Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-03-29
2001-03-20
Sisson, Bradley L. (Department: 1655)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S005000, C435S006120, C435S091100, C435S091200, C435S183000, C536S023720
Reexamination Certificate
active
06204028
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a fast and convenient method that can be used to screen thousands of compounds for their potential to perturb or disrupt DNA synthesis by selected human herpesvirus polymerase/processivity factor proteins. The method of the present invention is useful in identifying potential antiviral agents against selected human herpesviruses including, but not limited to, human herpesvirus-8 and human herpesvirus-6.
BACKGROUND OF THE INVENTION
The first step in determining the potential antiviral activity of compounds is to use an initial or primary screen to identify whether a particular compound has some degree of antiviral activity. Since primary screening often involves large numbers of compounds, it is preferred that the assay system be automated to reduce human resources required and supply costs. Initial screens are typically kept quite simple, using a single method, one virus isolate for each virus group and a single cell line, preferably of human origin. Two basic types of screening assays have been used, depending upon whether the particular virus replicates in tissue culture cells and produces some type of cellular destruction or morphological change (cytopathic effect; CPE). For viruses that produce CPE, such as herpes simplex virus, the most commonly used assay systems are those that measure inhibition of CPE, focus formation, or syncytial formation. Nucleic acid hybridization is also used for viruses such as cytomegalovirus that may take a long time in culture to produce CPE, or for viruses such as Epstein Barr virus that do not produce CPE in cell culture systems but do replicate their DNA. Other assay systems that do not depend on complete viral replication with the production of CPE monitor synthesis of specific gene products such as P24 for HIV and enzymes such as thymidine kinase, DNA polymerase, or reverse transcriptase.
Human herpesvirus 6 (HHV-6) is the causative agent of roseola infantum, a common disease of infancy characterized by high fever and skin rash. This herpesvirus has also been suggested to have a role in mononucleosis, multiple sclerosis, pneumonitis and bone marrow suppression in transplant rejections (Ablashi et al.
Human Herpesvirus-
6.
Perspectives in Medical Virology
, A. J. Zuckerman, Editor, 1992, Vol. 4, Elsevier, Amsterdam; Braun et al.
Clin. Microbiol. Rev.
1997 10:521-567). HHV-6 is tropic for CD4+ T cells, which are also the natural targets for HIV infectivity. Thus, in AIDS patients, HHV-6 may contribute to the attrition of T cells, which is consistent with its potential catalytic role in HIV infection (Ablashi et al.
Human herpesvirus-
6.
Perspectives in Medical Virology
. A. J. Zuckerman, Editor, 1992, Vol.4. Elsevier, Amsterdam.; Braun et al.
Clin. Microbiol. Rev.
1997 10:521-567; Lusso, P. and Gallo, R. C.
Immunol. Today
1995 16:67-71) and its actual detection in many different necropsied tissues (Corbelliono et al.
Lancet
1993 342:1242; Knox, K. K. and Carrigan, D. R.
Lancet
1994 343:577-578). HHV-6 has a linear DNA genome of 160 kbp which is now completely sequenced (Gompels et al.
Virology
1995 209:29-51); there are 119 open reading frames predicted, of which 67% have counterparts in human cytomegalovirus (HCMV). Although certain structural and functional genes have been identified (Braun et al.
Clin. Microbiol. Rev.
1997 10:521-567), little is known about the nature of many HHV-6 gene products and the manner in which the virus is regulated.
Human herpesvirus-8 (HHV-8) is a recently identified herpesvirus which is the apparent causative factor of Kaposil's sarcoma, the most common neoplasm of AIDS patients (Chang et al.
Science
1994 266:1865-1869). Accordingly, HHV-8 is oftentimes referred to as Kaposils sarcoma-associated herpesvirus (KSHV). KSHV is also found in Kaposi's sarcoma tumors of non-AIDS patients. The causative role of KSHV in formation of these neoplasms is strongly supported by seroconversion to viral antigens prior to the clinical appearance of Kaposi's sarcoma. KSHV is also associated with body cavity based lymphoma or pleural effusion lymphoma (Cesarman et al.
N. Engl. J. Med.
1995 332:1186-1191) and multicentric Castleman's disease (Soulier et al.
