Compounds, methods of screening, and in vitro and in vivo...

Details Compounds, methods of screening, and in vitro and in vivo... Compounds, methods of screening, and in vitro and in vivo...

2000-11-21

2003-08-12

Wortman, Donna C. (Department: 1648)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving virus or bacteriophage

C435S006120, C435S007100, C435S007210

Reexamination Certificate

active

06605426

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to the field of cell physiology, and more particularly, to apoptosis. More specifically, the present invention relates to methods for detecting the anti-apoptotic activity and function of viral polypeptides and their interactions with cellular polypeptides. The present invention further relates to viral polypeptides having anti-apoptotic activity and also the mechanism by which such viral polypeptides function to regulate or modulate apoptosis or cell death. Moreover, the present invention relates to compounds that regulate or modulate the anti-apoptotic activity of viral polypeptides, and expression of polynucleotides encoding such polypeptides. Such regulation or modulation of anti-apoptotic activity can lead to the restoration or induction of apoptosis in virally-infected cells and, consequently, inhibit or diminish viral replication.
The present invention relates to novel compounds that regulate or modulate apoptosis and/or anti-apoptotic activity, and uses and methods of screening for such compounds, e.g., 1) novel compounds having anti-apoptotic activity, such as anti-apoptotic polypeptides and the polynucleotides encoding such polypeptides, and in vitro and in vivo uses and methods of screening for such compounds; and 2) novel compounds that inhibit or diminish the anti-poptotic activity of anti-apoptotic polypeptides and/or expression of polynucleotides encoding such polypeptides, and in vitro and in vivo uses and methods of screening for such compounds; wherein the compounds include diagnostic and/or therapeutic compounds, and wherein the uses include diagnostic and/or therapeutic uses.
The polypeptides, and polynucleotides encoding such polypeptides, comprise viral polypeptides having anti-apoptotic activity, and/or polynucleotides encoding such polypeptides, respectively. Examples of such polypeptides are viral polypeptides of human cytomegalovirus (HCMV) having anti-apoptotic activity, such as pUL36, pUL37
S
, pUL37
M
, and pUL37
L
.
The compounds that regulate or modulate apoptosis and/or anti-apoptotic activity comprise polypeptide, polynucleotide (e.g., DNA and/or RNA), amino acid, nucleotide, and/or chemical compounds, including analogs and/or modified forms of such compounds, and/or synthetic and/or chemical compounds.
BACKGROUND OF THE INVENTION
“Apoptosis” refers to programmed cell death which occurs by an active, physiological process (Kerr, J. F., et al., 1972; Wyllie, A. H., 1980). Cells that die by apoptosis undergo characteristic morphological changes, including cell shrinkage and nuclear condensation and fragmentation. Apoptosis plays an important role in developmental processes, including morphogenesis, maturation of the immune system, and tissue homeostasis whereby cell numbers are limited in tissues that are continually renewed by cell division (Ellis, R. E., et aL, 1991; Oppenheim, R. W., et al., 1991; Cohen, J. J., et al., 1992; Raff, M. C. 1992). Moreover, apoptosis is an important cellular safeguard against tumorigenesis (Williams, G. T., 1991; Lane, D. P., 1993). Defects in the apoptotic pathway causing disregulated or aberrant apoptosis may contribute to the onset or progression of malignancies. Under certain conditions, cells undergo apoptosis in response to the forced expression of oncogenes, or other genes that drive cell proliferation (Askew, D., et al., 1991; Evan, G. I., et al., 1992; Rao, L., et al., 1992; Smeyne, R. J., et al., 1993).
A variety of diseases and degenerative disorders may involve aberrant or disregulated apoptosis, resulting in inappropriate or premature cell death or inappropriate cell proliferation (Barr, P. J., et al., 1994). For example, inhibition of cell death may contribute to disease in the immune system by allowing the persistence of self-reactive B and T cells, which consequently promotes autoimmune disease (Watanabe-Fukunaga et al., 1992). Moreover, cancer may result when cells that fail to die undergo further mutations leading to a transformed state of the cells (Korsmeyer, S. J., 1992).
