Borna disease virus (BDV) provides an important model for the study of viral persistence4n the central nervous system (CNS), a subject relevant to health given the evidence that viruses can contribute to slowly progressive human neurological disorders. BDV is a nonsegmented, negative-stranded (NNS) RNA virus that replicates and transcribes in the nucleus. Based on its unique genetic and biological features BDV is considered to be the prototype of a new type of animal viruses. BDV can infect humans and might be associated with certain mental disorders, providing further impetus for the investigation of the molecular mechanisms underlying BDV-cell interactions in the CNS. Paramount for these studies will be the establishment of an efficient reverse genetic system for the investigation of the cis-acting signals and trans-acting factors involved in the control of BDV gene expression, RNA replication and virion formation, which constitute the long-term goals of the proposed studies with the following specific aims: 1) Revisit previously determined 5' and 3'-end sequences of the BDV genome. BDV RNA prepared at 72-96 h after infection, during the linear phase of increase viral RNA synthesis, will be used to determine the genomic 5'and 3'-end sequences for different BDV strains. The information obtained will be used to generate BDV minigenomes containing the authentic viral termini. 2) Determine cis-acting sequences and experimental conditions required for efficient transcription and replication of BDV niinigenome mediated by helper virus infection. BDV minigenome RNA will be intracellularly synthesized using the poll expression system, and its expression by complementation with helper BDV examined. Requirements for minigenome expression imposed by RNA length, and cis-acting signals will be investigated. 3) Determine the identity and relative expression levels of BDV proteins required for efficient rescue of BDV minigenome. The profile of BDV protein expression at 72-96 hours after infection will be recreated by cotransfection with plasmids encoding BDV proteins. Conditions required for reconstitution of BDV transcription and RNA replication by intracellular coexpression of BDV minigenome and BDV proteins from separated plasmids will be determined.
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