The Borna Disease Virus 2 (BoDV-2) Nucleoprotein Is a Conspecific Protein That Enhances BoDV-1 RNA-Dependent RNA Polymerase Activity
The Borna Disease Virus 2 (BoDV-2) Nucleoprotein Is a Conspecific Protein That Enhances BoDV-1 RNA-Dependent RNA Polymerase Activity
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DOI：
10.1128/jvi.00936-21
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发表时间：
2021-11-01
影响因子：
5.4
通讯作者：
Tomonaga, Keizo
中科院分区：
文献类型：
--
作者：
Kanda, Takehiro;Horie, Masayuki;Tomonaga, Keizo
基于博尔纳病病毒1（BoDV-1）的基于RNA病毒的附加型载体（REVec）是一种有前途的病毒载体，其在转导细胞中实现稳定和长期的基因表达。然而，用于产生REVec的反向遗传学的繁琐程序是必须克服的挑战之一，以使REVec技术实用。在这项研究中，为了解决反向遗传学带来的问题，我们专注于BoDV-2，一种在哺乳动物1型正核糖核酸病毒中与BoDV-1同源的病毒。我们根据参考序列合成了BoDV-2核蛋白（N）和磷蛋白（P），并评估了它们对BoDV-1大蛋白（L）的RNA聚合酶活性和病毒复制的影响。在微复制子测定中，我们发现BoDV-2 N显著增强BoDV-1聚合酶活性，并且BoDV-2 P支持N进一步增强该活性。单一氨基酸取代测定鉴定了BoDV-2 N的位置30处的丝氨酸和BoDV-2 P的位置24处的丙氨酸为用于增强BoDV-1聚合酶活性的关键氨基酸残基。相反，在反向遗传学中，单独的BoDV-2 N足以增加REVec的拯救效率。我们表明，REVec可以直接从转染的293 T细胞中拯救，通过使用BoDV-2 N作为辅助质粒，而无需与Vero细胞共培养，并经过数周的传代。此外，携带BoDV-2 N的嵌合REVec在转导细胞中产生比野生型REVec高得多的转基因mRNA和基因组RNA水平。我们的研究结果不仅有助于改进的REVec系统，但也了解orthobornaviruspolymeraseactivity.Importance博尔纳病病毒1（BoDV-1），一个原型病毒的物种哺乳动物1 orthobornaviruses，是一个nonsegmented负链RNA病毒，持续在宿主细胞核。BoDV-1的核蛋白（N）可与基因组和反基因组病毒RNA结合，在病毒的转录和复制过程中起重要作用。在本研究中，我们证明了另一种基因型的BoDV-2的N可以参与BoDV-1的病毒核糖核蛋白复合物，并在BoDV-1微复制子试验和反向遗传学系统中增强BoDV-1聚合酶（L）的活性。表达BoDV-2N而不表达BoDV-1 N的嵌合重组BoDV-1显示出更高的转录和复制水平，而嵌合病毒的增殖和感染性颗粒产生与野生型BoDV-1的那些相当，这表明病毒在核中的复制水平不直接参与BoDV的子代病毒颗粒产生。我们的研究结果证实了RNA病毒聚合酶活性的分子机制，这将有助于进一步开发使用正RNA病毒的载体系统。
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, con-versely, 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 trans-duced 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 dem-onstrated that the N of BoDV-2, another genotype in the species Mammalian 1 orthobor-navirus, 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 infec-tious 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 mecha-nism of bornaviral polymerase activity, which will contribute to further development of vector systems using orthobornaviruses.