Functional characterization of Borna disease virus replication and persistence using recombinant viruses: Molecular analyses of the regulatory and anti-apoptotic activities of the accessory protein X.

在第一个资助期间，我们设法建立了一个协议，允许博尔纳病病毒（BDV）聚合酶复合物的成功体内重建。在该“微型复制子”测定中，编码BDV聚合酶亚基L、N和P的质粒与携带CAT报告基因的负义微型基因组RNA一起在BSR-T7细胞中表达。我们现在想用BDV微型复制子来回答有关病毒聚合酶复合物的组成和功能的基本问题。我们将确定BDV-N的p38和p39亚型的作用，我们将研究BDV-X和BDV-P的p16亚型抑制病毒聚合酶复合物活性的机制，我们将研究病毒因子对来自BDV第三转录单位的转录物剪接效力的影响。此外，我们将尝试完全从克隆的cDNA中建立BDV的拯救。一旦这项新技术开始工作，我们将尝试产生BDV突变体，例如，缺乏功能性X基因，以确定这种难以捉摸的病毒蛋白在细胞培养物以及小鼠和大鼠中的作用。

During the first funding period, we managed to establish a protocol that permits the successful in vivo reconstitution of the Borna disease virus (BDV) polymerase complex. In this "minireplicon" assay, plasmids encoding the BDV polymerase subunits L, N and P are expressed in BSR-T7 cells together with a negative-sense minigenome RNA harboring a CAT reporter gene. We now would like to use the BDV minireplicon to answer fundamental questions regarding the composition and function of the viral polymerase complex. We will determine the roles of the p38 and p39 isoforms of the BDV-N, we will study the mechanisms by which BDV-X and the p16 isoform of BDV-P inhibit the activity of the viral polymerase complex, and we will investigate the influence of viral factors on the splicing efficacy of transcripts from the third transcription unit of BDV. Further, we will try to establish the rescue of BDV entirely from cloned CDNA. As soon as this new technology is working, we will try to generate BDV mutants that, for example, lack a functional X gene in order to determine the role of this elusive viral protein in cell cultures as well as in mice and rats.