Rose rosette virus (RRV) is a negative-sense RNA virus with a seven-segmented genome that is enclosed by a double membrane. We constructed an unconventional minireplicon system encoding the antigenomic (ag)RNA1 (encoding the viral RNA-dependent RNA polymerase [RdRp]), agRNA3 (encoding the nucleocapsid protein [N]), and a modified agRNA5 containing the coding sequence for the iLOV protein in place of the P5 open reading frame (R5-iLOV). iLOV expression from the R5-iLOV template was amplified by activities of the RdRp and N proteins in Nicotiana benthamiana leaves. A mutation was introduced into the RdRp catalytic domain and iLOV expression was eliminated, indicating RNA1-encoded polymerase activity drives iLOV expression from the R5-iLOV template. Fluorescence from the replicon was highest at 3 days postinoculation (dpi) and declined at 7 and 13 dpi. Addition of the tomato bushy stunt virus (TBSV) P19 silencing-suppressor protein prolonged expression until 7 dpi. A full-length infectious clone system was constructed of seven binary plasmids encoding each of the seven genome segments. Agro-delivery of constructs encoding RRV RNAs 1 through 4 or RNAs 1 through 7 to N. benthamiana plants produced systemic infection. Finally, agro-delivery of the full-length RRV infectious clone including all segments produced systemic infection within 60 dpi. This advance opens new opportunities for studying RRV infection biology.

玫瑰花环病毒（RRV）是一种负义RNA病毒，具有由双膜包围的七段基因组。我们构建了一个非常规的微型复制子系统，该系统编码反基因组（ag）RNA1（编码病毒RNA依赖性RNA聚合酶[RdRp]），agRNA3（编码核衣壳蛋白[N]）和包含iLOV蛋白编码序列的修饰agRNA5代替P5开放阅读框（R5-iLOV）。R5-iLOV模板中的iLOV表达通过本底烟草中RdRp和N蛋白的活性而扩增树叶。将突变引入RdRp催化域，并消除了iLOV表达，这表明RNA1编码的聚合酶活性从R5-iLOV模板驱动了iLOV表达。复制子发出的荧光在接种后3天（dpi）最高，而在7和13 dpi时下降。番茄浓密特技病毒（TBSV）P19沉默抑制蛋白的添加将表达延长至7 dpi。全长感染性克隆系统由编码七个基因组片段中每个片段的七个二元质粒构建。将编码RRV RNA 1至4或RNA 1至7的构建体农业运输至本氏烟草植物产生全身性感染。最后，全长RRV感染性克隆（包括所有片段）的农用递送在60 dpi内产生了系统性感染。这一进步为研究RRV感染生物学开辟了新的机会。