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.

在第一个资助期内，我们设法建立了一个方案，使博纳病病毒（BDV）聚合酶复合物能够成功地在体内重组。在这个“微型复制子”实验中，编码BDV聚合酶亚基L、N和P的质粒与含有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微型复制子来回答关于病毒聚合酶复合体的组成和功能的基本问题。我们将确定BDV- n的p38和p39异构体的作用，我们将研究BDV- x和BDV- p的p16异构体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BDV第三个转录单位转录物剪接效果的影响。进一步，我们将尝试建立完全由克隆CDNA拯救BDV的方法。一旦这项新技术发挥作用，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.