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.

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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.