Owens R A, Carter B J
Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892.
J Virol. 1992 Feb;66(2):1236-40. doi: 10.1128/JVI.66.2.1236-1240.1992.
An adeno-associated virus (AAV) genome with a Lys-to-His (K340H) mutation in the consensus nucleotide triphosphate binding site of the rep gene has a dominant-negative DNA replication phenotype in vivo. We expressed both wild-type (Rep78) and mutant (Rep78NTP) proteins in two helper-free expression systems consisting of either recombinant baculoviruses in insect cells or the human immunodeficiency virus type 1 long terminal repeat promoter in human 293 cell transient transfections. We analyzed nuclear extracts from both expression systems for the ability to complement uninfected HeLa cell cytoplasmic extracts in an in vitro terminal resolution assay in which a covalently closed AAV terminal hairpin structure is converted to an extended linear duplex. Although both Rep78 and Rep78NTP bound to AAV terminal hairpin DNA in vitro, Rep78 but not Rep78NTP complemented the terminal resolution assay. Furthermore, Rep78NTP was trans dominant for AAV terminal resolution in vitro. We propose that the dominant-negative replication phenotype of AAV genomes carrying the K340H mutation is mediated by mutant Rep proteins binding to the terminal repeat hairpin.
在rep基因的共有核苷酸三磷酸结合位点具有赖氨酸到组氨酸（K340H）突变的腺相关病毒（AAV）基因组在体内具有显性负性DNA复制表型。我们在两种无辅助病毒表达系统中表达野生型（Rep78）和突变型（Rep78NTP）蛋白，这两种系统分别是昆虫细胞中的重组杆状病毒或人293细胞瞬时转染中的人免疫缺陷病毒1型长末端重复启动子。我们在体外末端分辨率测定中分析了来自这两种表达系统的核提取物补充未感染的HeLa细胞细胞质提取物的能力，在该测定中，共价闭合的AAV末端发夹结构被转化为延伸的线性双链体。虽然Rep78和Rep78NTP在体外均与AAV末端发夹DNA结合，但Rep78而非Rep78NTP补充了末端分辨率测定。此外，Rep78NTP在体外对AAV末端分辨率具有反式显性作用。我们提出携带K340H突变的AAV基因组的显性负性复制表型是由突变的Rep蛋白与末端重复发夹结合介导的。
