Indian Institute of Science, Bangalore, Karnataka, India.
PLoS One. 2012;7(2):e31190. doi: 10.1371/journal.pone.0031190. Epub 2012 Feb 15.
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.
豇豆花叶病毒（SeMV）是一种正链 RNA 病毒，属于 Sobemovirus 属。构建传染性克隆是解析病毒基因在体内功能的重要步骤。我们使用基于农杆菌的瞬时表达系统表明，SeMV icDNA 可在Sesbania grandiflora 和 Cyamopsis tetragonoloba 植物上感染。与 Sesbania grandiflora 相比，icDNA 在 Cyamopsis 植物上的感染效率明显更高。侵染后 6 天内可在侵染叶片中检测到外壳蛋白。通过 northern 分析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），表明已经发生了完整的复制。基于对 SeMV icDNA 侵染叶片中产生的子代 RNA 的基因组末端序列和其 3' 和 5' 末端缺失突变体的分析，我们提出了体内 3' 和 5' 末端修复的可能机制。ORF 2 编码的多蛋白中切割位点的突变导致 icDNA 完全丧失感染能力，这表明正确的多蛋白加工在所有四个切割位点对于病毒复制都很重要。互补分析表明 ORF 2 基因产物可以在反式中起作用。然而，当多蛋白 2a 和 2ab 的 N 端疏水区缺失时，ORF 2 基因产物的反式作用能力被废除，这表明这些产物必然在复制位点起作用，在那里它们被锚定在膜上。
