Henan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, China.
Viruses. 2017 Dec 25;10(1):6. doi: 10.3390/v10010006.
Cucumber green mottle mosaic virus (CGMMV) is one of the most devastating viruses infecting members of the family Cucurbitaceae. The assembly initiation site of CGMMV is located in the coding region of the coat protein, which is not only involved in virion assembly but is also a key factor determining the long-distance movement of the virus. To understand the effect of assembly initiation site and the adjacent region on CGMMV infectivity, we created a GTT deletion mutation in the GAGGTTG assembly initiation site of the infectious clone of CGMMV, which we termed V97 (deletion mutation at residue 97 of coat protein), followed by the construction of the V94A and T104A mutants. We observed that these three mutations caused mosaic after -mediated transformation in , albeit with a significant delay compared to the wild type clone. The mutants also had a common spontaneous E96K mutation in the coat protein. These results indicated that the initial assembly site and the sequence of the adjacent region affected the infectivity of the virus and that E96 might play an essential role in this process. We constructed two single point mutants-E96A and E96K-and three double mutants-V94A-E96K, V97-E96K and T104A-E96K-to further understand the role of E96 in CGMMV pathogenesis. After inoculation in , E96A showed delayed systemic symptoms, but the E96K and three double mutants exhibited typical symptoms of mosaic at seven days post-infection. Then, sap from CGMMV-infected leaves was mechanically inoculated on watermelon plants. We confirmed that E96 affected CGMMV infection using double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and sequencing, which further confirmed the successful infection of the related mutants, and that E96K can compensate the effect of the V94, V97, and T104 mutations on virus infectivity. In addition, Northern blotting showed that the accumulation of viral RNA corroborated the severity of the symptoms.
黄瓜绿斑驳花叶病毒（CGMMV）是侵染葫芦科植物的最具破坏性病毒之一。CGMMV 的装配起始位点位于外壳蛋白的编码区，该起始位点不仅参与病毒粒子的装配，而且还是决定病毒远距离运动的关键因素。为了了解装配起始位点及其相邻区域对 CGMMV 感染力的影响，我们在 CGMMV 感染性克隆的 GAGGTTG 装配起始位点上创建了 GTT 缺失突变，我们将其命名为 V97（外壳蛋白残基 97 缺失突变），随后构建了 V94A 和 T104A 突变体。我们观察到，这三个突变体在 后引起花叶，但与野生型克隆相比，有明显的延迟。突变体在外壳蛋白中也有一个共同的自发 E96K 突变。这些结果表明，起始装配位点和相邻区域的序列影响病毒的感染力，E96 可能在这个过程中发挥重要作用。我们构建了两个单点突变体 E96A 和 E96K 以及三个双突变体 V94A-E96K、V97-E96K 和 T104A-E96K，以进一步了解 E96 在 CGMMV 发病机制中的作用。在 接种后，E96A 表现出延迟的系统性症状，但 E96K 和三个双突变体在感染后 7 天表现出典型的花叶症状。然后，用 CGMMV 感染的 叶片汁液进行机械接种西瓜植株。我们通过双抗体夹心酶联免疫吸附测定（DAS-ELISA）、反转录-聚合酶链反应（RT-PCR）和测序证实 E96 影响 CGMMV 感染，进一步证实了相关突变体的成功感染，并且 E96K 可以补偿 V94、V97 和 T104 突变对病毒感染力的影响。此外，Northern 印迹显示病毒 RNA 的积累与症状的严重程度相符。
