Structural And Functional Study Of MERS Coronavirus Main Protease-N3Inhibitor Complex And Crystallographic Study Of SARS Coronavirus Helicase

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which spread in middle east since2012, has caused more than100infections, with a death rate around50%. MERS-CoV, together with Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Human Coronavirus NL63(HCoV-NL63), Human Coronavirus HKU1(HCoV-HKU1) has brought great threat to the health of human beings. However, there are still no vaccines or effective drugs for clinical use against them thusfar around the world.As a positive single-stranded RNA virus, MERS-CoV encodes around16non-structural proteins (nsps) which mediate viral replication-transcriptions process. Among them, the nsp5protein (also named as main protease) is pivotal and indispensible for virus surviral since it extensiely participates in the processing of virus-encoded polyproteins (pp1a, pp1ab) into the16nsps. The absence of cellular homologue of nsp5protein in human beings further makes it an ideal target for anti-viral drug design.In this paper, we firstly solved the three-dimensional crystal structure of MERS-CoV nsp5protein complexed with a michael inhibitor named N3at2.27-A resolution by molecular replacement method. Based on detailed analysis on their interaction profile, we proved that MERS-CoV nsp5protein is functionally active as a dimer, with the Cys-His dyad as its catalytic center. We furtther quantitatively evaluate the inhibition activity of N3by using in vitro fluorescence assay. These work provides a good structural basis for understanding the inhibition mechanism of N3and proved that N3is an effective inhibitor against MERS-CoV nsp5. Then, by anylasing the high conservitity and mild differences among the nsp5-N3complex structures from MERS-CoV, SARS-CoV, HCoV-HKU1and IBV, we hope our work can assist the subsequent inhibitor optimization work.The other work in this paper foucs on the crystallographic study of SARS-CoV nsp13protein. Charactered of Helicase and NTPase activity, the nsp13protein is pivotal during the transcription-replication process and thus a good antiviral drug target. In this paper, we successfully collected the diffraction data of SARS-CoV nsp13protein at2.9A, after testing a number of clones and trying various purification methods. This work lay a good foundation for the further structure solement of SARS-CoV nsp13.