Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1997-09-19
2001-04-17
Campbell, Eggerton A. (Department: 1656)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S006120
Reexamination Certificate
active
06218522
ABSTRACT:
TECHNICAL FIELD
The present invention relates to DNA molecules and novel proteins associated with the repression of gene expression. In particular, the present invention relates to a DNA molecule having a gene expression repressing function, derived from human T-cell leukemia virus type I, and a plasmid including the DNA molecule. The present invention also relates to a protein which binds to a transcriptional repressive region existing in the U
5
region of human T-cell leukemia virus type I gene LTR; a structural gene for the protein; an expression vector including the gene; a transformant into which the expression vector is introduced; and a process, using the above transformant, for producing a protein which binds to a transcriptional repressive region existing in the U
5
region of human T-cell leukemia virus type I gene LTR. Furthermore, the present invention relates to an antiviral agent containing the protein, and a method for detecting cancer that utilizes the expression of the protein as an indicator.
BACKGROUND ART
In 1980, Human T-cell leukemia virus type I (HTLV-
1
) became the first retrovirus discovered in humans. It has been revealed that infection by this virus causes diseases such as adult T-cell leukemia (ATL), HTLV-I associated myelopathy (HAM), and tropical spastic paraparesis (TSP), and that the symptoms of such diseases develop after a long period of time in the body, with an average latency of about 40 to 50 years after the infection with the virus. However, little is known about the mechanisms of its latent infection and onset.
The gene of HTLV-I includes a tax/rex region in addition to three regions which are common to animal retroviruses, i.e., a gag region, a pol region, and an env region. The tax/rex region, which is located downstream of env, is considered to have an important role in the expression of viral gene and the onset of ATL. The mRNA of tax/rex results after double splicings from a primary transcript of the HTLV-I gene, and includes two overlapping open reading frames. From one open reading frame is translated a 40 kilodalton protein called Tax, which acts on the LTR of the virus itself as well as promoters of various genes in cells to activate transcription. From the other open reading frame is translated a 27 kilodalton protein called Rex, which controls the processing of the viral RNA occurring within the nucleus after transcription, and positively acts on the transport to the cytoplasm of unspliced mRNA.
From a different start codon within the same open reading frame as that of the Rex protein, a 21 kD protein called p21X is translated. The inventors revealed that p21X is translated from the p21X mRNA which lacks the second exon through single splicing from the HTLV-I gene (Orita et al. FEBS Lett., 295, 127-134 (1991)). However, the functions of the protein are unknown. The inventors further discovered that the p21X mRNA is expressed by a mutant provirus having a deletion in a broad region encompassing the gag, pol, and env regions in a HTLV-I infected cell line (Orita et al. Nucleic Acids, Res., 21, 3799-3807 (1993)). Moreover, the inventors discovered that the p21X mRNA is also expressed by the abovementioned mutant provirus in peripheral blood lymphocytes of patients infected with HTLV-I. On the other hand, it is known that the expression of mRNA for the tax/rex region from a complete provirus is rarely observed in the peripheral blood of patients infected with HTLV-I, and is detectable in vivo only by the RT-PCR method, which is an ultra-sensitive detection method. However, once peripheral blood lymphocytes of patients infected with HTLV-I are transferred to a culture system in vitro, its expression is known to become high enough to be easily detected (Kiyokawa et al., Proc. Natl. Acad. Sci. USA., 82, 8359-8363 (1985)). Moreover, the expression of the p21X mRNA has also been found to be on the same level before and after the culture of the aforementioned peripheral blood lymphocytes of patients infected with HTLV-I (Orita et al., J. Gen. Virol., 73, 2283-2289 (1992)). Therefore, it was considered that, in vivo, the p21X mRNA is expressed without being repressed, whereas the expression of the mRNA for tax/rex is repressed.
It is considered that HTLV-I and human immunodeficiency virus (HIV), which is a kind of RNA virus, delays splicing reactions by making the splicing signal within a DNA sequence a non-optimal one, thereby providing time for the Rex protein of HTLV-I or the Rev protein of HIV to repress splicing reactions (Chang, D. D. and Sharp. P. A., Science, 249, 614-615 (1990)). It is considered that sufficient amounts of the Rex protein and the Rev protein need to be expressed and accumulated within the nucleus in order to be polymerized, before their functions can be exhibited to promote the repression of the splicing of the viral mRNA having RXE (Rex responsible element) or RRE (Rev responsible element) and the transport to the cytoplasm, thereby triggering the replication of the viruses (expression of the structural proteins) (Inoue et al., Proc. Natl. Acad. Sci. USA., 84, 3653-3657 (1987); Hidaka et al., EMBO J., 7, 519-523 (1988); Seiki et al. Proc. Natl. Acad. Sci. USA., 85, 7124-7128(1988); and Hanly et al. Genes Dev., 3, 1534-1544(1989)).
The above indicates that at least two regulatory factors, i.e., Tax and Rex proteins, are translated from the pX region characteristic of HTLV-I, and that these factors are necessary for the replication of HTLV-I. Tax is a transcription activation factor which acts on the LTR (long terminal repeat) and also activates various cellular genes. Tax also has an ability to transform certain types of cultured cells. Thus, the possibility of Tax being involved in the oncogenesis of a cell by HTLV-I is suggested.
The expression of the HTLV-I gene is known to be very low in periods of inapparent infection and to be low even after the onset of ATL. Therefore, in order to elucidate the mechanism of latent infection of HTLV-I, it is considered important to study the expression repression mechanism of the viral gene. It is known that the expression control of the HTLV-I gene is performed mainly on the LTR of HTLV-1. The LTR region is subdivided into three regions called U
3
, R, and U
5
. The U
3
region includes a sequence on which Tax acts, as well as sequences acted upon by CREB, ETS, AP1, etc., which are cellular transcription activation factors. The R region is known to include a sequence to which YB-1 binds to activate transcription (Kashanchi et al. J. Virol., 68(1): 561-565 (1994)). Moreover, the R and U
5
regions include a region which functions repressively with respect to the HTLV-I gene expression on the transcription level or post-transcriptionally (Xu et al., Mol. Cell. Biol., 14(8): 5371-5383 (1994); and Seiki et al., Virology, 176: 81-86 (1990)). Furthermore, the inventors recently discovered a novel transcriptional repressive sequence (U
5
repressive element; U
5
RE), and reported the existence of three proteins of 110 kDa, 80 kDa, and 70 kDa which specifically bind to U
5
RE (Okumura et al. FEBS Let., 356: 94-100 (1994)).
DISCLOSURE OF THE INVENTION
In order to elucidate the gene expression repression mechanism of human T-cell leukemia virus type I, the inventors conducted studies on gene sequences and proteins associated with expression repression.
First, the inventors predicted that a region which represses the expression of the viral gene exists in a region which is missing in a mutant provirus of human T-cell leukemia virus type I (HTLV-I) that is expressing p21X mRNA but exists in the genome of a complete provirus. Accordingly, the inventors incorporated a portion of a DNA sequence of the genome of the virus into a plasmid and conducted studies using an assay system that utilized the expression of the CAT gene as an indicator. As a result, the inventors discovered two gene expression repressive regions in the pol region.
Therefore, according to one aspect of the present invention, there is provided a DNA molecule having a gene expression repressing function derived from
Igarashi Hisanaga
Okumura Kouichi
Orita Satoshi
Saiga Akihiko
Sakaguchi Gaku
Campbell Eggerton A.
Fish & Neave
Haley Jr. James F.
Shionogi & Co. Ltd.
Weissman Jennifer T.
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