Molecular mechanism on the replication of Bombyx densovirus

家蚕致密核病病毒（山梨县分离物；BmDNV-2）是一种具有两节基因组的小球形病毒。为了阐明BmDNV-2的本质，本研究对病毒DNA复制机制、基因组组织和病毒蛋白功能进行了研究。病毒DNA的复制中间体（RI）分析表明，细小病毒复制的自引和发夹转移模型不适用于BmDNV-2的复制。基因组dna的核苷酸测序研究表明，与BmDNV-1和其他细小病毒不同，BmDNV-2末端序列不包含能够形成稳定发夹结构的大回文序列。利用计算机分析程序进行二次结构预测，证实了这一点。这些结果也表明BmDNV-2具有独特的复制机制，而不是自引和发夹转移机制。此外，序列分析表明BmDNV-2基因包含6个主要的开放阅读帧（orf），其中VD1和VD2分别有4个和2个。免疫化学研究表明，主要的四种病毒结构蛋白（VP1、2、3和4）编码在VD1的ORF2中。随后，以荧光素酶为报告基因，采用瞬时表达法研究了非结构蛋白（NSPs）的功能。在VD1的ORF3和orf4中分别编码的NSPs p37和p14反激活了主要结构蛋白基因的试探性启动子序列，表明这些蛋白参与了结构蛋白表达的调控。此外，p27是编码在VD2中的NSP，可以反激活所有的病毒启动子，这表明p27是BmDNV-2复制的关键调控蛋白。总之，本研究阐明了BmDNV-2的基因组结构和基因组组织。结果表明，BmDNV-2是一种新型单链DNA病毒，具有不同于细小病毒的独特复制机制。此外，对病毒蛋白的功能分析表明，病毒基因的表达受到病毒dna编码蛋白的复杂调控。p27将是BmDNV-2复制的关键调控蛋白，可能是决定宿主特异性的因素之一。少

Bombyx mori denso nucleosis virus (Yamanashi isolate ; BmDNV-2) is a small spherical virus with a bi-partite genome. In this study, to elucidate the nature of BmDNV-2, the viral DNA replication mechanism, genome organization and function of viral proteins were investigated. Analysis of the replicative intermediates (RI) of the viral DNA suggested the self-priming and hairpin-transfer model for parvovirus replication could not be applied to the replication of BmDNV-2. Nucleotide sequencing studies of the genomic DNAs showed that, unlike BmDNV-1 and the other parvoviruses the BmDNV-2 terminal sequences did not contained the large palindromic sequence which could form a stable hairpin structure. This was confirmed by the secondary structure prediction using a computer analyzes program. These results also suggested that the BmDNV-2 had a unique replication mechanisms other than the self-priming and hairpin-transfer mechanism.Furthermore, the sequence analyzes elucidated that the BmDNV-2 ge … More nome contained 6 major open reading frames (ORFs), 4 ORFs in VD1 and 2in VD2. Immunochemical studies demonstrated that the major four viral structural proteins (VP1,2,3 and 4) were encoded in the ORF2 of VD1. Followingly, the functions of the nonstructural proteins (NSPs) were investigated by transient expression assay using the luciferase as a reporter. The NSPs, p37 and p14 which were encoded in ORF3 and4 of VD1, respectively, transactivated the tentative promoter sequence for the major structural protein gene, suggesting that these proteins were concerned in the regulation of the expression fo structural proteins. In addition, p27 which was an NSP encoded in VD2 could transactivate all of the viral promoters implying that p27 is a key regulatory protein in the BmDNV-2 replication.In conclusion, this study elucidated the genome structure and the genome organization of the BmDNV-2. The results revealed that the BmDNV-2 is a new type of single-stranded DNA virus with a unique replication mechanism which was different from those of parvoviruses. Furthermore, the functional analyzes of the viral proteins suggested that the viral gene expression was regulated in the complicated manner by the proteins encoded in viral DNAs. And, p27 will be a key regulatory protein in the replication of the BmDNV-2 and may be one of the factor concerning in the determination of the host specificity. Less