Biswas Poulomi, Kundu Anirban, Ghosh Ananta Kumar
Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India.
J Gen Virol. 2015 Jan;96(Pt 1):95-105. doi: 10.1099/vir.0.069716-0. Epub 2014 Sep 16.
Cloning and sequencing of Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV) genome segment S4 showed that it consists of 3410 nt with a single ORF of 1110 aa which could encode a protein of ~127 kDa (p127). Bioinformatics analysis showed the presence of a 5' RNA triphosphatase (RTPase) domain (LRDR), a S-adenosyl-l-methionine (SAM)-binding (GxGxG) motif and the KDKE tetrad of 2'-O-methyltransferase (MTase), which suggested that S4 may encode RTPase and MTase. The ORF of S4 was expressed in Escherichia coli as a His-tagged fusion protein and purified by nickel-nitrilotriacetic acid affinity chromatography. Biochemical analysis of recombinant p127 showed its RTPase as well as SAM-dependent guanine N(7)-and ribose 2'-O-MTase activities. A MTase assay using in vitro transcribed AmCPV S2 RNA having a 5' G*pppG end showed that guanine N(7) methylation occurred prior to the ribose 2'-O methylation to yield a m(7)GpppG/m(7)GpppGm RNA cap. Mutagenesis of the SAM-binding (GxGxG) motif (G831A) completely abolished N(7)- and 2'-O-MTase activities, indicating the importance of these residues for capping. From the kinetic analysis, the Km values of N(7)-MTase for SAM and RNA were calculated as 4.41 and 0.39 µM, respectively. These results suggested that AmCPV S4-encoded p127 catalyses RTPase and two cap methylation reactions for capping the 5' end of viral RNA.
蓖麻蚕细胞质多角体病毒（AmCPV）基因组片段S4的克隆与测序表明，它由3410个核苷酸组成，有一个1110个氨基酸的单一开放阅读框，可编码一个约127 kDa的蛋白质（p127）。生物信息学分析显示存在一个5' RNA三磷酸酶（RTPase）结构域（LRDR）、一个S-腺苷-L-甲硫氨酸（SAM）结合（GxGxG）基序以及2'-O-甲基转移酶（MTase）的KDKE四联残基，这表明S4可能编码RTPase和MTase。S4的开放阅读框在大肠杆菌中表达为His标签融合蛋白，并通过镍-次氮基三乙酸亲和层析进行纯化。重组p127的生化分析显示其具有RTPase以及SAM依赖性鸟嘌呤N(7)-和核糖2'-O-MTase活性。使用具有5' G*pppG末端的体外转录AmCPV S2 RNA进行的MTase分析表明，鸟嘌呤N(7)甲基化先于核糖2'-O甲基化发生，产生m(7)GpppG/m(7)GpppGm RNA帽。SAM结合（GxGxG）基序（G831A）的诱变完全消除了N(7)-和2'-O-MTase活性，表明这些残基对加帽的重要性。通过动力学分析，计算出N(7)-MTase对SAM和RNA的Km值分别为4.41和0.39 μM。这些结果表明，AmCPV S4编码的p127催化RTPase和两个帽甲基化反应，用于封闭病毒RNA的5'末端。
