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Title:  Variant tax gene of bovine leukemia virus

United States Patent:  6,646,116

Issued:  November 11, 2003

Inventors:  Aida; Yoko (3-105, Shareru tsukuba matsushiro, 21-2, Matsushiro 4-chome, Tsukuba-shi, Ibaraki 305-0035, JP); Tajima; Shigeru (203, Sanraifu nakane, 1-38-7, Kannondai, Tsukuba-shi Ibaraki 305-0856, JP)

Assignee:  Riken (Saitama, JP); Aida; Yoko (Ibaraki, JP); Tajima; Shigeru (Ibaraki, JP)

Appl. No.:  415260

Filed:  October 12, 1999

Abstract

A variant tax gene of bovine leukemia virus enhancing ability to induce replication of bovine leukemia virus or a retrovirus related to the bovine leukemia virus which encodes a variant gene product containing one or more mutations selected from the group consisting of substitution of the 240th Ser for Thr, substitution of the 247th Asp for Gly, substitution of the 251st Thr for Ala, substitution of the 258th Asp for Gly, substitution of the 261st His for Arg, substitution of the 261st His for Tyr, and substitution of the 265th Ser for Gly.

SUMMARY OF THE INVENTION

The inventors of the present invention noted the fact that bovine individuals infected by BLV are classified into three characteristic pathological conditions, i.e., healthy individuals but positive to the antiviral antibody, those developing persistent lymphocytosis (PL) and those developing enzootic bovine leukosis, which is B lymphoma, after a long latent period, and the inventors made efforts to elucidate cause of the phenomenon at a genetic level. As a result, they found that various variants of the tax gene exist, and virus strains with a Tax protein including one or more particular mutations had enhanced transcription activity compared to wild-type virus strains.

The present inventors further continued researches, and found that mutations of the tax gene effected not only on the ability to activate the expression of the viral gene, but also on the strength of viral proliferation ability, and that viral strains having a particular variant tax gene had remarkably enhanced proliferation ability. The present invention was achieved on the basis of these findings.

The present invention thus provides a variant tax gene of bovine leukemia virus, which encodes a variant gene product containing one or more mutations selected from the group consisting of:

substitution of the 240th serine for threonine,

substitution of the 247th aspartic acid for glycine,

substitution of the 251st threonine for alanine,

substitution of the 258th aspartic acid for glycine,

substitution of the 261st histidine for arginine,

substitution of the 261st histidine for tyrosine, and

substitution of the 265th serine for glycine.

The present invention also provides a gene which contains the aforementioned variant tax gene and functions to enhance ability to induce replication of the bovine leukemia virus or a retrovirus related to the bovine leukemia virus (e.g., HTLV and HIV), preferably the bovine leukemia virus; and a gene which contains the aforementioned variant tax gene and functions to enhance ability to activate transcription of the bovine leukemia virus or a retrovirus related to the bovine leukemia virus, preferably the bovine leukemia virus.

According to another aspect of the present invention, there is provided a protein which is a product of a bovine leukemia virus tax gene and contains one or more mutations selected from the group consisting of:

substitution of the 240th serine for threonine,

substitution of the 247th aspartic acid for glycine,

substitution of the 251st threonine for alanine,

substitution of the 258th aspartic acid for glycine,

substitution of the 261st histidine for arginine,

substitution of the 261st histidine for tyrosine, and

substitution of the 265th serine for glycine.

The present invention also provides the aforementioned protein which enhances ability to induce replication of bovine leukemia virus or a retrovirus related to the bovine leukemia virus, preferably the bovine leukemia virus; and the aforementioned protein which enhances ability to activate transcription activity of bovine leukemia virus or a retrovirus related to the bovine leukemia virus, preferably the bovine leukemia virus.

According to preferred embodiments of the present invention, the aforementioned variant gene product is selected from those including substitution of the 240th amino acid, i.e., serine, for threonine, substitution of the 247th amino acid, i.e., aspartic acid, for glycine, or substitution of the 261st amino acid, i.e., histidine, for arginine. The gene of the present invention preferably encodes any one of the aforementioned preferred gene products.

According to another aspect of the present invention, there is provided a bovine leukemia virus having enhanced replication ability which contains the aforementioned gene. According to still another aspect of the present invention, there is provided a recombinant vector containing the aforementioned gene of the present invention which has enhancing ability to induce replication of bovine leukemia virus or a retrovirus related to the bovine leukemia virus, preferably the bovine leukemia virus.

According to still further aspects of the present invention, there are provided a method for producing a wild-type bovine leukemia virus, which comprises the steps of infecting a host cell introduced with a recombinant vector containing the aforementioned gene with a wild-type bovine leukemia virus, and culturing the resulting cell; and a method for enhancing ability to induce replication of bovine leukemia virus or a retrovirus related to the bovine leukemia virus, preferably a wild-type bovine leukemia virus, which comprises the step of expressing the aforementioned gene in a cell infected by the bovine leukemia virus or the retrovirus related to the bovine leukemia virus.

