Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3′ and 5′ End Repair and Role of Polyprotein Processing in Viral Replication
Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3′ and 5′ End Repair and Role of Polyprotein Processing in Viral Replication
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DOI：
10.1371/journal.pone.0031190
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发表时间：
2012-02-15
期刊：
影响因子：
3.7
通讯作者：
Savithri, Handanahal S.
中科院分区：
文献类型：
--
作者：
Govind, Kunduri;Makinen, Kristiina;Savithri, Handanahal S.
田菁花叶病毒（SeMV）是一种正链RNA病毒，属于Sobemovirus属。构建感染性克隆是研究病毒基因在体内功能的重要步骤。利用基于农杆菌的瞬时表达系统，我们表明SeMV icDNA对田菁和瓜尔豆植物具有感染性。与大花田菁相比，发现icDNA感染瓜尔豆属植物的效率显著较高。在渗透后6天内，在渗透的叶片中可以检测到外壳蛋白。在北方分析中可以检测到不同种类的病毒RNA（双链和单链基因组和亚基因组RNA），表明已经发生了完全复制。通过对SeMV icDNA渗入叶片的子代RNA及其3'和5'末端缺失突变体的基因组末端序列分析，提出了SeMV 3'和5'末端修复的可能机制。ORF 2编码的多聚蛋白中切割位点的突变导致icDNA感染的完全丧失，这表明在所有四个切割位点处正确的多聚蛋白加工对于病毒复制的重要性。互补分析表明ORF 2基因产物可以反式作用，但ORF 2基因产物的反式作用能力在多聚蛋白2a和2ab的N端疏水结构域缺失后被消除，这表明这些产物必然在复制位点起作用，在那里它们被锚定到膜上。
Sesbania mosaic virus (SeMV) is a positive stranded RNA virus belonging to the genus Sobemovirus. Construction of an infectious clone is an essential step for deciphering the virus gene functions in vivo. Using Agrobacterium based transient expression system we show that SeMV icDNA is infectious on Sesbania grandiflora and Cyamopsis tetragonoloba plants. The efficiency of icDNA infection was found to be significantly high on Cyamopsis plants when compared to that on Sesbania grandiflora. The coat protein could be detected within 6 days post infiltration in the infiltrated leaves. Different species of viral RNA (double stranded and single stranded genomic and subgenomic RNA) could be detected upon northern analysis, suggesting that complete replication had taken place. Based on the analysis of the sequences at the genomic termini of progeny RNA from SeMV icDNA infiltrated leaves and those of its 3' and 5' terminal deletion mutants, we propose a possible mechanism for 3' and 5' end repair in vivo. Mutation of the cleavage sites in the polyproteins encoded by ORF 2 resulted in complete loss of infection by the icDNA, suggesting the importance of correct polyprotein processing at all the four cleavage sites for viral replication. Complementation analysis suggested that ORF 2 gene products can act in trans. However, the trans acting ability of ORF 2 gene products was abolished upon deletion of the N-terminal hydrophobic domain of polyprotein 2a and 2ab, suggesting that these products necessarily function at the replication site, where they are anchored to membranes.