Intracellular immunization against Aujeszky's disease

使用病毒蛋白的反式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生殖系转化以抵抗病毒感染的新方法。为了获得伪狂犬病病毒（PRV）立即早期（IE）基因表达的有效阻遏物，编码IE蛋白（IE 180）的PRV IE基因的转显性基因和编码由IE 180的DNA结合结构域和尾-PRV IE 180是几种不同启动子的强反式激活因子，也是自身转录的抑制因子。为了定位IE 180的功能结构域，我们制备了各种截短突变体，并使用氯霉素乙酰转移酶测定来分析它们的转录调节活性。从1460个氨基酸多肽的羧基末端截短的突变体分析 ...更多信息 结果表明，具有氨基酸1至1063的多肽保留了重要的反式激活功能和自身调节潜力。另一方面，去除氨基酸1至132导致反式激活潜力的完全丧失，表明负责反式激活的结构域位于IE 180的氨基末端。额外的氨基末端截短至氨基酸427不影响自动调节活性，表明氨基酸428和1063之间的区域具有自动调节潜力。构建了PRV IE 180的DNA结合域与缺失HSV 1转录激活域的尾截短型病毒体蛋白Vmw 65的嵌合基因。嵌合基因产物在瞬时表达测定中抑制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，感染性病毒的合成受到抑制，稳定转染嵌合基因的HeLa细胞对PRV感染具有明显的抗性。在PRV感染的转化细胞中，PRV IE基因的转录被抑制，表明融合蛋白干扰了PRV IE基因的转录，从而产生了对PRV感染的抗性，表明转显性基因和嵌合基因是一种强有力的反式基因，可用于伪狂犬病的“细胞内免疫”。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