| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1992: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
"Intracellular immunization" using transdominant or dominant negative mutants, of viral proteins is considered to be a new approach to antiviral therapy in humans and to germ-line transformation in animals to conger resistance to virus infection. To obtain effective repressors of pseudorabies virus (PRV) immediate-early (IE) gene expression, a transdominant gene of PRV IE gene encoding an IE protein (IE180) and a chimeric gene encoding a fusion protein consisting of the DNA-binding domain of IE180 and tail-truncated Vmw65 lacking the transcription activation domain were constructed.
PRV IE180 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, weprepared various truncated mutants and analyzed their transcriptional regulatory, activities using the chloramphenicol acetyl transferase assay. Analysis of nutants truncated from the carboxy-terminal end of the 1460-amino acid polypeptide s
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howed that a polypeptide possessing amino acids 1 to 1063 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 the IE180. Additional amino-terminal truncation up to amino acid 427 did not affect the autoregulation activity, indicating that the region between amino acids 428 and 1063 has autoregulation potential. Finally, a CPK cell line stably transformed with the truncated gene showed resistance to PRV intection.
A chimeric gene encoding a fusion protein consisting of the DNA-binding domain of the IE180 of PRV and a tail-truncated virion protein, Vmw65, lacking the transcription activation domain of herpes simplex virus 1 was constructed. The chimeric gene product inhibited transcription from the PRV IE promoter in a transient expression assay. The denovo synthesis of infectious virus was suppressed following cotransfection with the chimeric gene expression plasmid and PRV genomic DNA.A HeLa cell line stably transformed with the chimeric gene showed remarkable resistance to PRV infection. In the transformed cells infected with PRV, the PRV IE gene transcription was repressed, indicating that the resistance to PRV infection was due interference with the IE gene transcription by the fusion protein.
These results indicate that the transdominant gene and the chimeric gene act as a powerful trans-gene for "intracellular immunization" against pseudorabies. Less
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