Abstract
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.
Highlights
The active transcription and replication complex of non-segmented negative strand RNA viruses, termed ribonucleoprotein complex [1], consists of the viral polymerase (L),1 the phosphoprotein (P), the nucleoprotein (N), and the viral RNA [2]
A requirement of P oligomers for viral RNA synthesis was described for P proteins of other viruses of the order Mononegavirales, including vesicular stomatitis virus [10, 11] and human parainfluenza virus type 3 [12]
Functional analysis of the BDV polymerase complex based on artificial minigenomes demonstrated that N, P, and L are sufficient for viral replication and transcription and that X has a negative regulatory function on polymerase activity [26, 27]
Summary
L, viral polymerase; P, phosphoprotein; N, nucleoprotein; M, matrix protein; G, glycoprotein; X, negative regulatory factor; BDV, Borna disease virus; ORF, open reading frame; GFP, green fluorescent protein; DMEM, Dulbecco’s modified Eagle’s medium; HA, hemagglutinin; wt, wild type. A requirement of P oligomers for viral RNA synthesis was described for P proteins of other viruses of the order Mononegavirales, including vesicular stomatitis virus [10, 11] and human parainfluenza virus type 3 [12]. We further showed that the viral negative regulatory factor X can interact with P1⁄7L and P1⁄7N complexes, and we provide evidence that P multimers are of critical importance for polymerase activity. Based on these various results, we suggest that P multimers act as central regulatory elements of assembly and activity of the polymerase complex
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