A cDNA Clone to Clover Yellow Vein Potyvirus Genome is Highly Infectious
A cDNA Clone to Clover Yellow Vein Potyvirus Genome is Highly Infectious
复制标题
三叶草黄脉马铃薯Y病毒基因组的cDNA克隆具有高度传染性
作者：
I. Uyeda;Tohru Takahashi;Yōko Takahashi
我们获得了高传染性的三叶草黄脉病毒（ClYVV）的 cDNA 克隆。对构建全长cDNA克隆的cDNA片段进行测序，并确定ClYVV RNA的完整核苷酸序列。病毒基因组长度为 9584 个核苷酸 (nt)，不包括聚腺苷酸 (poly(A)) 尾，并包含一个编码 3072 个氨基酸的大多聚蛋白的开放阅读框 (ORF)。 ORF 之前的非编码区长 190 nt。终止密码子后面是 175 nt 的序列。通过寻找马铃薯Y病毒组中的切割共有序列，在ClYVV多蛋白中发现了7个潜在的蛋白酶NIa、1个HC-pro和1个P1蛋白酶识别位点。 ClYVV NIa 蛋白酶的裂解二肽是 Q(E)/S(A,G)。除P3/6K1连接处外，F在切割位点的-2位上是保守的，并且在许多马铃薯Y病毒组中在-4位上保守的V根本不存在。确定了 ClYVV 的基因组结构，并将氨基酸序列与其他马铃薯病毒组的氨基酸序列进行了比较。通过组合cDNA片段构建了ClYVV的全长cDNA克隆，并将其置于花椰菜花叶病毒35S启动子的控制下。构建全长cDNA，使得转录起始位点处不存在额外的核苷酸，并且ClYVV cDNA克隆的3'端仅存在10个腺嘌呤残基。将环状质粒DNA机械接种到蚕豆幼苗上，引起全身感染，症状与野生型病毒相似，但较轻微。稀释至 500 pg/μl 的质粒能够诱发症状，证明这种全长 ClYVV cDNA 比迄今为止报道的任何其他感染性 cDNA 更具感染性。通过免疫电镜在受感染植物的粗汁液中观察到与 ClYVV 抗血清反应的丝状颗粒，并通过植物总 RNA 中 ClYVV 基因组 3' 非编码区的 RT-PCR 证明了基因组复制。
We obtained a highly infectious cDNA clone of clover yellow vein virus (ClYVV). The cDNA fragments, from which a full-length cDNA clone was constructed, were sequenced, and the complete nucleotide sequence of ClYVV RNA was determined. The viral genome is 9584 nucleotides (nt) in length excluding the poly(A) tail and contains one open reading frame (ORF) encoding a large polyprotein of 3072 amino acids. The non-coding region preceding the ORF is 190 nt long. The termination codon is followed by a 175-nt sequence. Seven potential protease NIa, one HC-pro and one P1 protease recognition sites were found in the ClYVV polyprotein by searching for cleavage consensus sequences among the potyvirus group. The cleavage dipeptides of ClYVV NIa protease are Q(E)/S(A,G). The F is conserved at the −2 position from the cleavage site except for at the P3/6K1 junction, and the V conserved at the −4 position among many potyviruses is not present at all. The genome organization of ClYVV was determined, and the amino acid sequence was compared with that of other potyviruses. The full-length cDNA clone of ClYVV was constructed by combining cDNA fragments and placed it under the control of the cauliflower mosaic virus 35S promoter. The full-length cDNA was constructed so that no extra nucleotide was present at the transcription initiation site and only 10 adenine residues were present at the 3′ end of the ClYVV cDNA clone. Mechanical inoculation of a circular-formed plasmid DNA onto broad bean seedlings led to systemic infection, and the symptoms were similar to those caused by the wild-type virus but rather mild. Plasmid diluted as low as 500 pg/µl was able to induce symptoms, demonstrating that this full-length ClYVV cDNA is more infectious than any other infectious cDNAs so far reported. Filamentous particles reacting with the antiserum to ClYVV were observed in the crude sap of infected plants by immunoelectron microscopy, and genome replication was demonstrated by RT-PCR of 3′ non-coding regions of ClYVV genome in total plant RNAs.
烟草静脉斑驳病毒多蛋白中辅助成分蛋白 N 末端或附近的体外裂解。
影响因子：
3.7
作者：
Mavankal,G;Rhoads,RE
通讯作者：
Rhoads,RE