Biochemical characterization of the major DNA-binding protein of murine cytomegalovirus
Date
1999-01-01
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ORCID
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Degree Level
Doctoral
Abstract
Murine cytomegalovirus (MCMV) has been extensively used as a model for Human cytomegalovirus (HCMV) infection due to its striking biological similarities to HCMV. The MCMV major DNA-binding protein (MDBP) is a non-structural protein produced during early times of viral infection. The protein encoded by the M57 open reading frame (ORF) of the viral genome has previously been shown to share significant amino acid sequence similarity to both HCMV single-stranded DNA-binding protein (ssDBP) and herpes simplex virus type 1 (HSV-1) ICP8. HSV-1 ICP8 is one of seven essential proteins required for viral DNA replication. In this study, I characterized the biochemical properties of the MCMV MDBP, especially the single-stranded DNA-binding domain and nuclear localization signals (NLSs) of the protein. To do this, the M57 ORF encoding MCMV MDBP was first cloned into an expression vector. The histidine-tagged MCMV MDBP fusion protein was produced and used to develop a panel of monoclonal antibodies (MAbs) which recognized epitopes located within the N- and C-termini of the MDBP. These MAbs were used to monitor the expression of wild type (wt) and mutant MDBPs during infection and transfection experiments, and facilitated the characterization of the biochemical properties of MCMV MDBP. Single-stranded DNA-cellulose column chromatography was carried out to assess the ssDNA-binding property of the wild type (wt) and mutant MDBPs; synthesized in an ' in vitro' coupled transcription-translation reaction. Nuclear localization signal regions were identified by transiently expressing the mutant MDBP gene in COS-1 cells and observing the subcellular localization of the expressed protein with indirect immunofluorescence. Gel filtration chromatography was performed to define the native molecular weight of the MDBP extracted from MCMV-infected cells. The results demonstrated that wt MDBP was eluted from the ssDNA-cellulose column at 250 mM NaCl. Deletion of a region containing a zinc finger-like motif completely abolished ssDNA-binding. However, the ssDNA-binding activity of the protein was found to reside largely in the C-terminal half of the protein. Partial deletion of a putative ssDNA-binding motif significantly affected ssDNA-binding. In indirect immunofluorescence experiments, the MDBP was localized to discrete areas of the nuclei of infected cells. In contrast, a more diffuse nuclear staining pattern was evident during transient expression of the protein in COS-1 cells. Although the C-terminus of the protein contains two clusters of basic residues capable of targeting a heterologous cytoplasmic protein to the nucleus, the N-terminus of the protein, which also contains such residues, appeared to constitute the major determinant for the nuclear localization of MCMV MDBP. Finally, while the protein migrated as a monomer with an apparent molecular weight of 133 K in SDS-PAGE, it was found mainly in high molecular weight complexes of in excess of 200 K in MCMV-infected cells. Collectively, these results indicate that MCMV MDBP is similar in many aspects but not identical to the previously reported HSV-1 counterpart, ICP8. (Abstract shortened by UMI.)
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Degree
Doctor of Philosophy (Ph.D.)
Department
Microbiology and Immunology
Program
Microbiology and Immunology