Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1998-05-18
2001-04-17
Wortman, Donna C. (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C514S012200
Reexamination Certificate
active
06218511
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel compositions and methods of screening for targets for antiviral chemotherapy having anti-apoptotic activity, and compositions and methods of screening for antiviral compounds that interfere with the anti-apoptotic activity of such targets. The targets comprise viral polypeptides having anti-apoptotic activity, and polynucleotides encoding such polypeptides. Examples of such targets are viral polypeptides of human cytomegalovirus (HCMV) having anti-apoptotic activity, such as pUL36, pUL37
S
, and pUL37
L
, and the polynucleotides encoding such polypeptides. The antiviral compounds comprise polypeptide, polynucleotide, DNA, RNA, amino acid, nucleic acid, and chemical compositions, including modified forms of such compositions, that interfere with the anti-apoptotic function of the target polypeptides and polynucleotides, leading to the induction of apoptosis and, consequently, the prevention or inhibition of replication.
BACKGROUND OF THE INVENTION
Human cytomegalovirus (HCMV) is widespread in human populations (Britt and Alford, 1996). Congenitally infected newborns and immunocompromised individuals, such as those undergoing organ transplants and those with malignancies receiving immunosuppressive chemotherapy, and particularly patients with AIDS, are at greatest risk of HCMV-induced diseases. These diseases range from developmental abnormalities, mental retardation, deafness, mononucleosis, chorioretinitis to fatal diseases like interstitial pneumonitis and disseminated HCMV infections (Huang and Kowalik, 1993; Britt and Alford, 1996).
Antiviral treatments seek to prevent or arrest viral replication. Today there are only very limited treatment options available for cytomegalovirus infections, and treatment is often associated with high toxicity and the generation of drug resistance (Hirsch, 1994; White and Fenner, 1994; Lalezari et al., 1997). Several potential drug development targets for herpesviruses have been identified (White and Fenner, 1994, page 267, Table 16.1). Currently available agents, Gancyclovir, Foscarnet (PFA, phosphonoformic acid) and Cidofovir all act as inhibitors of viral DNA polymerase.
Animals and humans have developed an intricate system of defenses against viral infections. One important defense mechanism activates the cellular suicide program, or apoptosis, in the virally-infected host cell, thereby preventing the replication of the virus. Conversely, viruses have evolved to overcome the defenses of the host. Of particular interest, in the context of this application, is that many viruses have evolved to carry genes that can prevent or retard the onset of apoptosis in the virally-infected cells. This inhibition of apoptosis by viral gene products is achieved by a variety of mechanisms, examples of which include: 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., J. Biol. Chem. 272, 9621-9624 (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). Some of these 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.
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 which induces several cellular anti-apoptotic genes, e.g., LMP-1 of Epstein-Barr virus (Henderson et al., 1991; Wang et al., 1996; Fries et al., 1996); 3) a polypeptide interacting with FLICE (also called caspase-8), e.g., Equine herpesvirus type 2 polypeptide E8 (Bertin et al., 1997; Hu et al., 1997); and 4) two polypeptides with anti-apoptotic properties with a yet poorly characterized mechanism, ICP4 and U
S
3 of HSV-1 (Leopardi and Roizman, 1996; Leopardi et al., 1997). However, little is known about the ability of HCMV to regulate apoptosis in HCMV-infected cells.
The HCMV genome (AD 169 strain) has been completely sequenced (Chee et al. 1990; Mocarski, 1996). From the sequence, 208 ORFs (open reading frames) of greater than 300 base pair lengths were predicted and given names. However, for many of these ORFs the predicted corresponding polypeptides have not been directly identified. Whether most of these genes are expressed and have any functional importance for HCMV replication remains unknown. In fact, a number of these ORFs were found to be dispensable for the replication and/or propagation of HCMV in cultured cells (Mocarski, 1996).
Although Zhu et al. (1995) proposed that two polypeptides encoded in the HCMV genome, IE1 and IE2, have an anti-apoptotic activity in HeLa cells, these data were not independently confirmed by other researchers. Moreover, the present inventors demonstrated that IE1 and IE2 did not display any anti-apoptotic activity (see below). No other HCMV anti-apoptotic genes have been identified yet, and no homology to any of the known anti-apoptotic polypeptides has been found in the HCMV genome.
Furthermore, little was known about the UL36 and UL37 genes of HCMV. On the basis of DNA sequence analysis and RNA transcription studies, it was predicted that UL36 has two exons which encode a polypeptide product pUL36, and that UL37 encodes two polypeptide products; pUL37
S
encoded by the first exon, and pUL37
L
encoded by all three exons (Chee et al., 1990; Tenney and Colberg-Poley, 1991a,b). The expression of pUL37
L
in HCMV-infected cells has been detected. However, up until now, it has not been clear whether the hypothetical polypeptide pUL37
S
was expressed in HCMV-infected cells.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of methods for detecting the anti-apoptotic function of viral polypeptides and their interactions with cellular polypeptides. The present invention further relates to the discovery of several viral polypeptides having anti-apoptotic activity and also the mechanism by which such viral polypeptides function to inhibit apoptosis. In particular, interference with the anti-apoptotic function of viral polypeptides, and the polynucleotides encoding such polypeptides, can lead to the restoration or induction of apoptosis in virally-infected cells and, consequently, prevent or inhibit viral replication. Thus, these findings provide an approach for the rational design of antiviral drugs that interfere with the anti-apoptotic function of viral polypeptides and polynucleotides, thereby leading to the regulation of apoptosis, including the restoration or induction of apoptosis.
Accordingly, the object of the present invention is to provide compositions and methods of screening for targets for antiviral chemotherapy, having anti-apoptotic activity, and compositions and methods of screening for antiviral compounds that interfere with the anti-apoptotic function of such viral targets.
An embodiment of the present invention provides methods for identifying viral polypeptides havin
Bartle Laura
Goldmakher Viktor S.
Skaletskaya Anna
Apoptosis Technology, Inc.
Sughrue Mion Zinn Macpeak & Seas, PLLC
Wortman Donna C.
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