Department of Biotechnology, Indian Institute of Technology, Kharagpur, Kharagpur 721302, West Bengal, India.
Virology. 2010 Aug 15;404(1):21-31. doi: 10.1016/j.virol.2010.04.019. Epub 2010 May 20.
Genome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123 kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123 kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37 degrees C at pH 6.0 in the presence of 3 mM MgCl(2). Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents.
家蚕多粒包埋病毒基因组片段 2（S2）被转化为 cDNA 并进行了克隆和测序。S2 由 3798 个核苷酸组成，含有一个长开放阅读框，编码一个 1116 个氨基酸的蛋白质（123 kDa）。BLAST 和系统发育分析显示，AmCPV S2 与其他昆虫细胞质多角体病毒的 RNA 依赖性 RNA 聚合酶（RdRp）具有 29%的序列同一性和密切的亲缘关系，表明所有昆虫细胞质多角体病毒具有共同的起源。通过杆状病毒重组体在家蚕细胞中表达 S2 的 ORF 作为 123 kDa 可溶性 His 标记融合蛋白，该蛋白在体外 RNA 聚合酶测定中显示 RdRp 活性，而没有任何内在末端转移酶活性。在 pH6.0 下，在 3 mM MgCl2 的存在下，在 37°C 时观察到最大活性。定点突变证实了保守的 GDD 基序的重要性。这是首次对细胞质多角体病毒 RdRp 的功能特征进行描述，这可能导致抗病毒药物的开发。
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