Interactions of the Borna Disease Virus P, N, and X Proteins and Their Functional Implications

Abstract

Borna disease virus (BDV) causes persistent central nervous system infection and behavioral disturbances in warm-blooded animals. Protein interaction studies were pursued to gain insight into the functions of the putative nucleoprotein (N), phosphoprotein (P), atypical glycoprotein (gp18), and X protein (X) of BDV. Coimmunoprecipitation experiments indicated that N and P, and P and X, form complexes in infected cells. Twohybrid analyses confirmed interactions between P and P, P and X, and P and N, but not between P and gp18, N and gp18, X and gp18, or X and N. Analysis of P truncation mutants identified three nonoverlapping regions important for oligomerization (amino acids (aa) 135-172), and binding to X (aa 33-115) or N (aa 197-201). Coexpression of X stimulated oligomerization of P but decreased N-P complex formation. Immunocytochemistry of transfected noninfected CHO cells demonstrated that the distribution of X is dependent upon the presence of P-X expressed alone was found predominantly in the cytoplasm whereas coexpression of X and P resulted in nuclear localization. Immunocytochemistry of infected cells revealed nuclear colocalization of P and X. Interactions of P, N, and X may have implications for regulation of BDV transcription/replication and ribonucleoprotein assembly. Borna disease virus (BDV)1 is a nonsegmented, negativestrand RNA virus that infects a broad range of warm-blooded animal species to cause disturbances of movement and behavior (1-5). The antigenome of BDV contains at least six major open reading frames (ORFs) corresponding to the nucleoprotein (N), phosphoprotein (P), atypical glycoprotein (gp18), type I membrane glycoprotein (G), polymerase, and X-protein (X). Both N and P are abundant in infected cells. N is translated from a monocistronic RNA representing the first transcription unit. P is translated from an RNA representing the second transcription unit that contains coding sequences for both P and X. gp18, G, and polymerase are translated from RNAs generated from the third transcription unit by alternative splicing of one or two introns and are present only at low levels in infected cells (6). BDV replicates in the nucleus of infected cells (7-9). Thus, transport of viral RNA and protein between nucleus and cytoplasm is an essential feature of its life cycle. Nuclear localization signals are proposed for the P (10), N (11), and polymerase proteins (12, 13). In infected cells and tissues, N colocalizes with P and was recently reported to colocalize with X (14).The phosphoproteins of nonsegmented, negative-strand RNA viruses are essential for virus transcription and replication (7). Their phosphorylation by cellular kinases influences the ability of phosphoproteins to form homomultimers, bind other viral proteins, and serve as transcriptional activators. Here, we characterize the interactions between X, P, and N in vitro and in vivo and define X as a positive regulator of P-P multimerization. EXPERIMENTAL PROCEDURESCell Lines-COS-7 and C6 cells were maintained in Dulbecco's modifie...

Overlap of Interaction Domains Indicates a Central Role of the P Protein in Assembly and Regulation of the Borna Disease Virus Polymerase Complex

“…The cDNAs encoding N-terminally truncated P proteins were amplified from pCA-P using primers P 56 (ϩ, BamHI), P 94 (ϩ, BamHI), P 135 (ϩ, BamHI), P 155 (ϩ, BamHI), and P 172 (ϩ, BamHI), which all insert a BamHI restriction site and an initiation codon directly upstream of the indicated N-terminal residue, in combination with primer pCA(Ϫ, KpnI). The PCR fragments were BamHI/KpnIdigested and ligated into BamHI/KpnI-opened pGal4/N plasmid (24), thereby replacing the N ORF with the different P ORFs.…”

“…The X interaction domain is located between P residues 72 and 87 (30), and the N interaction domain includes P residues 197-201 (24). Co-transfection of pCA-N (encoding BDV-N) reduced luciferase activity some 5-fold (Fig.…”

“…A Monomeric Variant of BDV-P Retains the Ability to Interact with L, N, and X-Mammalian two-hybrid analyses suggested that BDV-P can oligomerize (24). To determine whether P monomers can interact with other viral proteins, we mutated a leucine zipper motif in the oligomerization domain of P ( 3A) by inserting two point mutations that replaced leucine 141 and methionine 148 by glycine residues.…”

“…Similar to other members of the family Mononegavirales, BDV-P can form oligomers and is able to interact with N and L (24,25). BDV-P also interacts with the viral X (negative regulatory factor) protein (24). The domains mediating the interactions with N and X were mapped, whereas the domain of P required for the interaction with L was unknown.…”

“…The viral genome includes six open reading frames (ORFs) encoding N, X, P, matrix protein (M), glycoprotein (G), and L (22,23). Similar to other members of the family Mononegavirales, BDV-P can form oligomers and is able to interact with N and L (24,25). BDV-P also interacts with the viral X (negative regulatory factor) protein (24).…”