ALPHAVIRUS CAPSID PROTEASE AND ITS INHIBITORS

Abstract

Alphaviruses are distributed worldwide and has acquired infamy by being health threat to humans and livestock. Almost 20 different alphaviruses are categorized as medically important viruses as the infection accompanies arthritis, encephalitis, rashes and fever. Alphavirus infection is transmitted to humans majorly by the mosquito bite hence alphavirus is categorized among arbovirus. Ross River virus (RRV), Sindbis virus (SINV), Semliki Forest virus (SFV) and Venezuelan Equine encephalitis virus (VEEV) are exploited as viral vectors for gene delivery therefore serve as important bait in gene therapy. Aura virus (AURAV) is an alphavirus which displays similarity with SINV in terms of structure organization and gene sequence. The nucleotide sequence of promoter of AURAV bears striking similarity with the promoter of SINV. AURAV has not yet gained notoriety for causing infections in humans but it is serologically similar to pathogenic WEEV and SINV so it is quite relevant to study the virus. Chikungunya virus (CHIKV) has created menace time and again worldwide especially in tropical and temperate region. The re-emergence of CHIKV is a matter to be pondered upon especially for developing nations where basic sanitation facilities get compromised because of their economy crunch. The prime source of virus spread is by mosquito bite. CHIKV infection is accompanied by fever, severe joint pain, muscle pain, headache, and nausea. Alphavirus is categorized in the group IV Togaviridae family of viruses. Structurally alphaviruses are enveloped viruses which exhibits T=4 icosahedral symmetry. Diameter of alphavirus is approximately 70nm. A mature virion of alphavirus acquires lipid membrane from the host. Alphavirus exhibits protruded spikes which are generated by the assembly of trimer of heterodimer of the viral glycoprotein E1 and E2. The envelope E1 and E2 proteins localised in transmembrane has its C-terminal exposed to cytoplasm via which nucleocapsid interacts and budding of virus is promoted. The genome of alphavirus is a 49S single-stranded, positive-sense, RNA approximately 12 kb in length. Along with the genomic RNA, alphaviruses make 26S subgenomic RNAs which encode for the viral structural proteins. Translation of the subgenomic RNA generates structural polyprotein and capsid protein (CP) is part of it. The CP cleaves itself from the N-terminus of structural polyprotein owing to its cis-proteolytic activity which is confined in the C-terminus subdomain. Post cleavage of the CP from its polyprotein, the autoproteolytic activity halts as the C-terminus Trp (conserved) residue binds to the S1 specificity (active site) pocket blocking the active site of the serine CP protease. The cleaved structural polyprotein is processed by the host cell protease to generate E3, E2, 6K and E1 II structural proteins. All these structural proteins along with CP play a vital role in the virus budding and encapsidation of the viral genome RNA. The cleaved CP interacts with the genomic RNA through its N-terminal domain which is laden with basic amino acidresidues. This interaction is highly specific and mediates encapsidation of genomic RNA and nucleocapsid (NC) assembly. The N-terminal domain of CP is highly disordered and till date no crystal structure comprising the domain has been determined yet. There is alarming need to study the N-terminal domain of alphavirus CP for understanding RNA and capsid interactions at the atomic level. The NC is enclosed by the phospholipid bilayer comprising trimeric spikes constituted by the assembly of heterodimer of E1 and E2. The CP has a small hydrophobic pocket present in the C-terminal domain via which the cytoplasmic tail of E2 (cdE2) binds and promotes the budding of virus. Since CP plays a prominent role in the viral life cycle by being involved in viral RNA encapsidation, structural polyprotein processing and in virus budding, therefore is a potential target for antiviral drug development. Chapter 1 is about literature reviewed on alphavirus. It briefs about the alphavirus life cycle, genome organisation of alphavirus, functional significance of non-structural and structural proteins of alphavirus. The chapter details about CP, its structural and functional role in viral assembly and budding. The chapter focuses on capsid protease as potential antiviral drug target and brief significance of repurposing of FDA approved drugs. Chapter 2 comprises of three sections, where the first section details about the expression, purification