Active borna disease virus polymerase complex requires a distinct nucleoprotein-to-phosphoprotein ratio but no viral X protein.
Analysis of the composition and regulation of the Borna disease virus (BDV) polymerase complex has so far been limited by the lack of a functional assay. To establish such an assay on the basis of an artificial minigenome, we constructed expression vectors encoding either nucleoprotein (N), phosphoprotein (P), X protein, or polymerase (L) of BDV under the control of the chicken beta-actin promoter. A Flag-tagged version of L colocalized with virus-encoded N and P in characteristic nuclear dots of BDV-infected cells and increased viral N-protein levels in persistently infected Vero cells. Vector-driven expression of L, N, and P in BSR-T7 cells together with a negative-sense BDV minigenome carrying a chloramphenicol acetyltransferase (CAT) reporter gene resulted in efficient synthesis of CAT protein. Induction of CAT protein synthesis strongly depended on a 10- to 30-fold molar excess of the N-encoding plasmid over the P-encoding plasmid. Cotransfection of even small amounts of plasmid encoding the viral X protein reduced CAT synthesis to background levels. Thus, the N-to-P stoichiometry seems to play a central role in the regulation of the BDV polymerase complex. Our data further suggest a negative regulatory function for the X protein of BDV.
Schneider
Urs
U
Department of Virology, University of Freiburg, D-79104 Freiburg, Germany. ursschn@ukl-freiburg.de
Naegele
Melanie
M
Staeheli
Peter
P
Schwemmle
Martin
M
D000818:Animals; D001891:Borna disease virus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D002522:Chlorocebus aethiops; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D009698:Nucleoproteins / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D012093:Replicon; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014773:Virology / Q000379:methods
D009698:Nucleoproteins; D010750:Phosphoproteins; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; D012324:RNA-Dependent RNA Polymerase