A reverse genetics system for Borna disease virus

Abstract

Borna disease virus (BDV) is an enveloped virus. Its non-segmented, negative-stranded RNA genome has the coding capability for six main polypeptides and has an organization characteristic of members of the order Mononegavirales. However, based on its unique genetics and biological features, BDV is considered to be the prototypic member of a new virus family, Bornaviridae. Here, the establishment of a reverse genetics system for BDV is described. Intracellular synthesis of a BDV RNA analogue or minigenome (MG) from a plasmid was driven by RNA polymerase I. Co-transfection with plasmids expressing the BDV polymerase (L), nucleoprotein (N) and phosphoprotein (P) under the control of RNA polymerase II allowed for BDV MG replication and expression. This process depended on a delicate N : P ratio, whereas the L : P ratio was less critical. Two isoforms of N, Np40 and Np38, are present in BDV-infected cells but only Np40 was strictly required for virus polymerase activity. BDV p10 polypeptide encoded by the P gene exhibited a strong inhibitory effect on BDV MG expression.Borna disease virus (BDV) causes CNS disease that is frequently manifested by behavioural abnormalities (Ikuta et al., 2002;Pletnikov et al., 2002;Rott & Becht, 1995). Evidence indicates that the natural host range of BDV, as well as its prevalence and geographical distribution, are very broad (Hatalski et al., 1997;Ikuta et al., 2002;Richt et al., 1997;Richt & Rott, 2001;Rott & Becht, 1995; Staeheli et al., 2000). Moreover, serological data and molecular epidemiological studies indicate that BDV can infect humans and might be associated with certain neuropsychiatric disorders (Billich et al., 2002;Carbone, 2001;Planz et al., 2002;Richt et al., 1997;Richt & Rott, 2001;Rott & Becht, 1995; Staeheli et al., 2000). Both virus and host factors contribute to a variable period of incubation, as well as significant heterogeneity, in the symptoms and pathology associated with BDV infection (Gonzalez-Dunia et al., 1997;Hatalski et al., 1997;Ikuta et al., 2002;Richt et al., 1997;Rott & Becht, 1995; Staeheli et al., 2000).BDV is an enveloped virus with a non-segmented, negativestranded RNA genome. Its genome is about 8?9 kb long, the smallest among known negative-stranded RNA viruses, and has an organization similar to that of other mononegaviruses (de la Torre, 1994;Schneemann et al., 1995). Six major ORFs are found in the BDV genome sequence (de la Torre, 1994;Schneemann et al., 1995). Based on their positions in the viral genome (39-N-p10/P-M-G-L-59), together with their biochemical and sequence features, these polypeptides are the counterparts of the nucleoprotein (N), phosphoprotein (P), transcriptional activator, matrix (M) protein, surface glycoprotein (G) and polymerase (L), respectively, found in other negative-stranded RNA viruses (Tordo et al., 1992). BDV has the property, unique among known animal negative-stranded RNA viruses, of a nuclear site for the replication and transcription of its genome (Briese et al., 1992;. In addition, BDV uses a remark...

The X Protein of Borna Disease Virus Serves Essential Functions in the Viral Multiplication Cycle

“…We assume that the apparent strict requirement of this nonstructural protein of BDV is related to the previously noted ability of X to regulate viral polymerase activity (7,8,13). In this context, it is of interest to note that the smear-like appearance of RNA derived from BDV-LRD-⌬X-X5Ј-infected Vero cells ( Fig.…”

“…The sixth protein, denominated X or p10, is a 10-kDa nonstructural polypeptide (15) that associates with the viral polymerase complex in infected cells (17) but is not required for the formation of an active polymerase complex in artificial minireplicon systems (7,13). X can specifically bind to viral polymerase cofactor P with the help of an interaction domain located near its N terminus (18).…”

Functional Characterization of the Genomic Promoter of Borna Disease Virus (BDV): Implications of 3′-Terminal Sequence Heterogeneity for BDV Persistence

“…We have established an RNA polymerase I/polymerase II system for intracellular reconstitution of BDV RNA replication and transcription (30). In this system, a BDV RNA analog, or minigenome, is intracellularly synthesized by the cellular RNA polymerase I.…”

“…In this system, a BDV RNA analog, or minigenome, is intracellularly synthesized by the cellular RNA polymerase I. This BDV minigenome RNA contains the BDV 5Ј and 3Ј untranslated regions cis-acting sequences required for RNA synthesis mediated by the BDV polymerase (30,39,40,42). Encapsidation of the polymerase I-derived BDV minigenome RNA by plasmid-supplied BDV N and P generates a template that is recognized by the intracellularly reconstituted BDV polymerase to direct synthesis of fulllength antiminigenome RNA (replicate) and a subgenomic mRNA (transcript) that codes for the chloramphenicol acetyltransferase (CAT) reporter gene (30).…”

“…This BDV minigenome RNA contains the BDV 5Ј and 3Ј untranslated regions cis-acting sequences required for RNA synthesis mediated by the BDV polymerase (30,39,40,42). Encapsidation of the polymerase I-derived BDV minigenome RNA by plasmid-supplied BDV N and P generates a template that is recognized by the intracellularly reconstituted BDV polymerase to direct synthesis of fulllength antiminigenome RNA (replicate) and a subgenomic mRNA (transcript) that codes for the chloramphenicol acetyltransferase (CAT) reporter gene (30). Using this system, we have documented that BDV L, N, and P are the minimal viral trans-acting factors required for efficient RNA synthesis mediated by the BDV polymerase (30).…”