Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus genome segment 9
Molecular cloning and characterization of Antheraea mylitta cytoplasmic polyhedrosis virus genome segment 9
复制标题
DOI：
10.1099/0022-1317-83-6-1483
复制
发表时间：
2002-06-01
影响因子：
3.8
通讯作者：
Ghosh, AK
中科院分区：
文献类型：
--
作者：
Qanungo, KR;Kundu, SC;Ghosh, AK
对3个家蚕质型多角体病毒（CPV）分离物AmCPV、AaCPV和ApCPV的11个片段的RNA基因组中的第9个片段进行了cDNA转化、克隆和序列测定。在每种情况下，该基因组片段由1473个核苷酸组成，具有一个1035 bp的长ORF，编码345个氨基酸的蛋白质，称为NSP38，分子量为38 kDa。二级结构预测显示，中心和末端结构域中存在9个α螺旋，与蓝舌病毒和传染性囊病病毒RNA聚合酶的RNA结合基序具有局部相似性。这三株CPV的核苷酸序列具有99.6%的相同性，但与公共数据库中的任何其他核苷酸或蛋白质序列均未发现相似性。AmCPV cDNA的ORF在E. coli中表达，并制备了多克隆抗体。免疫印迹以及免疫荧光分析与抗NSP38抗体表明，该蛋白不存在于多角体或未感染的细胞，但存在于AmCPV感染的宿主中肠细胞。NSP38通过杆状病毒表达载体在昆虫细胞中表达为可溶性蛋白，并显示具有结合poly（rI）-（rC）琼脂糖的能力，其被AmCPV病毒RNA竞争性去除。这些结果表明，NSP38在病毒感染的细胞中作为非结构蛋白表达。通过与病毒RNA结合，它可能在基因组RNA功能和包装的调节中发挥作用。
Genome segment 9 of the 11-segment RNA genomes of three cytoplasmic polyhedrosis virus (CPV) isolates from Antheraea mylitta (AmCPV), Antheraea assamensis (AaCPV) and Antheraea proylei (ApCPV) were converted to cDNA, cloned and sequenced. In each case, this genome segment consists of 1473 nucleotides with one long ORF of 1035 bp and encodes a protein of 345 amino acids, termed NSP38, with a molecular mass of 38 kDa. Secondary structure prediction showed the presence of nine alpha-helices in the central and terminal domains with localized similarity to RNA-binding motifs of bluetongue virus and infectious bursal disease virus RNA polymerases. Nucleotide sequences were 99.6% identical between these three strains of CPVs, but no similarity was found to any other nucleotide or protein sequence in public databases. The ORF from AmCPV cDNA was expressed as a His-tagged fusion protein in E. coli and polyclonal antibody was raised against the purified protein. Immunoblot as well as immunofluorescence analysis with anti-NSP38 antibody showed that the protein was not present in polyhedra or uninfected cells but was present in AmCPV-infected host midgut cells. NSP38 was expressed in insect cells as soluble protein via a baculovirus expression vector and shown to possess the ability to bind poly(rI)-(rC) agarose, which was competitively removed by AmCPV viral RNA. These results indicate that NSP38 is expressed in virus-infected cells as a non-structural protein. By binding to viral RNA, it may play a role in the regulation of genomic RNA function and packaging.