MOLECULAR CHARACTERIZATION OF AURA VIRUS CAPSID PROTEIN

Abstract

Alphaviruses, the members of Togaviridae family, are enveloped, arthropod borne, singlestranded RNA viruses. The alphavirus genus includes Aura virus, Sindbis virus, Chikungunya virus (CHIKV), Semliki Forest virus and Venezuelan equine encephalitis virus (VEEV). The recent outbreaks of Chikungunya epidemic pose a threat to human health and hence, the development of an effective antiviral drug to combat alphavirus infections has become a high priority. We have structurally and biochemically investigated the properties of capsid protein (CP) from Aura and Chikungunya virus for the development of novel strategies to identify inhibitors against alphavirus infection. The CP is a multifunctional protein and performs several functions including structural polyprotein processing, genomic RNA encapsidation, nucleocapsid formation and virus budding for the alphavirus propagation. The CP interacts with the E2 glycoprotein cytoplasmic tail which leads to virus budding. In order to analyze the interaction strategy, the CP from Aura virus (AVCP) has been cloned, expressed and purified to homogeneity. The crystal structure of the AVCP is determined. Also, the homology models of the cytoplasmic tails of glycoproteins were generated. The crystal structure of AVCP and the molecular models of E1 and E2 cytoplasmic tails are fitted into the cryo-electron microscopic (cryo-EM) density map of VEEV. The fitting studies reveal the presence of the helix-loop-helix motif in the cytoplasmic tail and the loop region of the helix-loop-helix motif is predicted to bind the hydrophobic pocket of the CP. The disulfide bridge, ionic and hydrophobic interactions between the two opposite helices stabilize the motif. The conserved Pro405 residue in the loop region of the helix-loop-helix motif of E2 tail binds and makes direct molecular interactions in the hydrophobic pocket. The crystal structure of AVCP in complex with dioxane has been determined and molecular contacts of the ligand with the protein have been analyzed. The dioxane molecule binds to the CP hydrophobic pocket to which E2 tail binds and is found to be present exactly at the same position where Pro405 is predicted to bind to the pocket. Molecular docking studies for various dioxane based compounds were performed and docked structures were analyzed for studying the binding mode of potential inhibitory dioxane derivatives. Additionally, another crystal structure of AVCP in complex with piperazine also shows the similar binding pattern of the ligand to the hydrophobic pocket. Interestingly, structural analysis of AVCP in complex with picolinic acid identifies a ligand binding site on the surface of CP, which is separate from the hydrophobic pocket. ii The alphavirus CP possessing serine protease activity after cleaving itself off from the rest of the polyprotein by cis-proteolysis becomes enzymatically inactive for the rest of the virus life cycle. Structural analysis of the trans-proteolytic active form of AVCP has been performed. The C-terminal truncated AVCP (last two residues including Trp267 deleted) has been cloned, expressed and purified. The crystal structure of the active AVCP has been determined and compared with various forms (inactive and substrate bound inactive) of the CP from different alphaviruses. The comparative structural analysis shows the differences in the C-terminal residues which are missing in the active form, highly flexible in substrate bound form and remains intact in Trp bound post-cleavage inactive form. The active site, oxyanion hole and the specificity pockets show marked variations in different enzymatic forms. The hydrophobic pocket of the different forms of the protein also shows conformational switching. Additionally, the trans-protease activity has been performed using the fluorogenic peptide substrate and the kinetic parameters were determined. The FRET (fluorescence resonance energy transfer) based trans-protease activity assay has also been optimized for CHIKV capsid protease. The assay can be developed further for high throughput inhibitor screening against alphavirus infection. Thus, the structural details and the in vitro FRET based protease assay provides the worthful guidelines for the structure based designing and testing of the protease inhibitors of the CP from alphavirus which will lead to the inhibition of the first step of structural polyprotein processing. The thesis consists of five chapters which include the structural and biochemical characterization of the CP from Aura virus, a member of the genus alphavirus. The molecular characterization of the CP involves the 3D structural analysis, in silico modeling, cryo-EM fitting and biochemical studies. The trans-proteolytic enzyme activity of alphavirus CP has been determined and characterized from Aura virus and CHIKV. Chapter 1 reviews the literature. It describes the alphavirus life cycle, its transmission and the structure of the overall virion. The alphavirus genome organization and the polyprotein processing including both structural and non-structural polyprotein have been described in detail. The nucleocapsid assembly and the virus budding process have also been depicted. The chapter also describes the overall structure of CP and its involvement in the process of virus budding, genomic RNA encapsidation and nucleocapsid assembly. The CP has been studied as a proteolytic enzyme and different inhibition strategies for alphavirus capsid protease are discussed in the chapter. iii Chapter 2 describes the structural analysis of the AVCP and insight into the capsidglycoprotein interaction. The recombinant CP has been produced in E. coli and purified to homogeneity by affinity and size exclusion