Identification of alternative splicing and negative splicing activity of a nonsegmented negative-strand RNA virus, Borna disease virus

Abstract

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that belongs to the Mononegavirales. Unlike other animal viruses of this order, BDV replicates and transcribes in the nucleus of infected cells. Previous studies have shown that BDV uses RNA splicing machinery for its mRNA expression. In the present study, we identified spliced RNAs that use an alternative 3' splice site, SA3, in BDV-infected cell lines as well as infected animal brain cells. Transient transfection analysis of cDNA clones of BDV RNA revealed that although SA3 is a favorable splice site in mammalian cells, utilization of SA3 is negatively regulated in infected cells. This negative splicing activity of the SA3 site is regulated by a putative cis-acting region, the exon splicing suppressor (ESS), within the polymerase exon of BDV. The BDV ESS contains similar motifs to other known ESSs present in viral and cellular genes. Furthermore, our results indicated that a functional polyadenylation signal just upstream of the BDV ESS is also involved in the regulation of alternative splicing of BDV. These observations represent the first documentation of complex RNA splicing in animal RNA viruses and also provide new insight into the mechanism of regulation of alternative splicing in animal viruses.

Figure 1

Detection of a 3′ splice site of BDV. (A) SA2 splicing of BDV. The outline of the BDV genome is indicated at the top. Arrows indicate approximate positions of primers used for RT-PCR. Positions of 5′ and 3′ splice site (SS) and a polyadenylation site (T3) are indicated. (B and C) Thermostable RT-time-release PCR of BDV RNA. RT-PCR was performed with S-1 and A-12 (B) or S-1 and A-9 (C) primer pairs. The images of agarose gels were captured electronically, and the pixels were inverted. Lanes: 1, MDCK/BDV; 2, MDCK; 3, C6BV; 4, C6; 5, OL/BDV; 6, OL; 7, BDV-infected rat brain; 8, uninfected rat brain; 9, MDCK/BDV RT(−). (D) Splicing patterns of BDV. Approximate location of a newly identified BDV intron and 3′ splice site (SA3) are shown. Lengths of PCR products are indicated at the left of the patterns in parentheses. (E) Comparison of the BDV intron III splice sequence with mammalian consensus sequence. A branchpoint nt is indicated by an arrow. Y, pyrimidine; R, purine; N, any base.

Figure 2

RNase protection and RNA blot assays of BDV RNA. (A) Schematic maps of BDV ORFs are shown at the top. The riboprobes for RPA and RNA blotting [Rp1 (nt 4560–4766) and Rp3 (nt 4075–4480)] are indicated by black and shaded bars, respectively. Expected sizes of protected fragments are indicated at the right of the patterns in parentheses. (B) RPA for SA3 splicing RNAs. Lanes: 1, RNase-untreated riboprobe; 2, uninfected rat brain; 3, BDV-infected rat brain, 4, OL cells; 5, OL/BDV cells. % of SA3 spliced RNAs is shown at the bottom of the lanes. (C) RNA blotting for SA3 spliced RNAs. Poly(A)⁺ RNAs from OL/BDV (lanes 1, 3, 5, and 7) and OL cells (lanes 2, 4, 6, and 8) were hybridized with Rp1 (lanes 1, 2, 5, and 6) or Rp3 (lanes 3, 4, 7, and 8) antisense probe. Lanes 5 to 8 represent an extended exposure (4 days) of the lanes 1 to 4. Appropriate sizes of the fragments are shown.

Figure 3

SA3 splicing is inefficient in persistently BDV-infected cells. (A) Schematic representation of BDV cDNA expression plasmid pCD2.1. Positions of splice sites and polyadenylation signal are indicated. Arrows show primers for RT-PCR. (B and C) Splicing analysis of cDNA expression plasmid. Arrows indicate SA2 or SA3 spliced products in the cells. Primer pairs used for analysis are indicated at the bottom. Lanes: (B) 1, pCD2.1-transfected COS-7 cells; 2, mock-transfected COS-7 cells; 3, OL/BDV cells; 4, OL cells. (C) 1, pCD2.1-transfected OL cells; 2, mock-transfected OL cells; 3, pCD2.1-transfected OL/BDV cells; 4, mock-transfected OL/BDV cells.

Figure 4

The BDV genome contains elements with negative regulatory effects on SA3 splicing. (A) A series of cDNA expression plasmid. Deletion sites are indicated by nt numbers. The putative ESS position is shown by a box. (B) RT-PCR analysis of deletion mutants. The mutants were transfected into COS-7 cells and SA3 splicing was detected by RT-PCR by using S-1/A-2 primer pairs. (C) RPA of deletion mutants. RNAs from the mutant-transfected cells were hybridized with the riboprobe shown in Fig. 2. Percent of SA3 splicing was indicated. Mutants used for the analysis are shown at the top of the lanes. Cr, mock-transfected COS-7 cells.

Figure 5

Detection of a functional polyadenylation signal upstream of the ESS region. (A) Sequences of the BDV ESS region. Schematic map of BDV ORFs is shown at the top. Splice and polyadenylation sites of BDV are shown by bars. Primers for 3′RACE analysis and putative elements of BDV ESS are also indicated. A polyadenylation signal (t6) and possible motifs for ESS are boxed. (B) 3′RACE analysis of BDV. RNAs from BDV-infected cells were subjected to 3′RACE analysis by using the primers shown in A. lanes: M, marker; 1, MDCK/BDV cells; 2, MDCK cells; 3, C6BV cells; 4, C6 cells; 5, OL/BDV cells; 6, OL cells.

Figure 6

The SA3 spliced RNA forms ORFs. (A) Schematic diagram of the new predicted BDV ORFs generated by SA3 splicing. Splice and polyadenylation sites in the BDV genome are indicated. ORFs and amino acid numbers of G^-, S1 and S2 ORFs predicted by the BDV genome sequence are indicated. (B) Translation of predicted S1 protein in cDNA transfected COS-7 cells. Lanes: 1, pcORFx1-FLAG; 2, pS1-FLAG; 3, Mock. Appropriate sizes of molecular weight are shown.