Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-11-29
2001-05-22
Wortman, Donna C. (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C435S007210
Reexamination Certificate
active
06235467
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods for identifying substances capable of regulating the cell cycle. It further relates to the use of said substances in treating or preventing viral infection, cancer or cell death.
BACKGROUND TO THE INVENTION
Herpes simplex virus (HSV) has a virulence determining locus in the long repeat region of its genome (Ackermann et al., 1986; Chou and Roizman, 1990; McGeoch et al., 1991; Dolan et al., 1992). The virulence phenotype has been specifically assigned to the RL1 gene and its encoded protein ICP34.5 (McKie et al., 1994). Null mutants in ICP34.5 are totally avirulent in mice (Taha et al., 1989a, b; Chou et al., 1990; MacLean et al., 1991) and the function of the protein in vitro has been shown to be cell type and cell state specific, depending on the stage in the cell cycle and the differentiation state (Brown et al., 1994).
One ICP34.5 function demonstrated in a human neuroblastoma cell line is the preclusion of host cell protein synthesis shut-off via the protein kinase PKR pathway following HSV infection (Chou and Roizman, 1992; Chou et al., 1995). This response to expression of ICP34.5 is however not ubiquitous and the precise molecular functions of ICP34.5 remain unknown.
A 63 amino acid carboxy terminal domain of ICP34.5 has been shown to share significant homology (McGeoch and Barnett, 1991) with the carboxy domain of the mouse myeloid differentiation protein MyD116 (Lord et al., 1990) and the hamster growth arrest and DNA damage gene GADD34 (Fornace et al., 1989) although the amino terminal parts of the proteins are quite diverse. The role of MyD116 and GADD34 in the cell appears to be in blocking growth and DNA replication following damage and thus they may act as tumour suppressor genes. The HSV type 1 (HSV1) strain 17 ICP34.5 protein comprises 248 amino acids whereas MyD116 and GADD34 are 657 and 590 amino acids respectively. Chou and Roizman (1994) have demonstrated that the carboxy terminus 63 amino acids are essential but not necessarily sufficient for the host cell shut-off phenotype of ICP34.5 and can be replaced by the homologous domain of
SUMMARY OF THE INVENTION
The present invention is based on the finding that ICP34.5 and MyD116 both interact, via their conserved domain, with proliferating cell nuclear antigen (PCNA). PCNA plays a role in several key cellular processes associated with cell cycle control and the maintenance of genome integrity. PCNA is involved in nucleotide excision repair where it associates with replication factor C and DNA polymerase &egr; to form a component part of the DNA repair complexes. It is also involved in DNA replication where it acts as a processivity factor for eukaryotic DNA polymerase &dgr;. Further, PCNA forms a complex with p21
C1P1
, an inhibitor of cyclin dependent kinases. The levels of p21 are up-regulated by the tumour suppressor p53, which is in turn activated by DNA damage and other forms of cellular stress.
Thus, the findings on which the present invention is based indicate that the role of HSV ICP34.5 may be to prevent host cell shut-down and/or cell death in response to cellular stress induced by viral infection, allowing viral replication to continue. In particular, an interaction between the C-terminus of ICP34.5 and PCNA may prevent or modify the interaction of PCNA with other components of the cell cycle control machinery which would normally result in host cell shut-down and/or cell death. One of these components may be MyD116/GADD34 and their homologues since ICP34.5 shares sequence homology with a region of MyD116/GADD34 and our results demonstrate that both ICP34.5 and MyD116 can bind PCNA.
Several possibilities arise from these findings. It may be possible to prevent viral propagation or the establishment of viral infection by disrupting the interaction between ICP34.5 and PCNA.
Thus the present invention provides a method for identifying a substance capable of disrupting an interaction between (i) a herpes simplex virus ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) proliferating cell nuclear antigen (PCNA) or a homologue thereof, or a derivative thereof, which method comprises:
(a) providing an HSV ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, as a first component;
(b) providing PCNA or a homologue thereof, or a derivative thereof, as a second component;
(c) contacting the two components with a substance to be tested under conditions that would permit the two components to bind in the absence of the said substance; and
(d) determining whether the said substance disrupts the interaction between the first and second component.
The method of the invention may further comprise:
(e
1
) administering a said substance which has been determined to disrupt the interaction between the first and second components to a mammalian cell; and
(f
1
) determining the effect of the said substance on the cell cycle of the said cell.
The ability of the substance to induce cell cycle arrest may be determined. The ability of the substance to induce cell death by apoptosis may be determined.
Alternatively, the method of the invention may further comprise:
(e
2
) administering a virus to a cell in the absence of a said substance which has been determined to disrupt the interaction between the first and second components;
(f
2
) administering the virus to the cell in the presence of the said substance; and
(g
2
) determining if the said substance reduces or abolishes the susceptibility of the cell to viral infection.
The invention further provides a substance capable of disrupting an interaction between (i) a herpes simplex virus ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) PCNA or a homologue thereof, or a derivative thereof, for use in treating the human or animal body by therapy or for use in diagnosis, whether or not practised on the human or animal body. Such a substance may thus be used in the prevention or treatment of viral infection. Preferably the target virus has homology to a herpes simplex virus. More preferably the target virus is a herpes simplex virus. Preferably the substance is identified by the method of the invention.
Since MyD116 and GADD34 have sequence homology with ICP34.5 and we have shown that MyD116 can bind to PCNA, it is likely that at least some of the activities of MyD116/GADD34 are mediated via similar interactions and pathways to ICP34.5. MyD116/GADD34 are thought to be involved in blocking cell growth and DNA replication following cellular stress, including DNA damage. Furthermore, we have shown that MyD116 is expressed in a range of different cell types of different species and that expression is not dependent on the differentiation state of the cell. Thus MyD116 is likely to have a conserved role in cell cycle regulation.
The invention therefore further provides a substance capable of disrupting between (i) a herpes simplex virus ICP34.5 polypeptide or a homologue thereof, or a derivative thereof, and (ii) PCNA or homologues thereof, or derivatives thereof, for use in regulating the cell cycle of a mammalian cell. Again, preferably the substance is identified by the method of the invention. The substance may be used for inducing growth arrest and/or cell death. In that event, the mammalian cell is typically a tumour cell.
One function of ICP34.5 appears to be to prevent cell death induced by viral infection. This may be achieved by competing with MyD116/GADD34 or their homologues for PCNA. It may therefore be possible to prevent cell death in non-infected cells by inhibiting the activity of MyD116/GADD34 or their homologues. Thus the substance above may alternatively be used for preventing cell death. Preferably the cell is then a cell of the central or peripheral nervous system of a mammal, especially a human.
The invention also provides a method of regulating the cell cycle in a mammalian cell, which method comprises administering to said cell a substance capable of disrupting an interaction between (i) a herpes simplex virus ICP34.5 polypeptide or
Brown Susanne M.
Harland June
Maclean Alasdair R.
Medical Research Council
Nixon & Vanderhye PC
Wortman Donna C.
Zeman Robert A.
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