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

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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.