The Borna Disease Virus 2 (BoDV-2) Nucleoprotein Is a Conspecific Protein That Enhances BoDV-1 RNA-Dependent RNA Polymerase Activity

Abstract

An RNA virus-based episomal vector (REVec) based on Borna disease virus 1 (BoDV-1) is a promising viral vector that achieves stable and long-term gene expression in transduced cells. However, the onerous procedure of reverse genetics used to generate a REVec is one of the challenges that must be overcome to make REVec technologies practical for use. In this study, to resolve the problems posed by reverse genetics, we focused on BoDV-2, a conspecific virus of BoDV-1 in the Mammalian 1 orthobornavirus . We synthesized the BoDV-2 nucleoprotein (N) and phosphoprotein (P) according to the reference sequences and evaluated their effects on the RNA polymerase activity of the BoDV-1 large protein (L) and viral replication. In the minireplicon assay, we found that BoDV-2 N significantly enhanced BoDV-1 polymerase activity and that BoDV-2 P supported further enhancement of this activity by N. A single amino acid substitution assay identified serine at position 30 of BoDV-2 N and alanine at position 24 of BoDV-2 P as critical amino acid residues for the enhancement of BoDV-1 polymerase activity. In reverse genetics, on the other hand, BoDV-2 N alone was sufficient to increase the rescue efficiency of the REVec. We showed that the REVec can be rescued directly from transfected 293T cells by using BoDV-2 N as a helper plasmid without cocultivation with Vero cells and following several weeks of passage. In addition, a chimeric REVec harboring the BoDV-2 N produced much higher levels of transgene mRNA and genomic RNA than the wild-type REVec in transduced cells. Our results contribute to not only improvements to the REVec system but also understanding of the molecular regulation of orthobornavirus polymerase activity. Importance Borna disease virus 1 (BoDV-1), a prototype virus of the species Mammalian 1 orthobornavirus , is a nonsegmented negative-strand RNA virus that persists in the host nucleus. The nucleoprotein (N) of BoDV-1 encapsidates genomic and antigenomic viral RNA, playing important roles in viral transcription and replication. In this study, we demonstrated that the N of BoDV-2, another genotype in the species Mammalian 1 orthobornavirus , can participate in the viral ribonucleoprotein complex of BoDV-1 and enhance the activity of BoDV-1 polymerase (L) in both the BoDV-1 minireplicon assay and reverse genetics system. Chimeric recombinant BoDV-1 expressing BoDV-2 N but not BoDV-1 N showed higher transcription and replication levels, whereas the propagation and infectious particle production of the chimeric virus were comparable to those of wild-type BoDV-1, suggesting that the level of viral replication in the nucleus is not directly involved in the progeny virion production of BoDVs. Our results demonstrate a molecular mechanism of bornaviral polymerase activity, which will contribute to further development of vector systems using orthobornaviruses.

Exogenous expression of both matrix protein and glycoprotein facilitates infectious viral particle production of Borna disease virus 1

“…Furthermore, we showed that simultaneous transfection of M and G expression plasmids with N, P and L helper plasmids by reverse genetics significantly increases the rescue efficiency of rBoDV-1. Together with our recent report that upregulation of viral polymerase activity does not necessarily increase infectious particle production [9], these observations suggest that the expression levels of M and G, which are constituents of the viral lipid envelope, determine the number of progeny virions in infected cells.…”

“…This feature also affects the rescue efficiency of recombinant BoDV-1 (rBoDV-1) by reverse genetics. To recover rBoDV-1 by reverse genetics, a vector plasmid transcribing the full-length antigenomic RNA of BoDV-1 and three helper plasmids expressing N, P and L were cotransfected into 293 T cells; however, this method allowed us to rescue only a small amount of infectious rBoDV-1 from transfected cells [9].…”

“…( b, c ) Comparison of viral RNA synthesis by RT-qPCR analysis. Total RNA was extracted from transfected 293 T cells at 48 hpt using a NucleoSpin RNA Plus Kit (Macherey-Nagle; #740984) according to the manufacturer’s instructions, and the copy numbers of genomic RNA ( b ) and GFP mRNA ( c ) were quantified by RT-qPCR as described previously [9]. ( d ) Detection of viral proteins by Western blot analysis.…”

“…Effect of exogenous expression of M and G by reverse genetics. As described previously [9], a vector plasmid and three helper plasmids expressing N, P and L were cotransfected into 293 T cells. In addition, 40 ng MΔSDSA and/or 10 ng GΔSDSA were simultaneously transfected as helper plasmids.…”

“…(b) Comparison of the expression level of M from Mwt and MΔSDSA expression plasmids by Western blot analysis. Whole-cell lysates were prepared from 293 T cells transfected with the indicated plasmid at 48 h post-transfection (hpt).Western blot analysis was conducted as described previously[9] using the antibodies indicated on the left of the panel. Alpha-tubulin was used as the loading control.…”