1. Figure 5: Expression and solubility tests for constructs of CoVs. Coronaviruses are complex, positive-sense RNA viruses that cause mild to severe respiratory tract infections. These viruses infect both humans and animals and have a complex infectious cycle including multiple RNA processing steps. We are studying a protein family that is distributed across several types of CoVs and has diverse biochemical functions: the macrodomains. Macrodomains are a key poly(ADP-ribose) (PAR) binding module and depending on the protein sequence, may also bind smaller molecules and catalyze enzymatic reactions. We are investigating predicted new, noncanonical macrodomains found in CoVs with little sequence similarity to known proteins. In the SARS-CoV, these proteins were found to bind G- quadruplex RNA. The macrodomain regions were predicted using bioinformatic methods such as profile/profile alignment and secondary structure prediction. Next using subcloning techniques, such as restriction enzyme cloning, we isolated specific DNA sequences. The specific DNA constructs were transformed into expression cells and expression of the desired proteins was achieved. The proteins expressed will be purified using column chromatography. Binding assays will be carried out to understand how macrodomains recognize nucleic acids. By examining the tertiary structure and biochemical functions of these coronavirus protein domains, we hope to further understand the relationships between the domains in the macrodomain protein family, as well as general principles of PAR and nucleic acid molecular recognition, protein sequence-structure-function relationships, and biochemical functions of the macrodomain regions in CoVs. Methods Abstract Conclusion Discussion Future Work Protein Purification Cloning Protein Expression References Viral nonstructural protein sequences were analyzed for the presence of macrodomains and 13 macrodomains were cloned into expression vectors. 8 macrodomains expressed in E. coli to date, with 2 expressing in the soluble fraction. By investigating the predicted macrodomain-containing regions of CoV nonstructural proteins, we were able to successfully produce multiple proteins in preparation for structural and biochemical studies. The proteins will be purified further using nickel affinity, ion exchange, and/or gel filtration chromatography to remove any remaining impurities. Circular dichroism (CD) spectroscopy will be used to analyze the secondary structure of our proteins, and nuclear magnetic resonance (NMR) will be used to determine the high-resolution structure of our proteins. Bioinformatic Analysis of Protein Sequences 1 st Nickel Column Dialysis 2 nd Nickel Column Heparin Sepharose Column Gel Filtration Column Ion Exchange Column Figure 7: Purification procedures for BtCoV HKU4 B-L. Option 3 was selected for consistent protein buffer conditions. Figure 1: Schematic drawings of various proteins as predicted by bioinformatics methods. Canonical macrodomains are indicated in blue. Purple and gold regions represent noncanonical, RNA-binding macrodomains. The canonical macrodomains MD1 and MD2 of PARP-9 and the noncanonical macrodomains MD2 of HCoV-NL63 and MERS-CoV were successfully cloned, as well as 9 different constructs from the macrodomain region of bat CoV HKU4. Constructs of NL63 MD2 and MERS MD2 successfully expressed in BL21 cells. NL63 MD2 appeared at 36 kDa instead of the expected 18 kDa, suggesting that this protein formed a dimer. HKU4 constructs M, B-S, and B-L expressed well in T7 Lys S cells. While both B-S and B-L were soluble in the lysate (L), M was insoluble in the pellet (P). HKU4 B-L was purified through the first nickel column. After running an SDS-PAGE gel, the protein strongly expressed while remaining fairly pure. Figure 6: SDS-PAGE gel of fractions from gel filteration column purification of B-L. Figure 8: 1 st nickel affinity column purification trace of HKU4 B-L Protein eluting from the Ni affinity column during imidazole gradient Figures 2-4: Agarose gels of various PCR-amplified constructs Figure 2 Figure 3 Figure 4 Bioinformatics Restriction Enzyme Cloning Transformation into E. coli 5α Cells Transformation into E. coli 5α Cells Transformation into BL21 & T7 LysS Expression Cells Plasmid DNA Miniprep Inoculation of 5- mL Culture Expression & Solubility Tests Large Culture Growth Protein Purification by Column Chromatography 1.Johnson MA, Chatterjee A, Neuman BW, Wüthrich K. 2010. SARS coronavirus unique domain: three-domain molecular architecture in solution and RNA binding. J. Mol. Biol. 400:724–742. 2.Peiris, J. S., Guan, Y. & Yuen, K. Y. (2004). Severe acute respiratory syndrome. Nat. Med. 10, S88–S97. 3.Poon, L. L., Guan, Y., Nicholls, J. M., Yuen, K. Y. & Peiris, J. S. (2004). The aetiology, origins, and diagnosis of severe acute respiratory syndrome. Lancet Infect. Dis.4, 663– 671. Bioinformatic Tools Used to Predict Macrodomain Regions FFAS: Alignment comparison with similar solved structures Jpred: Secondary structure predictions Expasy Translate: Translates DNA sequence into amino acids Expasy Protparam: Analysis of protein sequence properties 1. Results 2.3. Figure 10: Binding Assay of HKU4 showing that HKU4 has some interaction with DNA, and it binds to G- quadraplex RNA Cloning, Expression, and Characterization of Recombinant Coronavirus Macrodomains Figure 9: Gel Filteration column purification trace of HKU4 B-L Figure 11: 1D NMR of HKU4 showing that HKU4 is a folded protein.