STRUCTURAL AND FUNCTIONAL STUDIES OF CHIKUNGUNYA VIRUS CAPSID PROTEIN

Abstract

Alphaviruses are a group of single-stranded positive-sense RNA viruses that are transmitted by mosquitoes and infect vertebrates. Alphaviruses belong to the Togaviridae family and include many medically relevant viruses like Chikungunya virus (CHIKV), Ross River virus (RRV), Western Equine Encephalitis virus (WEEV), Venezuelan Equine Encephalitis virus (VEEV) etc. Alphavirus infections cause fever, rash arthritis and encephalitis; and continues to be a potential threat to humans. The alphavirus virion is approximately 70 nm in diameter and has host cell derived lipid membrane which is embedded with 80 spikes in T=4 icosahedral symmetry. Each spike is composed of trimers of E1 and E2 glycoprotein heterodimers. The positive strand RNA genome (~11.7 kb) is surrounded by 240 copies of the capsid protein that forms a nucleocapsid core. The genomic RNA of virion has a 5′ methylated cap and a 3′ polyadenylated tail similar to host mRNA and on entry into the cytoplasm of host cell, viral genomic RNA is directly translated to viral proteins. The interaction of capsid protein (CP) with the cytoplasmic endodomain of E2 glycoprotein (cdE2) facilitate the budding of virus particles from the plasma membrane of host cell. Till now no effective antivirals have been developed against any member of Alphavirus genus nor a vaccine is commercially available in the market Alphaviruses are classified into the New and the Old World viruses depending upon the mechanism by which they down regulate the transcription of host cell. The Old World viruses like Chikungunya virus (CHIKV), Semliki Forest virus (SFV), Sindbis virus (SINV) employ nsp2 protein for down regulating the cellular transcription, while the New World viruses like Venezuelan Equine Encephalitis Virus (VEEV), Western Equine Encephalitis Virus (WEEV) use capsid protein for shutting off the host transcription. Currently, the genus alphavirus is classified into seven antigenic complexes which includes both the New and Old World viruses. The La Reunion outbreak of chikungunya in 2005 infected about 40 % of the 785,000 population. This re-emergence of chikungunya infection poses a serious threat to the mankind and necessitates the study of alphaviral biology. The CP of alphaviruses is a multifunctional protein. It consists of two domains, the RNA binding N-terminal domain and the C-terminal protease domain. The C-terminal protease domain of alphavirus CP has a chymotrypsin-like serine protease scaffold that contains the catalytic triad residues: Ser, His and Asp similar to other serine proteases. The N-terminal domain, which is highly disordered is involved in binding to the genomic RNA, PPIs (protein-protein interactions) that lead to the CP dimerization and the inhibition of host cell transcription. The C-terminal CP has cis-autoproteolytic activity and it cleaves itself from the N-terminus of the structural polyprotein. Thus, it plays a critical role in initiation of the structural polyprotein processing andthe viral life cycle. After the cis-cleavage, the conserved C-terminal tryptophan residue of CP remains bound to the active site and blocks further CP protease activity. The molecular interaction of CP with the cdE2 facilitates the budding of virions from the plasma membrane of infected host cell. Previous studies have postulated that dioxane present in the CP-hydrophobic pocket of AVCP-dioxane complex structurally mimics the pyrrolidine ring of Pro405 residue of cdE2. This suggested that heterocyclic ring compounds similar to dioxane/compounds containing the pyrrolidine ring/ their derivatives can potentially bind in the CP hydrophobic pocket and disrupt its PPIs with cdE2. Picolinic acid (PCA), a pyridine containing compound is known to have antiviral, antimicrobial, cytotoxic and apoptotic properties. The binding of PCA to chikungunya virus capsid protein (CVCP) hydrophobic pocket was analysed by molecular docking, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and fluorescence studies. Moreover, PCA significantly inhibited CHIKV replication in infected Vero cells, decreasing viral mRNA and viral load as assessed by qRTPCR and plaque reduction assay, respectively. The thesis consists of four chapters which include the structural and functional characterization of chikungunya virus capsid protein (CVCP). The structural and functional studies performed in this study have characterized the CVCP at the molecular level. These studies include in silico protein modeling, molecular docking, screening of small heterocyclic compounds targeting CP-cdE2 interaction, 3D crystal structure determination and analysis, CVCP trans-protease activity assay, beside biochemical and biophysical elucidations and interpretations. Chapter 1 reviews the literature. It