Two amino acids near the N-terminus of Cucumber mosaic virus 2b play critical roles in the suppression of RNA silencing and viral infectivity
Two amino acids near the N-terminus of Cucumber mosaic virus 2b play critical roles in the suppression of RNA silencing and viral infectivity
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黄瓜花叶病毒 2b N 末端附近的两个氨基酸在抑制 RNA 沉默和病毒感染性中发挥着关键作用。
DOI：
10.1111/mpp.12270
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发表时间：
2016-02-01
影响因子：
4.9
通讯作者：
Wang, Xian-Bing
中科院分区：
文献类型：
--
作者：
Dong, Kai;Wang, Ying;Wang, Xian-Bing
黄瓜花叶病毒(CMV)2b主要通过结合不同大小的双链RNA(DsRNA)来抑制RNA沉默。然而，2b的生物活性形式仍然难以捉摸。在这里，我们证明了CMV 2b N端15个亮氨酸和18个蛋氨酸的单丙氨酸和双丙氨酸替代突变体在野生型植物中表现出极大的毒力减弱，但在RNA依赖RNA聚合酶6(RDR6)和Dird-like 4(DCL4)缺陷的突变植物中有效拯救。此外，2b而不是2blm(L15A/M18A)的转基因植株通过抑制抗病毒沉默而挽救了Δ-2b的高感染性。L15a、M18a或两者均可减弱2b对局部和系统转基因沉默的抑制活性。与2b对短dsRNAs和长dsRNAs的高亲和力相比，2blm与21bp的双链小干扰RNA(SiRNA)的结合能力显著降低，但对长dsRNAs的亲和力保持较强。在交联法中，2b在戊二醛处理后可以形成二聚体、四聚体和低聚物，而在 体外，2blm只形成二聚体，而不形成四聚体和低聚体。综上所述，这些发现表明CMV 2b的L15和M18是与ds-siRNAs高亲和力和寡聚活性所必需的，这是2b抑制抗病毒沉默活性所必需的。
Cucumber mosaic virus (CMV) 2b suppresses RNA silencing primarily through the binding of double-stranded RNA (dsRNA) of varying sizes. However, the biologically active form of 2b remains elusive. Here, we demonstrate that the single and double alanine substitution mutants in the N-terminal 15th leucine and 18th methionine of CMV 2b exhibit drastically attenuated virulence in wild-type plants, but are efficiently rescued in mutant plants defective in RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Moreover, the transgenic plants of 2b, but not 2blm (L15A/M18A), rescue the high infectivity of CMV-Δ2b through the suppression of antiviral silencing. L15A, M18A or both weaken 2b suppressor activity on local and systemic transgene silencing. In contrast with the high affinity of 2b to short and long dsRNAs, 2blm is significantly compromised in 21-bp duplex small interfering RNA (siRNA) binding ability, but maintains a strong affinity for long dsRNAs. In cross-linking assays, 2b can form dimers, tetramers and oligomers after treatment with glutaraldehyde, whereas 2blm only forms dimers, rather than tetramers and oligomers, in vitro. Together, these findings suggest that L15 and M18 of CMV 2b are required for high affinity to ds-siRNAs and oligomerization activity, which are essential for the suppression activity of 2b on antiviral silencing.