Stein D A, Skilling D E, Iversen P L, Smith A W
AVI BioPharma, Inc., Corvallis, OR 97333, USA.
Antisense Nucleic Acid Drug Dev. 2001 Oct;11(5):317-25. doi: 10.1089/108729001753231696.
Caliciviruses infect and cause disease in animals and humans. They are nonenveloped, positive-stranded RNA viruses with a genome of approximately 7.5 kb that encodes viral proteins in three open reading frames (ORF). Antisense oligomers targeting one of the three ORF of caliciviruses of the genus Vesivirus significantly inhibit viral replication in tissue culture. Porcine kidney and African green monkey kidney cells were infected with Vesivirus isolates SMSV-13 and PCV Pan-1. Phosphorodiamidate morpholino oligomers (PMO) with sequence complementary to the AUG translation start site regions of ORF1, ORF2, and ORF3 were evaluated for their effect on viral titer. Scrape-loading delivered PMO to 50%-70% of the cells of the two cell lines, as measured by fluorescence microscopy and flow cytometry. A PMO targeting ORF3 caused a significant increase in viral titer. A PMO targeting ORF2, a scrambled PMO control sequence, and an unrelated PMO antisense sequence did not alter viral titer. Various PMO sequences antisense to an upstream region of ORF1 were effective in reducing viral titer up to 80% in a dose-dependent and sequence-specific manner. The extent of viral titer reduction was proportional to the delivery of PMO to cells. These observations demonstrate that antisense PMO can disrupt caliciviral gene function in a nucleic acid sequence-specific manner and are potentially effective antiviral agents.
杯状病毒可感染动物和人类并引发疾病。它们是无包膜的正链RNA病毒，基因组约为7.5 kb，在三个开放阅读框（ORF）中编码病毒蛋白。靶向水疱性口炎病毒属杯状病毒三个ORF之一的反义寡聚物可显著抑制组织培养中的病毒复制。用分离出的水疱性口炎病毒SMSV - 13和猪杯状病毒Pan - 1感染猪肾细胞和非洲绿猴肾细胞。评估了与ORF1、ORF2和ORF3的AUG翻译起始位点区域序列互补的磷酰二胺吗啉代寡聚物（PMO）对病毒滴度的影响。通过荧光显微镜和流式细胞术检测，刮取加载法将PMO递送至两种细胞系50% - 70%的细胞中。靶向ORF3的PMO导致病毒滴度显著增加。靶向ORF2的PMO、随机对照PMO序列和无关的PMO反义序列均未改变病毒滴度。针对ORF1上游区域的各种反义PMO序列以剂量依赖性和序列特异性方式有效降低病毒滴度达80%。病毒滴度降低的程度与PMO向细胞的递送量成正比。这些观察结果表明，反义PMO可以以核酸序列特异性方式破坏杯状病毒基因功能，是潜在有效的抗病毒药物。