describes the genus alphavirus, life cycle, transmission, general structure of the virion. The genome organization of prototype alphavirus, structural and non-structural polyprotein processing have been described in details. The overall structure of the capsid protein and its multifunctional role in the virus life cycle like budding, autoproteolytic activity, nucleocapsid assembly and RNA encapsidation etc. have been described in details. Different strategies for CVCP protease inhibition and disruption of CP-cdE2 interactions have been discussed in the chapter. Chapter 2 describes the in silico analysis of CVCP. Homology modeling was done for CVCP protein (residue range: 113-261). The crystal structure of Semliki Forest virus capsid protein (PDBID: 1VCP) was the first hit with 93 % sequence identity to CVCP. Dioxane based derivatives targeting protein-protein interactions have been reported to possess antiviral activity against Sindbis Virus (SINV), the prototype alphavirus. The modeled CVCP structure was usedfor analyzing the binding of dioxane, proline and picolinic acid (PCA) into the conserved hydrophobic pocket of capsid protein. The recombinant CVCP has been produced in E. coli and purified using affinity chromatography and gel-filtration chromatography. The purified CVCP protein was used for biophysical studies. The binding of small heterocyclic compounds to purified protein was further analysed by ITC, SPR and fluorescence studies. The binding constant KD obtained for PCA was 2.1 × 10−7 M. Additionally, PCA inhibits the CHIKV replication in Vero cells, decreasing viral mRNA and viral load as assessed by qRT-PCR and plaque reduction assay, respectively. Chapter 3 describes the crystal structure determination and analysis of CVCP. The purified CVCP protein was used for crystallization and screened by using Hampton protein crystallization screens. The purified CVCP was crystallized and mountable crystals were obtained in 15 days. The addition of Octyl β-D-glucopyranoside to purified CVCP protein helped in improving the CVCP crystal quality. The crystals were diffracted at 2.0 Å, and data was collected at the home source (MCU, IIT Roorkee). The CVCP crystal data was solved using molecular replacement method and three dimensional structure analysis was done. The crystal structure reveals the presence of chymotrypsin-like structure having Greek key motif. A detailed analysis of CVCP protease active site and hydrophobic pocket has been done. Structural comparisons with crystal structure of other alphavirus capsid proteins have been done to identify residues of the conserved hydrophobic pocket and residues of the pocket that interacts with E1. Additionally, structural and sequential mapping of CP epitopes has been done. CVCP crystal structure was also used for structure-based identification of inhibitors targeting the conserved hydrophobic pocket of CVCP. Molecular docking of the compounds into the hydrophobic pocket of the CVCP crystal structure was performed. The binding affinity of these compounds to CVCP was further analyzed by surface plasmon resonance and fluorescence studies. Kinetic parameters of binding for these compounds to purified CVCP has been determined. Chapter 4 reports the trans-proteolytic activity of alphavirus capsid protease. Virus specific proteases are verified drug targets including the example of classical HIV protease. However, for screening and testing of protease inhibitors we need a high throughput screening (HTS) protease assay. CHIKV capsid protease is an attractive target for anti-CHIKV agents because of its important role in structural polyprotein processing and nucleocapsid core formation inside host cells. This assay involves the use of fluorogenic peptide substrates for developing HTS assay. The sequence of the peptide was derived from the capsid protease cleavage site that includes the conserved W/S residues. When the capsid protease cleavage siteincorporated in the fluorogenic peptide substrate is cleaved by purified and active capsid protease, the FRET decreases and the fluorescence emission intensity for the donor fluorophore increases. Therefore, the protease activity of purified CP was detected by monitoring the increase of fluorescence at 490 nm. This fluorogenic peptide assay could be used for high throughput screening (HTS) of inhibitors against CHIKV capsid protease. Kinetic parameters using fluorogenic peptide substrates for the chikungunya virus capsid protease were estimated. The effect of serine protease inhibitors (N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK) and Benzamidine hydrochloride) on CVCP protease activity has been determined. In conclusion, this chapter explains the development of a high-throughput method for testing and screening inhibitors against the proteolytic activity of CHIKV capsid protease.