The nucleolus is the site of Borna disease virus RNA transcription and replication

Abstract

Borna disease virus (BDV) is a neurotropic nonsegmented negative-strand RNA virus with limited homology to rhabdoviruses and paramyxoviruses. A distinguishing feature of BDV is that it replicates in the nucleus of infected cells. Strand-specific probes used for in situ hybridization of infected rat brain showed that there was differential localization of positive- and negative-strand RNAs within the nucleus of neurons. Within nuclei, sense-strand RNAs were preferentially localized within nucleolar regions while genomic-sense RNAs were found in both nucleolar and nonnucleolar regions. These results suggested a role for the nucleolus in BDV replication. Nucleoli isolated from persistently infected neuroblastoma cells contained both genomic and antigenomic BDV RNA species as well as an enrichment of the 39/38-kDa and gp18 BDV proteins. Since the nucleolus is the site of rRNA transcription, we examined BDV transcription in the presence of inhibitors of RNA polymerase I. Inhibition of RNA polymerase I did not affect levels of BDV transcription.

FIG. 1

In situ hybridization of BDV-infected rat brain 12 days postinfection. Sections were hybridized with single-stranded DNA probes that detect sense-strand RNA (a) and genomic RNA (b). (a) The nucleolus is densely labeled, while there are few grains in the remainder of the nucleus. (b) The nucleolus is spared, but grains are densely distributed at the periphery of the nucleolus and found more diffusely in the nucleoplasm of the neuron. The figure was prepared with Photoshop.

FIG. 2

Detection of RNA species in subcellular fractions of BDV-infected cells. RNA isolated from subcellular fractions was subjected to Northern blot analysis with probes specific for BDV genomic RNA (A), BDV sense RNA (B), and 18S rRNA (C). The locations of the full-length genomic and antigenomic RNAs are indicated in panels A and B. Panel B also shows the location of the abundant 0.85-kb mRNA. The location of the mature 18S rRNA species is indicated in panel C; asterisks show the precursor species detected in the nuclear and nucleolar fractions and in the TE wash of nucleoli. The figure was made from scanned fluorographs with Photoshop and Illustrator.

FIG. 3

Detection of protein species in subcellular fractions of BDV-infected cells. Equivalent levels of ³⁵S-incorporated radioactivity were used for immunoprecipitations of subcellular fractions by using antisera specific for BDV proteins (A) and for markers of nucleolar proteins (B23) and nonnucleolar nuclear proteins (SC-35) (B and C). (A) Immunoprecipitation of BDV proteins. (B) Immunoprecipitation of B23. (C) Immunoprecipitation of SC-35. The figure was made from scanned fluorographs with Photoshop and Illustrator.

FIG. 4

RNase protection analysis of RNA species synthesized in the presence of RNA Pol inhibitors. ³²P-labeled RNA was isolated from cells that were metabolically labeled during treatment with Pol inhibitors. The labeled RNA was hybridized with unlabeled antisense transcripts generated in vitro. Following digestion with RNase A, the protected fragments were resolved by gel electrophoresis. The specific full-length protected band is indicated by an asterisk in each panel. (A) BDV probe. (B) Actin probe. (C) 18S rRNA probe. For panel C, 1 μg of total labeled cellular RNA was used for hybridization. For panels A and B, 10 μg of total labeled cellular RNA was used for hybridization. The figure was made from scanned fluorographs with Photoshop and Illustrator. campto, camptothecin; Act D, actinomycin D.