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A Small Interfering RNA Cocktail Targeting the Nucleoprotein and Large Protein Genes Suppresses Borna Disease Virus Infection.

ABSTRACT: Recently, Borna disease virus (BoDV-1)-related fatal encephalitis human cases have been reported, which highlights the potential of BoDV-1 to cause fatal human diseases. To protect the infected brain from lethal damage, it is critical to control BoDV-1 as quickly as possible. At present, antivirals against BoDV-1 are limited, and therefore, novel types of antivirals are needed. Here, we developed a novel treatment using small interfering RNAs (siRNAs) against BoDV-1. We screened several siRNAs targeting the viral N, M, and L genes for BoDV-1-reducing activity. Among the screened candidates, we chose two siRNAs that efficiently decreased the BoDV-1 load in persistently BoDV-1-infected cells to prepare a siRNA cocktail (TD-Borna) for BoDV-1 treatment. TD-Borna successfully reduced the BoDV-1 load without enhancing the risk of emergence of escape mutants. The combination of TD-Borna and T-705, a previously reported antiviral agent against bornaviruses, decreased the BoDV-1 load more efficiently than TD-Borna or T-705 alone. Furthermore, TD-Borna efficiently decreased the BoDV-1 load in BoDV-1-infected neuron-derived cells, in which T-705 did not decrease the viral load. Overall, we developed a novel antiviral candidate against BoDV-1, TD-Borna, that can be used in combination with T-705 and is effective against BoDV-1 in neuron-derived cells, in which T-705 is less effective. Considering that BoDV-1 is highly neurotropic, TD-Borna can offer a promising option to improve the outcome of BoDV-1 infection.

SUBMITTER: Teng D

PROVIDER: S-EPMC6895540 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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A Small Interfering RNA Cocktail Targeting the Nucleoprotein and Large Protein Genes Suppresses Borna Disease Virus Infection.

Teng Da D Obika Shunsuke S Ueda Keiji K Honda Tomoyuki T

Frontiers in microbiology 20191129

Recently, Borna disease virus (BoDV-1)-related fatal encephalitis human cases have been reported, which highlights the potential of BoDV-1 to cause fatal human diseases. To protect the infected brain from lethal damage, it is critical to control BoDV-1 as quickly as possible. At present, antivirals against BoDV-1 are limited, and therefore, novel types of antivirals are needed. Here, we developed a novel treatment using small interfering RNAs (siRNAs) against BoDV-1. We screened several siRNAs t ...[more]

PMID: 31849913

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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.

Dataset Information

A Small Interfering RNA Cocktail Targeting the Nucleoprotein and Large Protein Genes Suppresses Borna Disease Virus Infection.

Publications

A Small Interfering RNA Cocktail Targeting the Nucleoprotein and Large Protein Genes Suppresses Borna Disease Virus Infection.

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