Intracellular immunization against Aujeszky's disease

利用病毒蛋白的跨显性或显性阴性突变体的“细胞内免疫”被认为是人类抗病毒治疗和动物种系转化以延长病毒感染抵抗力的新方法。为了获得有效抑制伪狂犬病毒（PRV）即刻早期（IE）基因表达的基因，构建了编码IE蛋白的伪狂犬病毒IE基因的跨显性基因（IE180）和编码由IE180的dna结合域和缺失转录激活域的尾部截断的Vmw65组成的融合蛋白的嵌合基因（chimeric gene）。PRV IE180是几种不同启动子的强反激活子，也是其自身转录的抑制子。为了绘制IE180的功能域，我们制备了各种截断突变体，并使用氯霉素乙酰转移酶测定分析了它们的转录调控活性。对含有1460个氨基酸的多肽羧基末端截短的nutants的分析表明，含有1 ~ 1063个氨基酸的多肽保留了显著的转激活和自调节电位功能。另一方面，去除氨基酸1 ~ 132导致完全丧失了转激活的潜力，这表明负责转激活的结构域位于IE180的氨基末端。对氨基酸427的氨基末端额外截断不影响自调节活性，表明428和1063之间的氨基酸区域具有自调节潜力。最后，用截断的基因稳定转化的CPK细胞系显示出对PRV感染的抗性。构建了由PRV IE180的dna结合域与缺乏单纯疱疹病毒1转录激活域的尾截断病毒粒子蛋白Vmw65组成的融合蛋白的嵌合基因。嵌合基因产物在瞬时表达实验中抑制了PRV IE启动子的转录。嵌合基因表达质粒与PRV基因组DNA共转染后，传染性病毒的脱氧合成受到抑制。嵌合基因稳定转化的HeLa细胞系对PRV感染表现出显著的抗性。在感染PRV的转化细胞中，PRV IE基因转录受到抑制，表明该融合蛋白干扰了IE基因转录，从而对PRV感染产生了抗性。这些结果表明，跨显性基因和嵌合基因作为一种强大的抗伪狂犬病“细胞内免疫”的转基因基因。少

"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 … More 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