Splicing-Dependent Subcellular Targeting of Borna Disease Virus Nucleoprotein Isoforms

Abstract

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.

The Borna Disease Virus 2 (BoDV-2) Nucleoprotein Is a Conspecific Protein That Enhances BoDV-1 RNA-Dependent RNA Polymerase Activity

“…While BoDV-2 N contains 6 amino acid differences and an amino acid deletion not shared with BoDV-1 N (Fig. 2A), in addition to NLS and NES, which determine the intracellular localization of N (21,22), four amino acid residues thought to interact with viral RNA (29) are completely conserved in BoDV-2 N. Moreover, a downstream start codon and splicing donor and acceptor sites that generate several isoforms of N are also completely conserved (30,31). Whereas differences at amino acid positions 52 and 91 are located in the P-binding domain (23), they did not induce any deleterious effect on polymerase activity in the minireplicon assay (Fig.…”

Exogenous expression of both matrix protein and glycoprotein facilitates infectious viral particle production of Borna disease virus 1

“…The expression of M and G is regulated by some transcriptional and post-transcriptional mechanisms in infected cells. First, the transcript encoding both the M and the G genes is transcribed as a polycistronic transcript that has the lowest expression level among the viral transcripts [18]. This polycistronic transcript then undergoes multiple alternative splicing events to mature into the separate mRNAs expressing M, G and L [19].…”

The hidden diversity of ancient bornaviral sequences from X and P genes in vertebrate genomes

“…First, X/P transcripts may be rarely reverse-transcribed and/or integrated into the host genome. However, the amount of X/P transcript is comparable to that of the N transcript in cells infected with Borna disease virus 1 (BoDV-1), a modern orthobornavirus [8], suggesting that there is enough chance to be integrated into the host genome. Further, we experimentally demonstrated that X/P mRNA of BoDV-1 could be integrated into the host genomes [9].…”

“…First, X/P mRNA, which encodes the X and P proteins, may have a low propensity for reverse-transcription and/or integration into the host genome. However, previous studies of Borna disease virus 1 (BoDV-1; genus Orthobornavirus ) showed that X/P mRNA is the most abundant transcript among the viral mRNAs (11, 12), which should therefore provide enough chances for integration. Moreover, we have previously demonstrated that the X/P mRNA of BoDV-1 can be reverse-transcribed and integrated into the genome of host cells (3, 7).…”