Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur 721302, India.
Virol J. 2014 Mar 21;11:53. doi: 10.1186/1743-422X-11-53.
Antheraea mylitta cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of Reoviridae family, infects non mulberry Indian silk worm, Antheraea mylitta, and contains eleven segmented double stranded RNA in its genome (S1-S11). Some of its genome segments (S1-S3, and S6-S11) have been previously characterized but genome segment encoding the viral guanylyltransferase which helps in RNA capping has not been characterized.
In this study genome segment 5 (S5) of AmCPV was converted to cDNA, cloned and sequenced. S5 consisted of 2180 nucleotides, with one long ORF of 1818 nucleotides and could encode a protein of 606 amino acids with molecular mass of ~65 kDa (p65). Bioinformatics analysis showed presence of KLRS and HxnH motifs as observed in some other reoviral guanylyltransferase and suggests that S5 may encodes viral guanylyltransferase. The ORF of S5 was expressed in E. coli as 65 kDa his tagged fusion protein, purified through Ni-NTA chromatography and polyclonal antibody was raised. Immunoblot analysis of virion particles with the purified antibody showed specific immunoreactive band and suggests p65 as a viral structural protein. Functional analysis showed that recombinant p65 possesses guanylyltransferase activity, and transfers GMP moiety to the 5' diphosphate (A/G) ended viral RNA after the formation of p65-GMP complex for capping. Kinetic analysis showed K(m) of this enzyme for GTP and RNA was 34.24 uM and 98.35 nM, respectively. Site directed mutagenesis at K21A in KLRS motif, and H93A or H105A in HxnH motif completely abolished the autoguanylylation activity and indicates importance of these residues at these sites. Thermodynamic analysis showed p65-GTP interaction was primarily driven by enthalpy (ΔH = -399.1 ± 4.1 kJ/mol) whereas the p65-RNA interaction by favorable entropy (0.043 ± 0.0049 kJ/ mol).
Viral capping enzymes play a critical role in the post transcriptional or post replication modification in case of RNA virus. Our results of cloning, sequencing and functional analysis of AmCPV S5 indicates that S5 encoded p65 through its guanylyltransferase activity can transfer guanine residue to the 5' end of viral RNA for capping. Further studies will help to understand complete capping process of cypoviral RNA during viral replication within the viral capsid.
野桑蚕质型多角体病毒（AmCPV）是呼肠孤病毒科的一种环状病毒，感染非桑蚕的野桑蚕，其基因组中含有 11 个分段的双链 RNA（S1-S11）。其部分基因组片段（S1-S3 和 S6-S11）已经得到了鉴定，但编码病毒鸟苷转移酶的基因组片段尚未得到鉴定，该酶有助于 RNA 加帽。
本研究将 AmCPV 的基因组片段 5（S5）转化为 cDNA，进行克隆和测序。S5 由 2180 个核苷酸组成，具有一个长的 1818 个核苷酸的开放阅读框，可编码一个 606 个氨基酸的蛋白质，分子量约为 65 kDa（p65）。生物信息学分析显示，在其他一些呼肠孤病毒鸟苷转移酶中观察到 KLRS 和 HxnH 基序，表明 S5 可能编码病毒鸟苷转移酶。在大肠杆菌中表达 S5 的 ORF 作为 65 kDa 的 his 标记融合蛋白，通过 Ni-NTA 层析纯化，并制备多克隆抗体。用纯化的抗体对病毒粒子进行免疫印迹分析显示出特异性的免疫反应带，表明 p65 是一种病毒结构蛋白。功能分析表明，重组 p65 具有鸟苷转移酶活性，可在 p65-GMP 复合物形成后，将 GMP 部分转移到 5'二磷酸（A/G）末端的病毒 RNA 上，进行加帽。动力学分析显示，该酶对 GTP 和 RNA 的 K(m) 分别为 34.24 μM 和 98.35 nM。在 KLRS 基序中的 K21A 以及 HxnH 基序中的 H93A 或 H105A 进行定点突变，完全消除了自身鸟苷转移酶活性，表明这些残基在这些位点的重要性。热力学分析表明，p65-GTP 相互作用主要由焓驱动（ΔH =-399.1 ± 4.1 kJ/mol），而 p65-RNA 相互作用由有利的熵驱动（0.043 ± 0.0049 kJ/mol）。
病毒加帽酶在 RNA 病毒的转录后或复制后修饰中起着关键作用。我们对 AmCPV S5 的克隆、测序和功能分析的结果表明，S5 通过其鸟苷转移酶活性编码的 p65 可以将鸟嘌呤残基转移到病毒 RNA 的 5'端进行加帽。进一步的研究将有助于了解野桑蚕质型多角体病毒 RNA 在病毒衣壳内复制过程中的完整加帽过程。
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