长链非编码RNA CTO-S对伪狂犬病毒复制的影响

Effect of long non-coding RNA CTO-S on the replication of pseudorabies virus

【目的】研究伪狂犬病毒(pseudorabies virus,PRV)的长链非编码RNA(long noncoding RNA,lncRNA)CTO-S对于病毒复制的影响。【方法】利用3′RACE和5′RACE鉴定CTO-S的全长序列。验证CTO-S是否具有编码多肽的能力。Red重组方法构建CTO-S缺失株以及回复突变株。分析野毒株、缺失株和回复突变株复制的差异。利用RNA pull down联合质谱的方法筛选与CTO-S互作的宿主蛋白。利用RNA免疫共沉淀(RNA immunoprecipitation,RIP)方法进行验证。最后利用四甲基偶氮唑盐(MTT)法检测野毒株、缺失株和回复突变株对细胞凋亡的影响。【结果】CTO-S全长267 bp,不具有编码多肽的能力。CTO-S缺失不影响PRV的复制。CTO-S与泛醇细胞色素c还原酶的组成部分cytochrome b-c1 complex subunit 1互作,诱导细胞凋亡。【结论】CTO-S对于PRV的复制是非必需的,CTO-S与泛醇细胞色素c还原酶的组成部分cytochrome b-c1 complex subunit 1互作,诱导细胞凋亡。

[Objective]This study aims to reveal the effect of the long non-coding RNA(lncRNA)CTO-S on the replication of pseudorabies virus(PRV).[Methods]We employed 3′RACE and 5′RACE to identify the full-length sequence of CTO-S,and verified whether CTO-S had the ability to encode polypeptides.After constructing the CTO-S deletion and complementation strains with the Red recombination method,we analyzed the differences in the replication of wild-type,deletion,and complementation strains.We used RNA pull down combined with mass spectrometry to screen out the host protein interacting with CTO-S,and then adopted RNA immunoprecipitation(RIP)method to verify the interaction between the screened protein and CTO-S.Finally,the methyl tetrazolium(MTT)method was used to detect the effects of wild-type,deletion,and complementation strains on cell apoptosis.[Results]CTO-S had a full length of 267 bp and did not have the ability to encode polypeptides.The deletion of CTO-S did not affect the replication of PRV.CTO-S interacted with cytochrome b-c1 complex subunit 1,a component of ubiquinol-cytochrome c reductase,and induced apoptosis.[Conclusion]CTO-S is not essential for the replication of PRV.It interacts with cytochrome b-c1 complex subunit 1,a component of ubiquinol-cytochrome c reductase,and induces cell apoptosis.