Structure and function of the RNA polymerase complex of Borna disease virus, a nuclear-replicating non-segmented negative-strand RNA virus.

Borna disease virus 1 (BoDV-1) is a non-segmented negative-strand (NNS) RNA virus that uniquely replicates in the nucleus of mammalian host cells, in contrast to most NNS RNA viruses that replicate in the cytoplasm. The mechanisms underlying nuclear replication of BoDV-1 and related bornaviruses with their RNA-dependent RNA polymerase (RdRp) complexes remain poorly understood. Here, we report the 2.8 Å cryo-EM structure of the BoDV-1 RdRp complex, comprising the large (L) protein and tetrameric phosphoprotein (P). The L protein features an N-terminal superdomain containing the RdRp and GDP polyribonucleotidyltransferase (PRNTase, mRNA-capping enzyme) domains, along with three C-terminal appendages, including a methyltransferase-like domain. The RdRp initiates de novo RNA synthesis internally at the genomic promoter, producing 5'-triphosphorylated transcripts corresponding to the 5' end of the anti-genome. P interacts with the fingers RdRp subdomain of L. Structure-guided mutagenesis shows that the residues involved in the L-P interaction are essential for efficient transcription initiation and, consequently, for viral gene expression. A flexible loop within the PRNTase domain, analogous to the rhabdovirus priming-capping loop, appears critical for transcription initiation. These findings provide the structural and functional insights into the BoDV-1 RdRp and support a shared evolutionary origin between nuclear and cytoplasmic NNS RNA viruses.