Identification of signal sequences that control transcription of borna disease virus, a nonsegmented, negative-strand RNA virus

Borna disease virus (BDV) is a nonsegmented negative-strand (NNS) RNA virus that is unusual because it replicates in the nucleus. The most abundant viral protein in infected cells is a 38/39-kDa doublet that is presumed to represent the nucleocapsid. Infectious particles also contain high levels of this protein, accounting for at least 50% of the viral proteins. The two forms of the protein differ by an additional 13 amino acids that are present at the amino terminus of the 39-kDa form and missing from the 38-kDa form. To examine whether this difference in amino acid content affects the localization of this protein in cells, the 39-and 38-kDa proteins were expressed in transfected cells. The 39-kDa form was concentrated in the nucleus, whereas the 38-kDa form was found in both the nucleus and cytoplasm. Inspection of the extra 13 amino acids present in the 39-kDa form revealed a sequence (Pro-Lys-Arg-Arg) that is very similar to the nuclear localization signals (in both sequence homology and amino-terminal location) of the VP1 proteins of simian virus 40 and polyomavirus. Primer extension analysis of total RNA from infected cells suggests that there are two mRNA species encoding the two forms of the nucleocapsid protein. In infected cells, the 39-kDa form is expressed at about twofoldhigher levels than the 38-kDa form at both the RNA and protein levels. The novel nuclear localization of the 39-kDa nucleocapsid-like protein suggests that this form of the protein is targeted to the nucleus, the site for viral RNA replication, and that it may associate with genomic RNA.

Section: Discussionmentioning

confidence: 77%

Section: Cell Fractionation Of Persistently Infected Sknsh Cellsmentioning

confidence: 98%

Molecular basis for the differential subcellular localization of the 38- and 39-kilodalton structural proteins of Borna disease virus

Pyper

Gartner

1997

“…The levels of splicing of NI-I and NI-II introns were estimated to be 1.6% and 6.8%, respectively (Fig. 1C), indicating that the expression levels of these splicing variants are not high enough to be detected by Northern blotting (10,27).…”

Section: Resultsmentioning

confidence: 99%

Splicing-Dependent Subcellular Targeting of Borna Disease Virus Nucleoprotein Isoforms

Kojima

Sato

Yanai

et al. 2019

Targeting of viral proteins to specific subcellular compartments is a fundamental step for viruses to achieve successful replication in infected cells. Borna disease virus 1 (BoDV-1), a nonsegmented, negative-strand RNA virus, uniquely replicates and persists in the cell nucleus. Here, it is demonstrated that BoDV nucleoprotein (N) transcripts undergo mRNA splicing to generate truncated isoforms. In combination with alternative usage of translation initiation sites, the N gene potentially expresses at least six different isoforms, which exhibit diverse intracellular localizations, including the nucleoplasm, cytoplasm, and endoplasmic reticulum (ER), as well as intranuclear viral replication sites. Interestingly, the ER-targeting signal peptide in N is exposed by removing the intron by mRNA splicing. Furthermore, the spliced isoforms inhibit viral polymerase activity. Consistently, recombinant BoDVs lacking the N-splicing signals acquire the ability to replicate faster than wild-type virus in cultured cells, suggesting that N isoforms created by mRNA splicing negatively regulate BoDV replication. These results provided not only the mechanism of how mRNA splicing generates viral proteins that have distinct functions but also a novel strategy for replication control of RNA viruses using isoforms with different subcellular localizations. IMPORTANCE Borna disease virus (BoDV) is a highly neurotropic RNA virus that belongs to the orthobornavirus genus. A zoonotic orthobornavirus that is genetically related to BoDV has recently been identified in squirrels, thus increasing the importance of understanding the replication and pathogenesis of orthobornaviruses. BoDV replicates in the nucleus and uses alternative mRNA splicing to express viral proteins. However, it is unknown whether the virus uses splicing to create protein isoforms with different functions. The present study demonstrated that the nucleoprotein transcript undergoes splicing and produces four new isoforms in coordination with alternative usage of translation initiation codons. The spliced isoforms showed a distinct intracellular localization, including in the endoplasmic reticulum, and recombinant viruses lacking the splicing signals replicated more efficiently than the wild type. The results provided not only a new regulation of BoDV replication but also insights into how RNA viruses produce protein isoforms from small genomes.

“…Whereas the 1.2-kb RNA, which encodes the first ORF (N) on the antigenome, is monocistronic, the other RNAs are multicistronic. The 0.8-kb RNA encodes both the P and X proteins (6,31,38). The 1.9-kb transcript includes the N ORF and P ORF as well as a putative leader sequence.…”

mentioning

confidence: 99%

“…The 1.9-kb transcript includes the N ORF and P ORF as well as a putative leader sequence. It is neither capped nor polyadenylylated and is likely to be the product of abortive replication (31). The 2.8and 7.1-kb RNAs are coterminal at the 5Ј terminus but differ in length due to transcriptional readthrough at termination site T3 (31).…”

mentioning

confidence: 99%

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

Schneider

Schwemmle

Lipkin

1997

Self Cite

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.