Lai V C, Zhong W, Skelton A, Ingravallo P, Vassilev V, Donis R O, Hong Z, Lau J Y
Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey 07033-0539, USA.
J Virol. 2000 Jul;74(14):6339-47. doi: 10.1128/jvi.74.14.6339-6347.2000.
Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, P(H/B) (wild-type HCV NS3 protease) and P(H/B(S139A)) (mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the P(H/B) clone yielded viable viruses, whereas the mutant clone, P(H/B(S139A)), failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV-Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV-Npro was highly defective in viral replication and growth, a finding consistent with the observed stability of the chimeric virus after serial passages.
瘟病毒独有的是，牛病毒性腹泻病毒（BVDV）基因组编码的N端蛋白是一种半胱氨酸蛋白酶（Npro），负责自我切割，从而释放核心蛋白（C）的N端。这种独特的蛋白酶对于病毒复制并非必需，其编码区可被直接框内融合到核心蛋白的泛素基因取代。为了开发一种能够评估抗丙型肝炎病毒（HCV）NS3蛋白酶抑制剂的抗病毒检测方法，构建了一种嵌合BVDV，其中Npro的编码区被与NS4A辅因子相连的HCV NS3蛋白酶结构域的编码区取代。这种与辅因子相连的HCV蛋白酶结构域通过HCV NS5A-NS5B连接位点与BVDV的核心蛋白框内相连，并模拟了Npro在释放BVDV核心以进行衣壳组装方面的蛋白水解功能。构建了一个带有无活性HCV NS3蛋白酶的类似嵌合构建体作为对照。然后将来自两个嵌合克隆P（H/B）（野生型HCV NS3蛋白酶）和P（H/B（S139A））（突变型HCV NS3蛋白酶）的基因组RNA转录本转染到牛细胞（MDBK）中。只有来自P（H/B）克隆的RNA转录本产生了有活力的病毒，而突变克隆P（H/B（S139A））没有产生任何感染迹象，这表明未加工的融合蛋白使BVDV核心蛋白在衣壳组装方面存在缺陷。与野生型BVDV（NADL）一样，嵌合病毒具有细胞病变效应，并在细胞单层上形成噬斑。序列和生化分析证实了嵌合病毒的身份，并进一步揭示了由于生长适应而产生的变异病毒。生长分析显示野生型和嵌合BVDV之间具有可比的复制动力学。最后，为了评估嵌合病毒的遗传稳定性，制备了一种缺失Npro的BVDV（BVDV-Npro，其中整个Npro编码区被删除）。尽管具有细胞病变效应，但BVDV-Npro在病毒复制和生长方面存在高度缺陷，这一发现与连续传代后观察到的嵌合病毒的稳定性一致。
