VIRAL PROTEINASES OF HERPES SIMPLEX TYPE 1 AND COXSACKIEVIRUS, RABBIT PGM AND

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。我们正在研究兔子和人类的PGM。这种酶是我们通过核磁共振研究的最大的蛋白质，它对细胞的能量产生至关重要。我们也对病毒蛋白酶感兴趣，例如人类疱疹病毒;它导致各种严重疾病。本论文对疱疹病毒二聚体丝氨酸蛋白酶（具有催化三联体Ser、His、His而不是Ser、His、Asp）的研究具有重要意义。本工作的目的是更好地了解这些新的蛋白酶的催化机制，这项工作可能导致新的抑制剂的开发。我们特别感兴趣的是组氨酸在催化位点的作用。我们已经克隆并过表达了来自单纯疱疹病毒1型（HSV-1）和卡波西肉瘤相关病毒（KSHV或疱疹病毒8型，疱疹病毒蛋白酶含有18个脯氨酸和几个半胱氨酸，因此需要重折叠技术以使蛋白质从包涵体中复性。其他小组发表的工作表明，这些蛋白质的产量很低，例如2至5毫克/升，这对我们的需求来说太低了。另外，这些蛋白酶具有自催化活性，因此我们必须开发改进的复性和蛋白纯化策略，以尽可能快地回收尽可能多的活性酶。我已经从小核糖核酸病毒科的柯萨奇病毒B3中纯化了3C半胱氨酸蛋白酶。我也克隆过表达2A蛋白酶。柯萨奇病毒与许多严重的人类疾病有关，如心脏病（心肌炎）、呼吸道感染和流感样疾病。2A和3C蛋白酶是单体，分别为16 kDa和20 kDa。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We are looking at rabbit and human PGM. This enzyme is the largest protein we are studying by NMR, and it is vital for energy production in cells.We are also interested in viral proteinases, for example human herpesviruses; which cause a wide variety of serious diseases. We are interested in studying the herpesvirus novel dimeric serine proteinases (which possess the catalytic triad, Ser, His, His instead of Ser, His, Asp) that are essential for viral matuartion. The goal of this work is to gain a better understanding of the catalytic mechanism of these novel proteinase, and this work could lead to the development of new inhibitors. We are particularly interested in the role of the histidines at the catalytic site. We have cloned and overexpressed serine proteinases from herpes simplex type-1 virus (HSV-1) and Kaposi sarcoma associated virus (KSHV or herpes virrus type 8 ie., HSV-8).The herpesvirus proteinases contain 18 prolines and several cysteines and therefo re require refolding techniques to renature the protein from inclusion bodies. Published work by other groups show that the yields of these proteins are low e.g. 2 to 5mg/liter, this is too low for our needs. So I have had to improve the expression and refolding/purification strategies Also, these proteinases possess autocatalytic activity, therefore it was essential for us to develop improved refolding and protein purification strategies, that recover as much active enzyme, as quickly as possible.I have purifed 3C cysteine proteinase from Coxsackievirus B3 of the Picornavirus family. I have also cloned, overexpressed 2A proteinase too. Coxsackieviruses have been implicated in many serious human diseases such as heart disease (myocarditis), respiratory infections and flu-like illnesses. The 2A and 3C proteinases are monomeric and 16 kDa and 20 kDa, respectively.