Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University College of Agriculture and Life Sciences, Ithaca, NY, 14853, USA.
J Gen Virol. 2019 Apr;100(4):709-720. doi: 10.1099/jgv.0.001234. Epub 2019 Feb 18.
Grapevine red blotch virus (GRBV) is type member of the newly identified genus Grablovirus. It possesses a single-stranded circular DNA genome of around 3200 nucleotides encoding three open reading frames (ORFs) in both the virion sense, the V1 (CP), V2 and V3, and complementary sense, C1 (RepA), C2 and C3. As shown for members of the genus Mastrevirus, the C1 and C2 ORFs are predicted to fuse through splicing to form a replication-associated protein (Rep). Data obtained using high-throughput sequencing (RNA-Seq) of three RNA-enriched populations, extracted from GRBV-infected grapevine (Vitis vinifera), confirmed the presence of the predicted C1-C2 intron (nts 2288-2450), but in addition identified a larger virion-sense intron (nts 251-589) spanning the V2 ORF. Evidence for both introns in a number of isolates was supported by bioinformatic analysis of publicly available datasets (n=20). These observations were further supported by RT-PCR analyses in both GRBV-infected grapevine and transient expression assays where GRBV genome segments were agro-inoculated onto Nicotiana benthamiana. The donor site of the virion-sense intron is located within two small ORFs, V0 and V02, while the acceptor site is two-thirds along the V2 ORF. Splicing at these positions is predicted to delete the N terminus of the encoded V2 protein. Comparative analyses of full-length GRBV sequences and the related tentative grabloviruses Prunus geminivirus A and wild Vitis virus 1 support the existence of both introns and V0. The probable regulatory role of these introns in the GRBV infection cycle is discussed.
葡萄卷叶红斑病毒（GRBV）是新鉴定的 Grablovirus 属的模式成员。它具有约 3200 个核苷酸的单链环状 DNA 基因组，在病毒义上编码三个开放阅读框（ORF），V1（CP）、V2 和 V3，以及互补义，C1（RepA）、C2 和 C3。与 Mastrevirus 属的成员一样，C1 和 C2 ORF 预计通过剪接融合形成复制相关蛋白（Rep）。使用从 GRBV 感染的葡萄（Vitis vinifera）中提取的三个富含 RNA 的群体的高通量测序（RNA-Seq）获得的数据证实了预测的 C1-C2 内含子（nt 2288-2450）的存在，但此外还鉴定了一个较大的病毒义内含子（nt 251-589）跨越 V2 ORF。对来自多个分离株的这两个内含子的证据得到了公共数据集（n=20）的生物信息学分析的支持。这些观察结果进一步得到了在 GRBV 感染的葡萄和瞬时表达测定中 RT-PCR 分析的支持，其中 GRBV 基因组片段被 agro-inoculated 到 Nicotiana benthamiana 上。病毒义内含子的供体位点位于两个小 ORF，V0 和 V02 内，而受体位点位于 V2 ORF 的三分之二处。这些位置的剪接预计会删除编码的 V2 蛋白的 N 端。对全长 GRBV 序列和相关的暂定 grabloviruses Prunus geminivirus A 和野生 Vitis virus 1 的比较分析支持这两个内含子和 V0 的存在。讨论了这些内含子在 GRBV 感染周期中的可能调节作用。
