Infectious diseases are identified as the third major cause of death in the United States. Development of effective and economic therapy of infectious diseases requires thorough understanding of the biology of these infectious agents and innovative approaches to the problems. My laboratory has been involved in X-ray crystallographic analysis of critical proteins and design of novel drugs based on the protein structure.
Vesicular stomatitis virus (VSV) is a typical negative stranded RNA virus. One of the viral-encoded proteins, nucleocapsid (N), is required for both virus assembly and replication. The viral RNA polymerase only recognizes the viral nucleocapsid protein-RNA complex (RNP) as the template. We developed a co-expression system to produce recombinant N and phoshoprotein (P) in one construct. In our E.coli expression system, N forms a soluble complex of 10 subunits and a 90-base RNA molecule in association with the P protein. After testing several different approaches, we were able to produce single crystals of the N-RNA complex that diffracted X-rays to 3.0 Å resolution at synchrotron sources. The 3D structure of the N protein-RNA complex has been achieved and the position of the RNA molecular has been identified. There are extensive interactions between the neighboring protein subunits, which makes the nucleocapsid protein forming a ribbon like structure. The RNA molecule is snuggled into the cleft formed within the protein subunit and between the boundaries of the subunit. The structure helps to explain how the RNA polymerase recognizes the nucleocpaid protein covered RNA template.
The threat of global pandemic influenza is escalating due to the wide spread of highly pathogenic avian influenza virus in poultry populations through out continents and increasing cases of human infection. We devised a plan to develop novel antivirals against influenza virus by targeting virus fusion. Membrane fusion is a vital step in virus entry and our preliminary compounds can block this step with an EC50 of less than 1 nM in plaque reduction assays. Studies of influenza virus induced membrane fusion and sensitivity of the hemagglutinin to protease cleavage in the presence of the inhibitors further confirmed that the protein targeted by the inhibitors is hemagglutinin that is responsible for virus fusion. Improvements of inhibitor properties and studies in animal models are in progress. Overall, we have developed a strategy to find inhibitor compounds that can inhibit influenza virus replication by interfering with virus fusion. This strategy has been attempted previously by other groups without advancing the inhibitors further. Our preliminary results indicated new promises, and viable clinical candidates are likely to emerge from this study, which may be synergistic or have the effect of combination therapy with the existing neuraminidase inhibitor drugs.
Ming Luo (b. 1959), Professor, received his Bachelor's Degree in Science from the Department of Chemistry, Wuhan University, China in 1982. His graduate work was to determine the three-dimensional structure of spherical viruses by X-ray crystallography. He completed his Ph.D. studies at the Department of Biological Sciences, Purdue University in 1987. His work on the Mengo virus structure established a novel approach to structure determination of spherical viruses. Besides work, he enjoys golf.