突变和重组可克服异源复制酶在病毒复制中的功能缺陷

Overcome the defect in virus replication conferred by heterogeneous combination of viral replicases through mutation and recombination

异源复制酶的组合无法有效地支持重组病毒的复制是雀麦花叶病毒科病毒存在的普遍现象。利用雀麦花叶病毒科黄瓜花叶病毒属的典型成员黄瓜花叶病毒(cucumber mosaic virus,CMV)和番茄不孕病毒(tomato aspermy virus,TAV)作为研究对象,分析2个复制酶的异源组合对重组病毒复制的影响。通过农杆菌浸润,将CMV RNA1(C1)和TAV RNA2(T2)进行异源组合,并将此与CMV RNA3(C3)或TAV RNA3(T3)混合侵染本生烟。接种后10 d,C1T2C3和C1T2T3接种的植物没有出现明显的病毒症状,通过Northern杂交,在系统叶中仅检测到微弱的病毒特异条带,这说明C1T2的异源组合无法高效地支持病毒的复制和侵染。当C1T2C3转接3代之后,接种的本氏烟植株表现明显的病毒症状,且病毒RNA的积累水平显著增加。病毒基因组RNA的测序结果显示,C1第1 390位、T2第1 695位发生点突变,而且C3的3'末端融合了T2的完整3'UTR序列。以上结果表明,通过病毒的突变或/和重组可有效提高异源复制酶对重组病毒的复制效率。

It is a common phenomenon that recombination of heterogeneous replicases cannot efficiently replicate recombinant virus in Bramoviridae. Here,the effects of heterogeneous combination of viral replicases on viral replication was investigated using cucumber mosaic virus（CMV） and tomato aspermy virus（TAV） as the research objectives,which are the typical species of the genus Cucumovirus,Bromoviridea. CMV RNA1（C1） and TAV RNA2（T2）were heterogeneously combined,mixed with either CMV RNA3（C3） or TAV RNA3（T3）,and then inoculated onto Nicotiana benthamiana plants by agroinfiltration. At 10 days post-infiltration,no obvious viral symptoms were observed on the plants inoculated with C1T2C3 or C1T2T3. Moreover,mild viral bands were detected in the upper systemic leaves of the plants by Northern blot. These results demonstrate that C1T2 heterogeneous combination could not support viral replication/infection efficiently. When C1T2C3 was passaged three times,the infected plants showed obvious viral symptoms and a high accumulation level of viral progeny RNAs. Sequencing data of viral genomic RNAs showed that C1 had a mutation at the position 1 390,and T2 had a mutation at the position 1 695. Moreover,the 3＇end of C3 was tagged with the whole 3＇ UTR of T2. Taken together,the recombinant virus C1T2C3 could overcome defects in virus replication conferred by the heterogeneous combination of their replicases by in vivo mutation and recombination.