The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity
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 determini...
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| Format: | Article |
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MDPI AG
2017-12-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/10/1/6 |
| _version_ | 1818066024223735808 |
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| author | Zhenwei Zhang Liming Liu Huijie Wu Lifeng Liu Baoshan Kang Bin Peng Qinsheng Gu |
| author_facet | Zhenwei Zhang Liming Liu Huijie Wu Lifeng Liu Baoshan Kang Bin Peng Qinsheng Gu |
| author_sort | Zhenwei Zhang |
| collection | DOAJ |
| description | 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 Agrobacterium-mediated transformation in Nicotiana benthamiana, 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 N. benthamiana, 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 N. benthamiana 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. |
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| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-12-10T15:01:12Z |
| publishDate | 2017-12-01 |
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| series | Viruses |
| spelling | doaj.art-eb277ce366704ca4bfbe3865b76c95e82022-12-22T01:44:10ZengMDPI AGViruses1999-49152017-12-01101610.3390/v10010006v10010006The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus InfectivityZhenwei Zhang0Liming Liu1Huijie Wu2Lifeng Liu3Baoshan Kang4Bin Peng5Qinsheng Gu6Henan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, ChinaHenan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, ChinaHenan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, ChinaHenan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, ChinaHenan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, ChinaHenan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, ChinaHenan Key Laboratory of Fruit and Cucurbit Biology, Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, ChinaCucumber 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 Agrobacterium-mediated transformation in Nicotiana benthamiana, 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 N. benthamiana, 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 N. benthamiana 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.https://www.mdpi.com/1999-4915/10/1/6cucumber green mottle mosaic virusassembly initiation siteinfectivitysystemic symptomcoat protein |
| spellingShingle | Zhenwei Zhang Liming Liu Huijie Wu Lifeng Liu Baoshan Kang Bin Peng Qinsheng Gu The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity Viruses cucumber green mottle mosaic virus assembly initiation site infectivity systemic symptom coat protein |
| title | The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity |
| title_full | The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity |
| title_fullStr | The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity |
| title_full_unstemmed | The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity |
| title_short | The 96th Amino Acid of the Coat Protein of Cucumber Green Mottle Mosaic Virus Affects Virus Infectivity |
| title_sort | 96th amino acid of the coat protein of cucumber green mottle mosaic virus affects virus infectivity |
| topic | cucumber green mottle mosaic virus assembly initiation site infectivity systemic symptom coat protein |
| url | https://www.mdpi.com/1999-4915/10/1/6 |
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