Bar-Magen Tamara, Spencer Eugenio, Patton John T
Laboratorio de Virologia, Facultad de Quimica y Biologia, Universidad de Santiago, Alameda 3363, casilla 33 correo 40, Santiago, Chile.
Virology. 2007 Dec 20;369(2):389-99. doi: 10.1016/j.virol.2007.07.029. Epub 2007 Sep 6.
Interactions between NSP5 and NSP2 drive the formation of viroplasms, sites of genome replication and packaging in rotavirus-infected cells. The serine-threonine-rich NSP5 transitions between hypo- and hyper-phosphorylated isomers during the replication cycle. In this study, we determined that purified recombinant NSP5 has a Mg2+-dependent ATP-specific triphosphatase activity that generates free ADP and Pi (Vmax of 19.33 fmol of product/min/pmol of enzyme). The ATPase activity was correlated with low levels of NSP5 phosphorylation, suggestive of a possible link between ATP hydrolysis and an NSP5 autokinase activity. Mutagenesis showed that the critical residue (Ser67) needed for NSP5 hyperphosphorylation by cellular casein kinase-like enzymes has no role in the ATPase or autokinase activities of NSP5. Through its NDP kinase activity, the NSP2 octamer may support NSP5 phosphorylation by creating a constant source of ATP molecules for the autokinase activity of NSP5 and for cellular kinases associated with NSP5.
NSP5与NSP2之间的相互作用驱动了病毒工厂的形成，病毒工厂是轮状病毒感染细胞中基因组复制和包装的场所。富含丝氨酸-苏氨酸的NSP5在复制周期中在低磷酸化和高磷酸化异构体之间转变。在本研究中，我们确定纯化的重组NSP5具有Mg2+依赖性的ATP特异性三磷酸酶活性，可产生游离的ADP和Pi（酶的Vmax为19.33 fmol产物/分钟/pmol酶）。ATP酶活性与低水平的NSP5磷酸化相关，提示ATP水解与NSP5自激酶活性之间可能存在联系。诱变表明，细胞酪蛋白激酶样酶使NSP5发生高磷酸化所需的关键残基（Ser67）在NSP5的ATP酶或自激酶活性中不起作用。通过其NDP激酶活性，NSP2八聚体可以通过为NSP5的自激酶活性以及与NSP5相关的细胞激酶创造恒定的ATP分子来源来支持NSP5的磷酸化。