Blood
1995 86:1276-1280). In Kaposi's sarcoma and pleural effusion lymphoma, malignant cells harbor the virus. Recently, KSHV was suggested to be involved in the bone marrow cancer, multiple myeloma and monoclonal gammopathy of undetermined significance (Rettig et al.
Science
1997 276:1851-1854; Said et al.
Blood
1997 90:4278-4282; Brooks et al.
J. Pathol.
1997 182:262-265; Levy. J. A.
Lancet
1997 349:558-563; Neipel et al.
J. Virol.
1997 71:4187-4192).
Accordingly, there is a need for rapid screening methods to identify potential antiviral agents specific against these and other human herpesviruses.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of screening compounds to identify potential antiviral agents against a selected herpesvirus which comprises measuring DNA synthesis by DNA polymerase and processivity factor of the selected human herpesvirus in the absence and presence of a compound wherein inhibition of DNA synthesis in the presence of the compound is indicative of antiviral activity against the selected human herpesvirus.
DETAILED DESCRIPTION OF THE INVENTION
Processivity factors associate with certain DNA polymerases, allowing them to synthesize extended stretches of DNA without dissociating from the template. Two such well-studied proteins, PCNA (proliferating cell nuclear antigen) of yeast and the &bgr; subunit of
Escherichia coli
(
E. coli
) DNA polymerase III holoenzyme, have been crystallized (Kong et al.
Cell
1992 69:425-437; Krishna et al.
Cell
1994 79:1233-1243). They confer processivity by forming topological rings encircling duplex DNA that tether their polymerases and slide along the DNA template. Several herpesvirus processivity factors have been identified, including UL42 of herpes simplex virus type 1 (HSV-1) (Gottlieb et al.
J. Virol.
1990 64:5976-5987), BMRF1 of Epstein-Barr virus (EBV) (Kiehl, A. and Dorsky, D. I.
J. Virol.
1995 69:1669-1677; Tsurumi et al.
J. Virol.
1993 67:7648-7653), and ICP36 of HCMV (Ertl, P. F. and Powell, K. L.
J. Virol.
1992 66:4126-4133; Mocarski et al.
Proc. Natl. Acad. Sci. USA
1985 82:1266-1270; and Weiland et al.
Virus Res.
1994 34:191-206). While the structure and mechanism by which these viral proteins confer processivity upon their polymerases remains unclear, herpesvirus processivity factors have been shown to be critical for viral replication (Johnson et al.
J. Virol.
1991 65:700-710; Ripalti et al.
J. Virol.
1995 69:2047-2057). Significantly, the UL42 null HSV-1 mutant failed to infect and replicate in Vero cells (Johnson et al.
J. Virol.
1991 65:700-710). Also, an antisense mRNA targeted against ICP36 strongly inhibited CMV replication (Ripalti et al.
J. Virol.
1995 69:2047-2057).
The 41 kD early antigen of human herpesvirus 6 (HHV-6), which exhibited nuclear localization and DNA-binding activity (Agulnick et al.
J. Gen. Virol.
1993 74:1003-1009) has now been confirmed to co-immunoprecipitate with a 110 kD protein identified to be the HHV-6 DNA polymerase (Pol-6) by an antibody raised against Pol-6. Co-immunoprecipitation experiments using anti-p41 and anti-Pol-6 antibodies confirmed that p41 complexed with Pol-6 in HHV-6 infected cells. In addition, both p41 and Pol-6 were synthesized in vitro and shown to form a specific complex. An in vitro polymerase assay using primed M13 single-stranded DNA template demonstrated that p41 functions as the processivity factor of HHV-6. Not only did p41 greatly increase the DNA synthesis activity of Pol-6, it allowed Pol-6 to synthesize DNA products corresponding to full-length M13 template (7,249 nucleotides), whereas Pol-6 alone could only synthesize DNA of less than 100 nucleotides. Moreover, the functional interaction between Pol-6 and p41 appears to be specific, since they failed to be
Lin Kai
Ricciardi Robert P.
Law Offices of Jane Massey Licata
Lu Frank
Sisson Bradley L.
The Trustees of the University of Pennsylvania
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