Mendez E, Ruggli N, Collett M S, Rice C M
Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
J Virol. 1998 Jun;72(6):4737-45. doi: 10.1128/JVI.72.6.4737-4745.1998.
Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10(5) PFU/microg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.
牛病毒性腹泻病毒（BVDV）NADL毒株最初是从一头患有致命黏膜病的动物体内分离出来的。该毒株在细胞培养中具有细胞病变效应，并产生两种含NS3的蛋白质形式：未切割的NS2-3和成熟的NS3。对于BVDV NADL来说，产生NS3是细胞病变型BVDV毒株的一个特征，据信这是由于在NS2编码区发生了一个270个核苷酸的细胞mRNA序列（称为cIns）的框内插入所致。在本研究中，我们在一个低拷贝数质粒载体中构建了BVDV NADL的稳定全长cDNA拷贝。通过转染MDBK细胞检测发现，从该模板转录的无帽RNA具有高度传染性（>10⁵ PFU/μg）。回收的病毒在蚀斑形态、生长特性、多聚蛋白加工和细胞致病性方面与BVDV NADL亲本相似。删除cIns消除了NS2/NS3位点的加工，并产生了一种对MDBK细胞不再具有细胞病变效应的病毒。这种缺失并不影响感染性病毒产生或病毒蛋白产生的效率，但它降低了病毒特异性RNA合成和积累的水平。因此，cIns不仅调节NS3的产生，而且相对于缺乏该插入片段的同基因非细胞病变衍生物，还上调了RNA复制。这些结果增加了BVDV NADL增强的RNA复制与病毒诱导的细胞致病性之间存在联系的可能性。
