Active borna disease virus polymerase complex requires a distinct nucleoprotein-to-phosphoprotein ratio but no viral X protein

Abstract

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.

FIG. 1.

BDV L with an N-terminal Flag tag colocalizes with viral P protein and increases N expression levels in BDV-infected cells. (A) Uninfected (a and b) and BDV-infected (c to h) Vero and BSR-T7 cells were transfected with pCA-flagL and analyzed for the cellular localization of Flag-tagged BDV L by confocal immunofluorescence microscopy. Cells were stained with a mouse MAb to the Flag epitope and rabbit antiserum to BDV P. Bound antibodies were visualized by fluorescence-labeled goat anti-mouse (green) and goat anti-rabbit(red) antibodies. Panels g and h show the overlay of panels c and e and panels d and f, respectively. (B) BDV-infected Vero cells were transfected with either pCA-flagGFP, pCA-flagP, or pCA-flagL. Four days later, the cells were simultaneously stained with a mouse MAb to the Flag epitope and rabbit antiserum to BDV N and analyzed by -immunofluorescence microscopy with two immunofluorescent stains. The BDV N expression levels of approximately 200 individual Flag-positive cells from each transfection were evaluated. These cells were assigned into three categories, depending on whether they expressed low (white bars), medium (gray bars,) and high (black bars) levels of N protein.

FIG. 2.

In vivo reconstitution of the BDV polymerase complex. (A) Schematic drawing showing the T7 RNA polymerase-driven expression cassettes used to generate BDV minigenomes and the expected transcription and replication intermediates. The unique restriction sites used for cloning of the minigenome expression constructs are indicated. Transcription of two vectors, pT7-gmgA and pT7-gmgC, by T7 RNA polymerase in BSR-T7 cells should yield RNAs in the genome negative (−)-sense orientation that differ only in the last nucleotide at the 3′ end (shown in bold type and underlined) of the fully processed molecule. Transcription is terminated by a T7 terminator sequence (φ), and the correct 3′ terminus of the minigenome RNA is produced by self-cleavage of the hepatitis delta virus ribozyme (δ) sequence. Both minigenome constructs contain the CAT gene (gray box) flanked by BDV 5′- and 3′-NCR of 78 and 54 or 53 nt, respectively. In the presence of the BDV proteins L, N, and P, the minireplicon should be recognized and copied by the reconstituted BDV polymerase complex, resulting in complementary minigenome RNA in the antigenome positive (+)-sense orientation, which in turn can serve as the template for the synthesis of more negative-sense RNA. Transcription of the minigenome by the reconstituted BDV polymerase complex should further yield capped and polyadenylated CAT mRNA. (B) Analysis of reporter gene expression by CAT ELISA. BSR-T7 cells in 12-well plates were transfected with 400 ng of either pT7-gmgA or pT7-gmgC together with the various expression plasmids indicated below the graph. Constant amounts of pCA-L and pCA-N and variable amounts of pCA-P were used. To control for specificity, plasmids encoding the various BDV-derived nucleocapsid components were replaced by the same amounts of plasmids encoding the analogous proteins from MV. For an internal control for transfection efficiency, 100 ng of a plasmid encoding the firefly luciferase under the control of a T7 RNA polymerase promoter was cotransfected. The total amount of transfected DNA was kept constant by adding the indicated amounts of pCA-flagGFP. Seventy-two hours after transfection, the cells were lysed and analyzed for CAT protein levels and luciferase activity by ELISA and luciferase-mediated light emission, respectively. CAT values were normalized for transfection efficacy.

FIG. 3.

Inhibitory effect of increasing concentrations of BDV P in the minireplicon assay. (A) Serial twofold dilutions of pCA-P were cotransfected with 400 ng of pT7-gmgA, 250 ng of pCA-N, 200 ng of pCA-L, and 100 ng of pBST7-luc. The total amount of transfected DNA was kept constant by adding the indicated amounts of pCA-flagGFP. To control for specificity, plasmid pCA-L was replaced by a plasmid that codes for the L protein of MV. CAT values were normalized for transfection efficacy. (B) Serial twofold dilutions of pCA-L were cotransfected with 400 ng of pT7-gmgA, 250 ng of pCA-N, 25 ng of pCA-P, and 100 ng of pBST7-luc. The total amount of transfected DNA was kept constant by adding the indicated amounts of pCA-flagGFP. To control for specificity, plasmid pCA-L was replaced by 200 ng of a plasmid that codes for the L protein of MV. CAT values were normalized for transfection efficacy.

FIG. 4.

The X protein of BDV exhibits potent inhibitory activity in the minireplicon assay. Serial twofold dilutions of pCA-X were cotransfected with 400 ng of pT7-gmgA, 200 ng of pCA-L, 250 ng of pCA-N, 25 ng of pCA-P, and 100 ng of pBST7-luc. The total amount of transfected DNA was kept constant by adding the indicated amounts of pCA-flagGFP. To control for specificity, plasmid pCA-L was replaced by 200 ng of a plasmid that codes for the L protein of MV. CAT values were normalized for transfection efficacy.

FIG. 5.

Analysis of BDV minireplicon-derived RNA by Northern blotting. BSR-T7 cells in six-well plates were either mock transfected (lane 1) or transfected with 1.5 μg of pT7-gmgA and the indicated amounts of pCA expression vectors (lanes 2 to 5). To control for specificity, plasmid pCA-L was replaced by 500 ng of a plasmid that codes for the L protein of MV. Ethidium bromide staining for rRNA verified that comparable amounts of RNA were loaded (top panel). Radiolabeled minus-strand minigenome RNA was used as the hybridization probe. (−), negative.