and crystallization of the C-terminal truncated; protease active form of chikungunya virus capsid protein (CVCP active, 110-259 amino acid residues). Due to the unsuccessful crystallization attempts of CVCP active, the conserved C-terminal residue W261 was computationally removed from the coordinate file of the crystal structure of CVCP native (inactive proteinPDB ID: 5H23) for making the active site accessible for the docking of compounds from the library of FDA approved drugs. The second part details about the in vitro study of selected compounds which were best hits based on the binding energies against the active site of CVCP. The inhibitory constants (Ki) of the compounds and their mode of inhibitions were determined using fluorogenic peptide substate having scissile bond W-S in a FRET- based protease assay. The 3rd section describes the antiviral effects of these CVCP inhibitors in the cell-based antiviral assays. From the library of FDA approved drugs, four compounds showed best hits against the active site of CVCP by molecular docking. These compounds inhibited the proteolytic activity of CVCP in the FRET based assay and showed III different mode of inhibitions; two compounds showed competitive while the rest two showed distinct mixed and non-competitive inhibition and the calculated Ki were in the micromolar range. In the cell-based antiviral assays, these compounds showed antiviral potential against CHIKV. Plaque reduction assay, q-RT-PCR, western blot analysis and immunofluorescence assay showed these compounds target the CHIKV life cycle at the later stage of cell infection. Chapter 3 attempts to study the structural and functional perspectives of the N-terminal domain of alphavirus CP. In number of crystal structures, the N-terminus of the alphavirus CP is disordered [1], [2], [3], [4], [5], [6].This chapter is categorised into three sections; the first part aims to crystallize and determine the three-dimensional structure of the N- terminal domain of CVCP and AVCP. This section includes the cloning and expression of AVCP19 (19-265 amino acid residues) and CVCP20 (20-259 amino acid residues). The purification of CVCP20 was unsuccessful but AVCP19 was purified by immobilized metal affinity chromatography (IMAC) using phosphate buffer. Co-crystallization AVCP19 with 12-mer DNA was done which was again not sucessful. Also co-crystallisation of AVCP80 (80-267 amino acid residues) done with 12-mer DNA is in accordance to the literature surveyed which shows in SINVCP, 81-113 residues are involved in specific binding of CP with the genomic RNA and mediates the formation of NCP cores [7]. The crystals diffracted to 1.2 Å but neither electron density of the N-terminal domain was found nor was the density for 12-mer DNA detected. The second section includes the biophysical studies of interaction of nucleic acid with purified AVCP19. The CP-DNA interactions were studied by size exclusion chromatography and differential scanning calorimetry (DSC). Size exclusion chromatography confirmed formation of CP-DNA complex and DSC showed shift in the Tm of AVCP19 bound DNA which suggested the interaction provides stability to the domain. The third section describes an attempt to study the effect of DNA binding on the proteolytic activity of active CVCP and AVCP. The conserved C-terminal Trp residue of both AVCP19 and CVCP20 constructs were deliberately removed while making the constructs to study the trans- protease activity of these in presence of DNA oligos. Chapter 4 concludes the thesis topic. The structure-assisted drug-repositioning approach has been used to identify potent antiviralmolecules for chikungunya therapeuticswhere the potential inhibitors of CHIKV CP protease were identified by virtual screening of compounds from the library of FDA approved drugs. The results of fluorogenic substarte peptide-based FRET assay and cell-based antiviral assays with the best hits compounds validated the above finding and led to the conclusion that alphavirus capsid protease could be a potential antiviral drug target. For IV the first time DNA-CPcomplex was purified by size-exclusion chromatography and stability of the complex was determined by DSC technique. The increased shift in the Tm of ‘DNA bound AVCP19’ showed the nucleic acid interaction with the N-terminal domain of alphavirus CP provides stability to the disordered region. Crystal structure of nucleic acid bound alphavirus CP can certainly help in studying the three-dimensional structure and molecular contact details of CP and nucleic acid.