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Borna disease virus possesses an NF-?B inhibitory sequence in the nucleoprotein gene.

ABSTRACT: Borna disease virus (BDV) has a non-segmented, negative-stranded RNA genome and causes persistent infection in many animal species. Previous study has shown that the activation of the I?B kinase (IKK)/NF-?B pathway is reduced by BDV infection even in cells expressing constitutively active mutant IKK. This result suggests that BDV directly interferes with the IKK/NF-?B pathway. To elucidate the mechanism for the inhibition of NF-?B activation by BDV infection, we evaluated the cross-talk between BDV infection and the NF-?B pathway. Using Multiple EM for Motif Elicitation analysis, we found that the nucleoproteins of BDV (BDV-N) and NF-?B1 share a common ankyrin-like motif. When THP1-CD14 cells were pre-treated with the identified peptide, NF-?B activation by Toll-like receptor ligands was suppressed. The 20S proteasome assay showed that BDV-N and BDV-N-derived peptide inhibited the processing of NF-?B1 p105 into p50. Furthermore, immunoprecipitation assays showed that BDV-N interacted with NF-?B1 but not with NF-?B2, which shares no common motif with BDV-N. These results suggest BDV-N inhibits NF-?B1 processing by the 20S proteasome through its ankyrin-like peptide sequence, resulting in the suppression of IKK/NF-?B pathway activation. This inhibitory effect of BDV on the induction of the host innate immunity might provide benefits against persistent BDV infection.

SUBMITTER: Makino A

PROVIDER: S-EPMC4649702 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Borna disease virus possesses an NF-ĸB inhibitory sequence in the nucleoprotein gene.

Makino Akiko A Fujino Kan K Parrish Nicholas F NF Honda Tomoyuki T Tomonaga Keizo K

Scientific reports 20150303

Borna disease virus (BDV) has a non-segmented, negative-stranded RNA genome and causes persistent infection in many animal species. Previous study has shown that the activation of the IκB kinase (IKK)/NF-κB pathway is reduced by BDV infection even in cells expressing constitutively active mutant IKK. This result suggests that BDV directly interferes with the IKK/NF-κB pathway. To elucidate the mechanism for the inhibition of NF-κB activation by BDV infection, we evaluated the cross-talk between ...[more]

PMID: 25733193

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Project description: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.

Project description:An RNA virus-based episomal vector (REVec) based on Borna disease virus 1 (BoDV-1) is a promising viral vector that achieves stable and long-term gene expression in transduced cells. However, the onerous procedure of reverse genetics used to generate an REVec is one of the challenges that must be overcome to make REVec technologies practical for use. In this study, to resolve the problems posed by reverse genetics, we focused on BoDV-2, a conspecific virus of BoDV-1 in the Mammalian 1 orthobornavirus. We synthesized the BoDV-2 nucleoprotein (N) and phosphoprotein (P) according to the reference sequences and evaluated their effects on the RNA polymerase activity of the BoDV-1 large protein (L) and viral replication. In the minireplicon assay, we found that BoDV-2 N significantly enhanced BoDV-1 polymerase activity and that BoDV-2 P supported further enhancement of this activity by N. A single amino acid substitution assay identified serine at position 30 of BoDV-2 N and alanine at position 24 of BoDV-2 P as critical amino acid residues for the enhancement of BoDV-1 polymerase activity. In reverse genetics, conversely, BoDV-2 N alone was sufficient to increase the rescue efficiency of the REVec. We showed that the REVec can be rescued directly from transfected 293T cells by using BoDV-2 N as a helper plasmid without cocultivation with Vero cells and following several weeks of passage. In addition, a chimeric REVec harboring the BoDV-2 N produced much higher levels of transgene mRNA and genomic RNA than the wild-type REVec in transduced cells. Our results contribute to not only improvements to the REVec system but also to understanding of the molecular regulation of orthobornavirus polymerase activity. IMPORTANCE Borna disease virus 1 (BoDV-1), a prototype virus of the species Mammalian 1 orthobornavirus, is a nonsegmented negative-strand RNA virus that persists in the host nucleus. The nucleoprotein (N) of BoDV-1 encapsidates genomic and antigenomic viral RNA, playing important roles in viral transcription and replication. In this study, we demonstrated that the N of BoDV-2, another genotype in the species Mammalian 1 orthobornavirus, can participate in the viral ribonucleoprotein complex of BoDV-1 and enhance the activity of BoDV-1 polymerase (L) in both the BoDV-1 minireplicon assay and reverse genetics system. Chimeric recombinant BoDV-1 expressing BoDV-2 N but not BoDV-1 N showed higher transcription and replication levels, whereas the propagation and infectious particle production of the chimeric virus were comparable to those of wild-type BoDV-1, suggesting that the level of viral replication in the nucleus is not directly involved in the progeny virion production of BoDVs. Our results demonstrate a molecular mechanism of bornaviral polymerase activity, which will contribute to further development of vector systems using orthobornaviruses.

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Borna disease virus possesses an NF-?B inhibitory sequence in the nucleoprotein gene.

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Borna disease virus possesses an NF-ĸB inhibitory sequence in the nucleoprotein gene.

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