The productive infection by certain viruses may depend on suppression of host cell death by anti-apoptotic viral gene products (Rao, L., et al., 1992; Ray, C. A., et al., 1992; White, E., et al., 1992; Vaux, D. L., et al., 1994), and inhibition of apoptosis can alter the course (i.e., lytic vs. latent) of viral infection (Levine, B., et al., 1993). Moreover, the widespread apoptosis of T lymphocytes triggered by HIV infection may, at least in part, be responsible for the immune system failure associated with AIDS (Gougeon, M., et al., 1993). The roles of apoptosis in normal and pathological cell cycle events are reviewed in Holbrook, N.J. et al., 1996. Importantly, apoptosis comprises an important antiviral defense mechanism in animals and humans by providing the means to rapidly eliminate virally infected cells and restrict viral propagation (O'Brien, 1998; Tschopp et al., 1998). Apoptosis of virally infected cells is triggered by killer cells of the immune system via Fas-ligand interaction with Fas and by granzyme-B-triggered caspase activation (Nagata and Golstein, 1995; Smyth and Trapani, 1998).
To counteract the host defense mechanism, many viruses encode genes that function to inhibit or diminish apoptosis in infected cells (O'Brien, 1998; Tschopp et al., 1998). This inhibition or diminution of apoptosis by viral gene products is achieved by a variety of mechanisms, including: 1) blocking and/or destruction of p53; 2) direct interaction with cellular polypeptides of apoptotic pathways, such as death-effector-domain-containing polypeptides [death-effector-domain motifs are defined in Hu et al., 1997], Bcl-2 family members, and caspases; or 3) by induction of cellular anti-apoptotic polypeptides (Pilder et al., 1984; Gooding et al., 1988; Clem et al., 1991; Hershberger et al., 1992; Brooks et al., 1995; Sedger and McFadden, 1996; Leopardi and Roizman, 1996; Leopardi et al., 1997; Razvi and Welsh, 1995; Teodoro and Branton, 1997; Vaux et al., 1994; Shen and Shenk, 1995; Duke et al., 1996; Vaux and Strasser, 1996; Thompson, 1995).
The prevalence and evolutionary conservation of anti-apoptotic viral genes suggests that suppression of apoptosis is a critical component of efficient viral propagation and/or persistence in vivo. In fact, some of the anti-apoptotic genes were found to be essential for the ability of the respective viruses to replicate and propagate. For example, mutants of human adenovirus that lack the expression of the E1B 19 kDa adenoviral analog of Bcl-2 induce massive apoptosis of infected cells (Teodoro and Branton, 1997) which, consequently, leads to reduced viral titers.
Human cytomegalovirus (HCMV) is widespread in human populations, and is of substantial clinical importance principally because of its pathogenicity in developing fetuses and immunocompromised individuals (Huang and Kowalik, 1993; Britt and Alford, 1996). In particular, those immunocompromised individuals undergoing organ and tissue transplants, or that have malignancies and are receiving immunosuppressive chemotherapy, or that have AIDS, are at greatest risk of HCMV-induced diseases. These diseases range from developmental abnormalities, mental retardation, deafness, mononucleosis, and chorioretinitis, to fatal diseases like interstitial pneumonitis and disseminated HCMV infections (Huang and Kowalik, 1993; Britt and Alford, 1996).
Human cytomegalovirus (HCMV) is a herpesvirus (Roizman, 1991). A number of herpesviruses were shown to induce an apoptotic host cell response, and to suppress this virus-induced apoptosis in the infected cells (Leopardi and Roizman, 1996; Leopardi et al., 1997; Bertin et al., 1997; Sieg et al., 1996). The genomes of several herpesviruses code for a variety of anti-apoptotic polypeptides such as: 1) Bcl-2 homologs, e.g., BHRF-1 of Epstein-Barr virus (Henderson et al., 1993), vbcl-2 of Kaposi's sarcoma-associated herpesvirus (Sarid et al., 1997), and ORF16 of herpesvirus Saimiri (Nava et al., 1997); 2) a polypeptide that indu

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