Related Experiment Videos
Characteristics of DNA-binding activity of human cytomegalovirus ppUL44
1Department of Microbiology, Seoul National University College of Medicine, and Institute of Endemic Diseases, Republic of Korea. hess@plaza.snu.ac.kr
Abstract:
Human cytomegalovirus (HCMV)-specific monoclonal antibody, SCMVM34, recognizes the early antigen encoded by UL44 of HCMV. This antigen is confined to the nucleus of HCMV-infected cells. This study was performed to characterize the DNA-binding activity of the protein encoded by UL44 of HCMV. The nuclear and cytoskeletal fraction of HCMV-infected cells was subjected to 0.4 M NaCl extraction, DEAE-Sephacel ion exchange chromatography, DNA-cellulose chromatography and SDS-PAGE analysis with monitoring of the reactive protein using SCMVM34 monoclonal antibody. The molecular weights of the resultant proteins were found to be 34, 40 and 52 kDa. The internal peptide fragments were isolated by tryptic digestion and reverse-phase HPLC. The internal amino acid sequence analysis of the peptides from the HPLC profile revealed that the antigen recognized by SCMVM34 monoclonal antibody was ppUL44. The reactive antigen began to be eluted from 250 mM NaCl (Tris-HCl pH 7.4) in DNA cellulose. The 34 kDa protein seems to bind to DEAE more tightly than the 52 kDa protein. The surface charge of 34 kDa might be more basic. Conclusively, the antigen recognized by SCMVM34 was the protein encoded by HCMV UL44, which was localized in the nuclei after HCMV infection, and was the DNA-binding protein with the characteristic that the surface charge of the molecule was more basic, as the molecular weights of the protein were decreased.
Insights
Human cytomegalovirus UL44 protein, recognized by SCMVM34 antibody, is a nuclear DNA-binding protein. Its basic surface charge increases as molecular weight decreases.
Area of Science:
- Virology
- Molecular Biology
- Immunology
Background:
- Human cytomegalovirus (HCMV) is a significant pathogen.
- HCMV-specific monoclonal antibody SCMVM34 targets an early antigen encoded by UL44.
- This antigen is localized to the nucleus of infected cells.
Purpose of the Study:
- To characterize the DNA-binding activity of the HCMV UL44 protein.
- To identify the specific protein recognized by the SCMVM34 antibody.
- To investigate the biophysical properties of the UL44 protein.
Main Methods:
- Extraction of nuclear and cytoskeletal fractions from HCMV-infected cells.
- Chromatographic techniques including DEAE-Sephacel ion exchange and DNA-cellulose chromatography.
- SDS-PAGE, tryptic digestion, and reverse-phase HPLC for protein analysis and sequencing.
Main Results:
- The SCMVM34 antibody recognized ppUL44, a protein encoded by HCMV UL44.
- Proteins with molecular weights of 34, 40, and 52 kDa were identified.
- The UL44 protein demonstrated DNA-binding activity, eluting at 250 mM NaCl from DNA cellulose.
- A more basic surface charge was observed for the 34 kDa protein compared to the 52 kDa protein.
Conclusions:
- The SCMVM34 antibody recognizes the HCMV UL44 protein, a nuclear DNA-binding protein.
- The UL44 protein exhibits varying molecular weights and a basic surface charge that correlates with decreased molecular weight.