Characteristics of DNA-binding activity of human cytomegalovirus ppUL44.
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.
Hwang
E S
ES
Department of Microbiology, Seoul National University College of Medicine, and Institute of Endemic Diseases, Republic of Korea. hess@plaza.snu.ac.kr
Kim
J
J
Jong
H S
HS
Park
J W
JW
Park
C G
CG
Cha
C Y
CY
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000956:Antigens, Viral / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D003587:Cytomegalovirus / Q000378:metabolism*; D003586:Cytomegalovirus Infections / Q000821:virology*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D007118:Immunoassay; D008969:Molecular Sequence Data; D014764:Viral Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; D004268:DNA-Binding Proteins; C072217:ICP36 protein, Cytomegalovirus; D014764:Viral Proteins; D004247:DNA