chromatography methods. The crystallization of the purified protein was performed and the crystal structure was determined. The crystal structure reveals the presence of chymotrypsin-like structure having Greek key motif. Subsequently, the homology models of E1 and E2 glycoproteins were made. The crystal structure of AVCP and the homology models of E1 and E2 glycoproteins cytoplasmic tails were fitted into the cryo-EM structure of VEEV. The fitted structure was analyzed for the molecular interactions of the capsid and glycoproteins. The structure shows the presence of helix-loop-helix motif of which the loop region binds to the hydrophobic pocket of the CP. The Cys-Cys disulfide bridge present in between the two helices has been proposed to stabilize this helix-loop-helix motif. Likewise, the Tyr401 ionic and hydrophobic interactions with the opposite helix also seem to be stabilizing the motif. The loop residue Pro405 binds directly to the hydrophobic pocket and might be involved in the capsid-glycoprotein interaction during virus budding process. Chapter 3 describes the structural analysis of AVCP in complex with potential CP inhibitory molecules. The crystal structure of AVCP in complex with dioxane, piperazine and picolinic acid was determined and analyzed for the ligand binding studies. For preparation of the ligand bound crystals, the native protein crystals were soaked into the reservoir buffer containing various ligands. The structural analysis of complexes reveals the presence of dioxane in the hydrophobic pocket of the CP. This dioxane-bound structure was fitted into the cryo-EM structure of VEEV along with the homology models of E1 and E2 cytoplasmic tails as described in chapter 2. The dioxane molecule was found to be present exactly at the same position where Pro405 of E2 tail binds to the pocket. Thus, it can be concluded that the dioxane molecule mimics the pyrollidine ring of the proline and might compete with E2 tail for the binding to the hydrophobic pocket. The piperazine also binds at the same position in the hydrophobic pocket where the dioxane binds and shows similar interactions with the pocket residues. Interestingly, the picolinic acid binds on the surface of the capsid surrounded by the neighboring symmetry related molecules. Bound picolinic acid also shows interactions with the symmetry CP molecules as well. Other potential antiviral compounds based on dioxane molecule have been designed which contain two dioxane moieties. The docking of these compounds shows that the two ring structures bind into two separate grooves of the CP hydrophobic pocket. iv Chapter 4 demonstrates the structural insight into the active form of the capsid protease from Aura virus and the comparative studies with the different forms of the enzyme. As the Trp267 binds to the specificity pocket after the cis-autoproteolytic cleavage and inhibits transprotease activity by blocking the binding of substrate to the specificity pocket. Therefore, the last two residues including Trp267 were deleted from the capsid construct to activate the capsid protease by unblocking the active site. The C-terminal truncated construct was cloned into the expression vector, expressed in E. coli and purified using the affinity and gel filtration chromatography. The crystals of the active truncated form were produced and the crystal structure was determined. The structure was compared with the other enzymatic forms revealing the differences in the C-terminal loop after His261, the specificity pockets and the active site residues. The oxyanion hole consists of a conserved water molecule which is absent in the post-cleavage form where water molecule is replaced by the scissile bond residues. The hydrophobic pocket also shows the differences in between the active and inactive forms stating the conformational switching in the pocket from binding with the other capsid monomers to interaction with the cytoplasmic tail of E2 glycoprotein. This indicates that the conformational changes in the hydrophobic pocket can lead to switching of the CP function from nucleocapsid formation to virus budding. The different specificity pockets show conformational variations in the active and inactive state. Thus, the chapter describes the 3D structure of active CP that has been previously proposed to be natively unfolded protein. Chapter 5 reports the trans-proteolytic activity of alphavirus capsid protease. The AVCP was characterized for its trans-proteolytic activity using the fluorogenic peptide substrate having EDANS (fluorophore) and DABCYL (quencher) at the ends. The peptide sequence that separates these tags includes the sequence derived from the scissile bond of the capsid protease. After the cleavage of the peptide in presence of active CP, the fluorescence intensity is expected to increase due to the disruption of FRET (Fluorescence resonance energy transfer). Kinetic parameters using fluorogenic peptide substrates for the Aura virus capsid protease were estimated; and a KM value was found to be 2.63 ± 0.62 μM, and a Kcat/KM value was 4.97 X 104 M-1 min-1. Additionally, for the activity analysis of the CHIKV CP (CVCP), cloning, expression and purification of CVCP to homogeneity was performed. The KM and Kcat/KM values of purified CVCP were determined using fluorogenic peptide substrate and were found to be 1.27 ± 0.34 μM and 5.5 X 104 M-1 min-1 respectively. The CVCP was characterized for the optimization of pH and NaCl concentration. The v effect of glycerol on the enzyme activity has also been determined. In conclusion, this chapter explains the development of high throughput screening method for proteolytic activity assay of alphavirus capsid protease that can be used for screening of alphavirus specific protease inhibitors