BEST MODE FOR CARRYING OUT THE INVENTION

The tax gene of the bovine leukemia virus has already been known, and the full sequence is disclosed in Proc. Natl. Acad. Sci. USA, 81, pp.4741-4745, 1984. The genes of the present invention are variants of the tax gene, which encode variant gene products of the bovine leukemia virus tax gene that contain, in the product of the bovine leukemia virus tax gene, substitution of the 240th amino acid, i.e., serine, for threonine; substitution of the 247th amino acid, i.e., aspartic acid, for glycine; substitution of the 251st amino acid, i.e., threonine, for alanine; substitution of the 258th amino acid, i.e., aspartic acid, for glycine; substitution of the 261st amino acid, i.e., histidine, for arginine; substitution of the 261st amino acid, i.e., histidine, for tyrosine; or substitution of the 265th amino acid, i.e., serine, for glycine. The gene of the present invention may contain two or more of these mutations in combination.

The gene of the present invention may be either DNA or RNA. The gene of the present invention can readily be obtained by, for example, the PCR (polymerase chain reaction) utilizing suitable primers Specific examples of the preparation are explained in detail in examples of the specification. The gene product of the gene of the present invention can be produced by introducing a vector for gene expression that contains the gene of the present invention into a host cell, and culturing the resulting transformed cell. A type of the host cell is not particularly limited. For example, microbial cells such as Escherichia coli, yeast, insect cells, animal cells and the like can be used.

The gene of the present invention has a function to enhance ability to induce the replication of the bovine leukemia virus, and also a function to enhance ability to induce transcription activity of the bovine leukemia virus. These functions of the gene of the present invention can readily be verified according to the methods specifically explained in the examples of the specification. In addition, the gene of the present invention has a function to enhance ability to induce replication of retroviruses related to the bovine leukemia virus, for example, human immunodeficiency virus (HIV), and a function to enhance ability to induce the transcription activity of such retroviruses.

The gene of the present invention encompasses any modified gene which includes substitution, insertion, and/or deletion of one or several nucleotides and has function to enhance ability to induce the replication or transcription activity that is substantially similar to that of the aforementioned specified genes. The gene product of the present invention encompasses any protein which is encoded by the aforementioned specified gene or the aforementioned modified gene and functions to enhance ability to induce the replication or transcription activity for the bovine leukemia virus or retroviruses related thereto.

Such modified genes can be obtained by subjecting, for example, Escherichia coli cells having a wild-type gene to a treatment to cause mutations by using a reagent such as N-nitro-N'-nitro-N-nitrosoguanidine, and recovering genes encoding modified proteins from the cells. Gene products of the resulting genes can be produced by a conventional method for gene expression. In addition, deletion, substitution, or addition of nucleotides may be directly introduced by, for example, direct treatment of the gene of the present invention with a reagent such as sodium sulfite, or alternatively, by the site-specific mutagenesis (Kramer, W. et al., Methods in Enzymology, 154, 350, 1987), the recombinant PCR (PCR Technology, Stockton press, 1989) or the like. Whether or not a modified gene has the desired promoting activity on virus replication can be readily determined by the method described in the examples of the specification.

Although the mode of using the gene of the present invention is not particularly limited, the gene can be used in, for example, a method for producing a recombinant virus having enhanced replication ability which comprises the steps of removing the tax gene from the bovine leukemia virus, and then introducing the gene of the present invention as foreign gene; a method for increasing replication rate of the bovine leukemia virus which comprises the steps of incorporating the gene of the present invention into a vector for gene expression, and then introducing the resulting recombinant vector into a BLV-infected cell; a method for increasing replication rate of the wild-type virus which comprises the steps of introducing the resulting recombinant vector into various cultured cells to allow expression of gene products in the host cells, and infecting the host cells with a wild-type virus or the like. However, methods of using the gene of the present invention are not limited to those examples.

The gene of the present invention is useful for studies of the replication mechanism of the bovine leukemia virus and retroviruses related thereto. The gene is also useful for studies to establish a method for preventive and therapeutic treatment of bovine leukemia, and methods for preventive and therapeutic treatment of infectious diseases caused by retroviruses related to the bovine leukemia virus. In the same manner, the gene product of the gene of the present invention is useful for studies of the replication mechanism of the aforementioned viruses, and studies to establish methods for preventive and therapeutic treatment of the aforementioned diseases.

Claim 1 of 1 Claim

What is claimed:

1. An isolated and purified variant bovine leukemia virus tax gene, which encodes a variant gene product containing one or more mutations selected from the group consisting of:

substitution of the 240th serine for threonine,

substitution of the 247th aspartic acid for glycine,

substitution of the 251st threonine for alanine,

substitution of the 258th aspartic acid for glycine,

substitution of the 261st histidine for arginine,

substitution of the 261st histidine for tyrosine, and

substitution of the 265th serine for glycine.




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