Qiu W, Scholthof K B
Department of Plant Pathology and Microbiology, Texas A&M University, College Station, Texas 77843, USA.
J Virol. 2000 Mar;74(5):2247-54. doi: 10.1128/jvi.74.5.2247-2254.2000.
Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3' untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5' UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5' UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential "trigger" for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus.
卫星黍花叶病毒（SPMV）依赖其辅助病毒黍花叶病毒（PMV）来提供用于复制和移动的反式作用蛋白。SPMV的824个核苷酸（nt）基因组拥有一个编码17.5 kDa衣壳蛋白（CP）的开放阅读框，已证明该蛋白对SPMV复制并非必需。为了定位复制和移动所需的顺式作用RNA元件，构建了一套完整的SPMV cDNA缺失突变体。结果表明，263 nt的3'非翻译区（UTR）加上CP终止密码子上游73 nt以及5' UTR中的前16 nt是SPMV RNA扩增和/或系统传播所必需的。5' UTR内17至67 nt的区域可能在RNA积累中起辅助作用，一个包含68至104 nt的片段似乎以宿主依赖的方式参与SPMV RNA的系统移动。出乎意料的是，在与PMV以及两个SPMV突变体之一共感染的粟植株中，缺陷RNA（D-RNA）从头积累：SPMV-91不能表达17.5 kDa的CP，SPMV-GUG表达低水平的17.5 kDa CP。从感染植株中分离出源自SPMV-91的D-RNA，并用作模板生成cDNA克隆。源自这个399 nt cDNA的RNA转录本在与PMV共同接种的粟植株中进行复制和移动。这种D-RNA的特性提供了生物学证据，证明通过反向遗传策略鉴定的关键RNA结构域对SPMV复制和移动至关重要。结果还表明，自发D-RNA积累的潜在“触发因素”可能与17.5 kDa SPMV CP的缺失或积累减少有关。这是与卫星病毒相关的D-RNA的首次报道。
