オーエスキー病ウイルス前初期蛋白の構造と機能 Mapping of Functional Domains of Aujeszky's Disease Virus Immediate-early Protein

博士論文

The 180 kilodalton immediate-early protein (IE180) of Aujeszky's disease virus functions as a strong transactivator of several different promoters and also as a repressor of its own transcription. To map the functional domains of IE180, a series of truncated mutants were prepared. Their transcriptional regulatory activities were analyzed using the chloramphenicol acetyl transferase (CAT) assay for transinduction of a thymidine kinase promoter-CAT gene construct and a glycoprotein X promoter-CAT gene construct and negative regulation of an IE promoter-CAT gene construct. IE180 is localized predominantly in the nuclei of infected cells. To define the nuclear localization signals within IE180, cells transfected with each truncated mutant was analyzed by indirect immunofluorescence. Analysis of mutants truncated from the carboxyterminal end of the 1,460-amino acid polypeptide showed that a polypeptide possessing amino acids 1 to 1,081 retained significant functions of transactivation and autoregulation potential. On the other hand, removing amino acids 1 to 132 resulted in a complete loss of transactivation potential, indicating that the domain responsible for transactivation is located in the amino-terminal end of IE180. Additional amino-terminal truncation up to amino acid 453 did not affect the autoregulation activity, indicating that the region between amino acids 454 and 1081 has autoregulation potential. Indirect immunofluorescence analysis of the truncated mutants showed that two regions including a short sequence of basic amino acid residues (RRKRR) were associated with the nuclear localization of IE180. To assess which region substantially functions as the signal for nuclear localization of IE180 molecule, two deletion mutants lacking each region were constructed. A mutant lacking amino acids 333 to 575 was detected in the nuclei of the transfected cells, whereas the other mutant lacking amino acids 900 to 950 was detected mainly in the cytoplasm. These results suggest that the region of amino acids 900 to 950 is responsible for nuclear localization of IE180.