Pfister T, Jones K W, Wimmer E
Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794-5222, USA.
J Virol. 2000 Jan;74(1):334-43. doi: 10.1128/jvi.74.1.334-343.2000.
Protein 2C(ATPase) of picornaviruses is involved in the rearrangement of host cell organelles, viral RNA replication, and encapsidation. However, the biochemical and molecular mechanisms by which 2C(ATPase) engages in these processes are not known. To characterize functional domains of 2C(ATPase), we have focused on a cysteine-rich motif near the carboxy terminus of poliovirus 2C(ATPase). This region, which is well conserved among enteroviruses and rhinoviruses displaying an amino acid arrangement resembling zinc finger motifs, was studied by genetic and biochemical analyses. A mutation that replaced the first cysteine residue of the motif with a serine was lethal. A mutant virus which lacked the second of four potential coordination sites for zinc was temperature sensitive. At the restrictive temperature, RNA replication was inhibited whereas translation and polyprotein processing, assayed in vitro and in vivo, appeared to be normal. An intragenomic second-site revertant which reinserted the missing coordination site for zinc and recovered RNA replication at the restrictive temperature was isolated. The cysteine-rich motif was sufficient to bind zinc in vitro, as assessed in the presence of 4-(2-pyridylazo)resorcinol by a colorimetric assay. Zinc binding, however, was not required for hydrolysis of ATP. 2C(ATPase) as well as its precursors 2BC and P2 were found to exist in a reduced form in poliovirus-infected cells.
小核糖核酸病毒的蛋白质2C（ATP酶）参与宿主细胞器重排、病毒RNA复制和衣壳化过程。然而，2C（ATP酶）参与这些过程的生化和分子机制尚不清楚。为了表征2C（ATP酶）的功能结构域，我们聚焦于脊髓灰质炎病毒2C（ATP酶）羧基末端附近一个富含半胱氨酸的基序。通过遗传和生化分析对这一区域进行了研究，该区域在肠道病毒和鼻病毒中高度保守，其氨基酸排列类似于锌指基序。将该基序的第一个半胱氨酸残基替换为丝氨酸的突变是致死性的。一个缺少锌的四个潜在配位位点中的第二个位点的突变病毒对温度敏感。在限制温度下，RNA复制受到抑制，而在体外和体内检测的翻译和多聚蛋白加工似乎正常。分离出一个基因组内的第二位点回复突变体，其重新插入了缺失的锌配位位点，并在限制温度下恢复了RNA复制。通过比色法在4-（2-吡啶偶氮）间苯二酚存在下评估，富含半胱氨酸的基序在体外足以结合锌。然而，ATP水解并不需要锌结合。在脊髓灰质炎病毒感染的细胞中，2C（ATP酶）及其前体2BC和P2以还原形式存在。
