment of the invention, a divalent cation is administered to a subject in order to inhibit picornavirus replication. The divalent cation competitively binds to the metal binding sites on the 3D
pol
enzyme. Certain metals such as magnesium, manganese, zinc and cobalt are known to bind to 3D
pol
and stimulate the uridylylation/elongation reaction. Other divalent cations, for example, nickel and calcium inhibit the uridylylation/elongation reactions and may therefore be used to compete for the metal binding sites on the 3D
pol
enzyme. One skilled in the art can test different divalent cations for their ability to bind to the 3D
pol
enzyme and inhibit the nucleotidylylation/elongation reactions by substituting a divalent cation for magnesium or manganese in the assays provided in the working examples.
In another aspect of the present invention, ribo and deoxyribo nucleotide analogs of UTP are administered to a subject in order to inhibit picornavirus genome replication. In this embodiment, the ribo and deoxyribo nucleotides competitively bind to VPg, 3D
pol
and the polyA tail of the virus. In one embodiment, 2′,3′-Dideoxy-5-fluoruridine triphospate is used as an inhibitor of RNA replication. In another embodiment, 2′,3′-Dideoxy-3′fluorouridine triphoshate is used as an inhibitor of RNA replication. In still another embodiment, 2′,3′-Dideoxyuridine triphosphate is used as an inhibitor of RNA replication. In yet another embodiment of the present invention, 2′,5′-Dideoxyuridine triphosphate is used as an inhibitor of RNA replication.
The present invention also provides in vitro assays useful in screening potential inhibitors of picornaviral RNA replication. The assays provided herein are particularly useful for identifying compositions which inhibit the nucleotidylation of picornaviral VPgs and VPg-dependent elongation.
A first method of identifying an inhibitor of picornaviral replication comprises the steps of incubating for a time and under conditions sufficient to detect uridylylation of picornaviral VPg, a first reaction mixture comprising a buffer with an approximate pH of 7.0-8.0 and a buffer concentration of approximately 10-50 mM; a divalent metal in the form of at least one of approximately 0.1-0.5 mM manganous acetate, approximately 1-7 mM magnesium acetate, approximately 0.1-0.5 mM cobaltous acetate, or approximately 0.1-0.5 mM zinc acetate;
Paul Aniko V.
Rieder Elizabeth
Wimmer Eckard
Schwartzman Robert A.
Scully Scott Murphy & Presser
The Research Foundation of State University of New York
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