Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot virus
Nucleotide sequence and infectious transcripts from a full-length cDNA clone of the carmovirus Melon necrotic spot virus
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DOI：
10.1007/s00705-002-0927-y
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发表时间：
2003-03-01
影响因子：
2.7
通讯作者：
Aranda, MA
中科院分区：
文献类型：
--
作者：
Díaz, JA;Bernal, JJ;Aranda, MA
我们研究了在西班牙收集的 MNSV 分离株 (MNSV-Maalpha) 的生物学和分子特征，并生成了全长 cDNA 克隆，从中可以产生感染性 RNA 转录本。 MNSV May 的宿主范围似乎仅限于葫芦科植物，与 MNSV Dutch 没有什么不同 [4, 21]。然而，两种分离株可能引起的症状类型存在差异。使用锚定T7 RNA启动子序列的5'端引物和3'端引物通过逆转录聚合酶链式反应(RT PCR)直接扩增并克隆MNSV-Maalpha的全长cDNA。从该 cDNA 克隆转录的无帽 RNA 具有传染性，当机械接种到甜瓜、黄瓜或西瓜植物上时，所引起的症状与病毒 RNA 引起的症状难以区分。 MNSV-Malpha5 的完整基因组序列是从全长 cDNA 克隆推导出来的。它长 4271 nt，与 MNSV-Dutch 类似，由 5' 和 3' 非翻译区 (UTR) 以及编码 29、89,42 和两个小 7 kDa 蛋白的 5 个开放阅读框 (ORF) 组成。发现了 MNSV-Maalpha 和其他已测序的 MNSV 分离株之间的一个显着差异，对于 MNSV May，在基因组中连续的两个小 ORF 中的第一个以真正的终止密码子终止，而对于 MNSV-Dutch 和其他已测序的 MNSV 分离株，它以琥珀密码子终止。这表明可以从 MNSV-Dutch 和其他 MNSV 基因组表达的推定 p14 通读蛋白不能从 MNSV-Maalpha 基因组表达。此外，核苷酸和氨基酸序列比较显示 MNSV-Maalpha 与其他已知的 MNSV 分离株有较远的关系。
We have studied the biological and molecular characteristics of a MNSV isolate collected in Spain (MNSV-Maalpha) and generated a full-length cDNA clone from which infectious RNA transcripts can be produced. The host range of MNSV May appeared to be limited to cucurbits and did not differ from that of MNSV Dutch [4, 21]. However, differences were observed in the type of symptoms that both isolates could induce. A full-length cDNA of MNSV-Maalpha was directly amplified by reverse-transcription polymerase chain reaction (RT PCR) using a 5'-end primer anchoring a T7 RNA promoter sequence and a 3'-end primer, and cloned. Uncapped RNAs transcribed from this cDNA clone were infectious and caused symptoms indistinguishable from those caused by viral RNA when mechanically inoculated onto melon, cucumber or watermelon plants. The complete genome sequence of MNSV-Malpha5 was deduced from the full length cDNA clone. It is 4271 nt long and, similarly to MNSV-Dutch, consists of 5' and 3' untranslated regions(UTRs) and five open reading frames (ORFs) coding for 29, 89,42 and two small 7 kDa proteins. One notable difference between MNSV-Maalpha and other sequenced MNSV isolates was found, as for MNSV May the first of the two small ORFs, which are contiguous in the genome, terminates with a genuine stop codon, whereas for MNSV-Dutch and other sequenced MNSV isolates it terminates with an amber codon. This suggested that the putative p14 readthrough protein that could be expressed from the MNSV-Dutch and other MNSV genomes could not be expressed from the MNSV-Maalpha genome. Also, the nucleotide and amino acid sequences comparisons showed a distant relationship of MNSV-Maalpha with other known MNSV isolates.