Performing Department
Biochemistry & Molecular Biology
Non Technical Summary
The main goal of this research is to provide detailed molecular basis by which a number of important viral and host proteins function, and to provide important clues on how to design novel anti-viral regents key to the immunity of livestock animals. The outcome will be helpful for development of treatments and preventions against a number of infectious diseases that target livestock animals and are economically devastating.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The main goal of this research is to provide detailed molecular basis by which a number of important viral and host proteins function, and to provide important clues on how to design novel anti-viral regents key to the immunity of livestock animals. The outcome will be helpful for development of treatments and preventions against a number of infectious diseases that target livestock animals and are economically devastating. Infectious animal diseases are destructing the health of livestock and agricultural economy. Porcine reproductive and respiratory syndrome (PRRS) is a major swine disease that costs swine producers in the United States approximately $700 million annually(Neumann, Kliebenstein et al., 2005). PRRS virus (PRRSV)-infected pigs are susceptible to pneumonia and reproductive losses (Cho & Dee, 2006, Lowe, Husmann et al., 2005). Pseudorabies virus (PrV) causes fatal swine disease that is characterized by neurological symptoms and death in young piglets, and respiratory and reproductive disorders in older pigs. The acute phase of the infection with a high abortion rate and neonatal mortality causes significant economic losses(Hahn, Fadl-Alla et al., 2010, Kluge, Beran et al., 1999). Foot-and-mouth disease virus (FMDV) causes a highly contagious disease in cloven-hoofed animals, which is highly infectious and a major plague of animal farming. Although immunization of animals with vaccines that are conventionally attenuated viruses helps provide some protections, there are still disadvantages such as the risk of reversion to virulence (Vilnis, Sussman et al., 1998, Zuckermann, 2000) and the risk of interference with efficient antigen presentation (Zuckermann, 2000). Attempts to enhance the immunogenicity of vaccines have been actively sought, among which administration of immunomodulatory molecules such as cytokine IL-18 has been of considerable interests. IL-18 is a potent interferon gamma (IFN-g) inducing factor, which plays a central regulatory role in host immune response to viral and microbial infections(Vivier, Tomasello et al., 2008). It was also shown that IL-18 may be useful as a therapeutic agent for the enhancement of immune responses and as a vaccine adjuvant, especially in neonatal piglets(Muneta, Goji et al., 2002). In fact, treatment of animals with exogenous IL-18 was reported to confer protections against viral infections, such as PRRSV(Shen, Jin et al., 2007), PrV(Kim, Kim et al.) and FMDV(Shi, Wang et al., 2007). It was also shown that IL-18 may be useful as a therapeutic agent for the enhancement of immune responses and as a vaccine adjuvant, especially in neonatal piglets(Muneta et al., 2002). Poxviruses including swinepox virus (Afonso, Tulman et al., 2002) adopt unique host immune evasion mechanisms, one of which is inhibiting IL-18 singling by viral protein IL-18BPs. In addition, two intracellular inhibitors of type I IFN antiviral activities, K1 and C7 from vaccinia virus (VACV), were discovered (Meng, Jiang et al., 2009, Meng, Schoggins et al., 2012). Further more, poxviruses use a unique yet unclear mechanism for membrane biogenesis, including key proteins A6 and H7. Knocking out A6 or H7 will be lethal for viral replication in mammalian cells. Revealing the mechanism by which these host range proteins target and inhibit animal host immunity proteins will provide key information on further development of anti-viral drugs. Finally, an essential host molecular chaperone, Hsp90, is nearly universally required for viral replication (reviewed in (Geller, Taguwa et al., 2012)) and development of cancer. Therefore, inhibiting Hsp90 by novel inhibitors will present an important way to fight viral infection and cancer.
Project Methods
Recombinant protein expression and purification. We have extensive experience in large-scale protein expression and purification, attested by a number of crystal structures determined in our previous and current lab. The detailed procedures have been published (Deng, Ernst et al., 2005, Deng, Lewis et al., 2008, Deng, Schnaufer et al., 2004).Crystallization procedures Step 1. Proteins will be screened in a small subset of 48 to 96 conditions with 200 to 500 nanoliter drops to investigate their overall solubility.Step 2. Trials will be set up using commercially available kits (Emerald Cryo I&II, Wizard I&II, Salt Rx, Pact Screen, Index Screen, Crystal Screen I&II, Crystal Screen Cryo and Peg Ion Screen; a total of about 800 conditions), in some cases supplemented by in-house made random screens.Step 3. Based on the results from the two previous steps, follow-up matrices will be designed.Steps 4 and more. Successive generations of follow-up matrices will be set up to further optimize the size and diffraction quality of protein crystals.Structure determinations We have ample experience with flash freezing of crystals for cryo-protection as evidenced by many structures that we reported.Model building and structure analysis The programs like ARP/wARP (Perrakis, Morris et al., 1999) and RESOLVE (Terwilliger, 2003) will be used for automatic tracing. Intermediate models will be improved using interactive graphics programs such as XTALVIEW (McRee, 1999), O (Jones, Zou et al., 1991), and COOT (Emsley & Cowtan, 2004).Analysis of the structures will be carried out by programs in the lab or on the web such as DALI (Holm & Sander, 1993) for searching related protein structures, GRASP (Nicholls, Sharp et al., 1991) for analyzing electrostatic and other surface characteristics, ACCESS for calculating buried solvent accessible surfaces (Leslie, 1992), LIGPLOT (Wallace, Laskowski et al., 1995) for analyzing ligand-protein interactions.Binding affinity assays BIACORE binding assay To facilitate BIAcore analysis, we have developed a facile procedure for coupling protein to sensor chips (Meng, Leman et al., 2007, Xiang & Moss, 2001)Virtual Docking and IL-18 BioassyWe have described the experimental procedures in details in the earlier publications (Krumm, Meng et al., 2017, Meng et al., 2007, Xiang & Moss, 1999).Structure based mutagenesis: Based on the expected structures, amino acids at the protein interface or drug binding site whose side chains are predicted to contribute to the binding specificity and affinity will be mutated to Ala.