Lackner T, Müller A, Pankraz A, Becher P, Thiel H-J, Gorbalenya A E, Tautz N
Institut für Virologie (FB Veterinärmedizin), Justus-Liebig-Universität Giessen, Frankfurter Strasse 107, 35392 Giessen, Germany.
J Virol. 2004 Oct;78(19):10765-75. doi: 10.1128/JVI.78.19.10765-10775.2004.
Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp) strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection. At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the pathogenicity of BVDV.
瘟病毒属于黄病毒科，其基因组是一条正链单链RNA，编码一种大的多聚蛋白，该多聚蛋白进一步加工成成熟蛋白。瘟病毒牛病毒性腹泻病毒（BVDV）的非细胞病变（noncp）毒株可建立持续性感染。在持续感染的动物中，noncp BVDV偶尔会在病毒非结构蛋白2（NS2）中发生突变，从而产生细胞病变（cp）BVDV变体，并最终导致致命疾病的发作。cp BVDV感染的一个分子标志物是复制性NS3蛋白酶/解旋酶的高水平表达，它与NS2一起源自NS2-3。在这里，我们提供了证据，证明NS2中一种新鉴定的半胱氨酸蛋白酶可对NS2-3进行自我加工，该蛋白酶与丙型肝炎病毒和GB病毒的NS2-3自蛋白酶有远缘关系。这种自蛋白酶在BVDV感染中的重要作用得以确立，这意味着NS3在瘟病毒RNA复制中具有不可或缺的功能，而其NS2-3前体无法提供该功能。因此，与当前的范式相反，我们在感染早期检测到noncp BVDV中的NS2-3几乎完全裂解。在感染后6至9小时，noncp BVDV的NS2-3自我加工减少到几乎检测不到的水平，但cp BVDV仅适度下降。病毒RNA合成速率与noncp和cp BVDV感染细胞中不同的NS3水平严格相关，这表明NS2自蛋白酶参与RNA复制控制。NS2-3自我加工的生物型特异性调节表明NS2自蛋白酶在BVDV致病性中起关键作用。
