Implication of a cis-acting element in the cytoplasmic accumulation of unspliced Borna disease virus RNAs

Abstract

Borna disease virus (BDV), the prototype of a new family within the order Mononegavirales, is unusual in its nuclear localization for replication and transcription and use of RNA splicing for gene expression. The BDV antigenome contains three transcription units and six major open reading frames. Multicistronic RNAs containing two introns are elaborated from the third transcription unit. Differential splicing of the two introns and cytoplasmic accumulation of the unspliced and partially spliced RNA are critical for the balanced expression of the putative matrix protein, glycoprotein, and polymerase. To investigate the mechanisms for cytoplasmic expression of unspliced and partially spliced BDV transcripts, the levels of these transcripts were measured in the cytoplasm of infected COS-7 cells and noninfected COS-7 cells transfected with plasmids containing 2.8-kb cDNA inserts representing either wild-type or mutant BDV RNA from the third transcription unit. Analysis of truncation mutations allowed the identification of a cis-acting element present within the 3 end of the BDV 2.8-kb transcript that facilitated the cytoplasmic accumulation of unspliced BDV transcripts through nucleocytoplasmic transport. The nucleocytoplasmic transport activity was not dependent on the presence of BDV proteins. Gel-shift assays revealed that the cis-acting element binds specifically to host cytoplasmic and nuclear proteins.

Authentic Borna disease virus transcripts are spliced less efficiently than cDNA-derived viral RNAs

“…In this study we were unable to confirm the results of our earlier report wherein the unspliced plasmid-encoded 2·8 kb RNA predominantly accumulated in the cytoplasm due to an RNA element located in the 3′ non-coding region (Schneider et al ., 1997 b ). Since we used the same detection technique (RPA), plasmid (pRc2.8) and cell type (COS-7 cells), we propose that the differences may relate to the fact that the RNA samples in this study were subjected to DNase treatment before analysis by RPA.…”

“…Plasmids pRPAwt and pRPAmt were used for in vitro transcription of the wild-type RNase protection assay (RPA) probe (RPAwt) or the mutant RPA probe (RPAmt) and were generated as follows: To create pRPAwt, the Hin dIII– Eco RI fragment of pRPA-1 (Schneider et al ., 1997 b ), encoding part of the BDV M/G-ORFs corresponding to nucleotides 1975–2510 of BDV strain He/80 (Cubitt et al ., 1994 a ), was cloned into pGEM-4 (Promega). Plasmid pRPAmt was derived from pRPAwt by replacing six nucleotides of exon 2 (ccagag to gttaac) of the M/G-ORFs corresponding to nucleotides 2216–2221 of BDV strain He/80 using the Quick-mutagenesis protocol (Roche Biochemicals).…”

“…Plasmid pRPAmt was derived from pRPAwt by replacing six nucleotides of exon 2 (ccagag to gttaac) of the M/G-ORFs corresponding to nucleotides 2216–2221 of BDV strain He/80 using the Quick-mutagenesis protocol (Roche Biochemicals). To clone the mutated DNA into plasmid pRc2.8 encoding the 2·8 kb RNA of BDV (Schneider et al ., 1997 b ), the Bbs I– Bst 1107I fragment of pRPAmt, harbouring the respective region, was first subcloned into plasmid pBS-XbaI containing the Xba I fragment of pRc2.8, resulting in plasmid pBS-XbaI/mt. In a final step, the Xba I fragment of pRc2.8 was replaced with the corresponding fragment of pBS-XbaI/mt, resulting in plasmid pRc2.8mt.…”

“…It is unclear whether virus-specific factors/elements are involved in the regulation of splicing. First indications that such factors might be implicated emerged in a recent study where a cis -acting RNA element located within the 3′ non-coding region of the 2·8 kb RNA was found to be important for efficient cytoplasmic accumulation of unspliced RNA (Schneider et al ., 1997 b ).…”

“…Little is known about the cytoplasmic levels of the various splice forms of BDV RNAs (Cubitt et al ., 1994 b ; Schneider et al ., 1994 , 1997 b ). It is unclear whether virus-specific factors/elements are involved in the regulation of splicing.…”