Overlap of interaction domains indicates a central role of the P protein in assembly and regulation of the Borna disease virus polymerase complex.
The active polymerase complex of Borna disease virus is composed of the viral proteins N, P, and L. The viral X (negative regulatory factor) protein acts as a regulator of polymerase activity. Interactions of P with N and X were previously studied, but interactions with L were poorly defined. Using a mammalian two-hybrid system, we observed that L specifically interacts with P but not with N, X, or itself. Mapping of the L-binding domain in the P molecule revealed that it overlaps with two adjacent domains required for multimerization and interaction with N. Competition experiments showed that the interaction between L and P was inefficient when N was present, indicating that L may preferentially interact with free P in infected cells. Interestingly, a multimerization-defective P mutant maintained the ability to interact with L, N, and X but failed to support reporter gene expression from an artificial Borna disease virus minigenome. Furthermore, dominant negative effects on minigenome activity were only observed when P mutants with an intact multimerization domain were used, suggesting that P multimers, rather than monomers, exhibit biological activity. P mutants lacking functional interaction domains for L or N still formed complexes with these viral proteins when wild-type P was available as a bridging molecule, indicating that P multimers have the potential to act as scaffolds on which the RNA polymerase complex is assembled.
Schneider
Urs
U
Department of Virology, University of Freiburg, Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany.
Blechschmidt
Kareen
K
Schwemmle
Martin
M
Staeheli
Peter
P
D001667:Binding, Competitive; D001891:Borna disease virus / Q000201:enzymology*; D002460:Cell Line; D002850:Chromatography, Gel; D018076:DNA, Complementary / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D005799:Genes, Dominant; D006801:Humans; D009154:Mutation; D010750:Phosphoproteins; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques; D014764:Viral Proteins / Q000737:chemistry*; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000502:physiology
D018076:DNA, Complementary; C496537:P protein, Borna disease virus; D010750:Phosphoproteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D012321:DNA-Directed RNA Polymerases