|Kristalle Cruz, 2nd Year
Graduate Program: Chemistry
Advisor: Jeanne Hardy, PhD.
Education: University of the Philippines Los Banos, Chemistry, BS 2013
Chikungunya and Mayaro viruses are emerging global threats which have caused millions of cases in over 50 countries in the past 10 years alone. Chikungunya and Mayaro are both mosquito-transmitted alphaviruses which cause acute fever and severe arthralgia that may last up to several months. Chikungunya virus (CHIKV) is endemic to Africa and Asia but local transmission of the virus had been reported in Italy and France and most recently, last December 2013, in the Caribbean Islands, the first local transmission in the Western Hemisphere. The Centers for Disease Control and Prevention (CDC) has reported more than 230 imported cases of CHIKV infection in the continental United States as well as locally acquired cases in Florida. Spread of CHIKV can be attributed to the increasing number of infected tourists and the expanding geographic range of A. aegypti and A. albopictus mosquitos which are the primary vectors of CHIKV. Mayaro virus (MAYV) has the same symptoms as CHIKV and is endemic to the Amazon and Central America and due to its transmission method, the virus is a potential global pathogen next in line after Zika, Dengue and CHIKV.
Vaccines are already being developed for CHIKV but none for MAYV. Vaccine development just like the case for dengue proves to be difficult because of the existence of different serotypes for a certain virus. Even if vaccine have been developed, antivirals are still of great importance especially in treating patients who have already been infected. Viral-encoded proteases have been the targets of highly effective antivirals for the past three decades. These proteases catalyze the processing of either non-structural or structural proteins necessary for the replication or assembly of the virus. Examples of this kind of antivirals are those being used for the treatment of HIV and hepatitis C virus. In order to develop effective protease-directed antivirals to treat CHIKV or MAYV infection, it is essential that we have a thorough understanding of the structure and function of the proteases from these viruses. The overall goal of my PhD project is to provide the essential structural insights for CHIKV and MAYV proteases and use that insight to develop effective inhibitors for these two proteases.
The proteases of both CHIKV and MAYV can be found on the C-terminal side of the non-structural protein region 2 (nsP2pro) of the non-structural polyprotein (nsP) 1-4 complex. nsP2pro cleaves the nsP complex into mature nsPs that forms the viral replicase responsible for the replication of the viral RNA genome. Crystal structure of CHIKV nsp2pro are already available on Protein Data Bank but structural analysis of the active site of the protease in complex with its substrate is still not yet available. In addition, the structure of MAYV nsP2pro is still not available. Cleavage site of CHIKV nsP2pro are DAGA↓GIIET, TRAGC↓APSYR and DRAGG↓YIFSS while MAYV nsP2pro cleavage sites are FRAGA↓GVVET, QAAGC↓APAYA and GRAGA↓YIFSS which represent the junction between non-structural polyproteins. The fact that the protein cleaves after a series of hydrophobic residues is somewhat unusual and therefore a closer look on the protein-substrate complex will definitely give a better understanding on the factors at work on the polyprotein processing of CHIKV nsP2pro.
Together with X-ray crystallography, which provides the enzyme-inhibitor complex structure and therefore make the analysis of the interacting residues between the enzyme and substrate possible, NMR experiment is also a valuable tool that can provide insight on the structural changes associated with the protein’s function. Protein dynamics studied through NMR has a wide range of application such as protein-protein interaction, ligand binding and target recognition. We have also established a collaboration with two labs at the University of Alberta which will allow us to determine the sequence specificity for proteases of both CHIKV and MAYV. Together, these insights will allow us to conduct effective drug discovery efforts on these proteases in two critical emerging diseases.
Development of antivirals, especially for CHIKV and MAYV, which proved to be an emerging global threat is critical for human health. Research in the area of antiviral development is of great interest in the biotechnology industry since many viruses are susceptible to genetic change that can result in drug or vaccine resistance. Nowadays, biotechnology companies as well as academic researchers are developing new approaches to combat viral infections. Therefore, skills that will be learned in conducting this research, together with an industrial internship will definitely be helpful for me to gain professional experience in a biotech research field and expand career opportunities. In addition, an industrial internship would also widen my knowledge in various instrumentations and techniques that can be applied in my current research and future research work.