IDENTIFICATION AND CHARACTERIZATION OF ANTIVIRAL MOLECULES AGAINST ALPHA VIRUSES

Abstract

Alphaviruses are single-stranded, positive-sense, enveloped viruses. The alphavirus genus includes chikungunya virus (CHIKV), Aura virus (AURV), Sindbis virus (SINV), Semliki Forest virus (SFV) etc. The recent outbreaks of CHIKV are a cause of significant public health and economic burden worldwide. Hence, the development of antiviral molecules to combat alphavirus infections is necessary. In this study, we have explored new approaches for the development of antivirals against alphavirus infections. Alphaviruses display complex glycans on their surface which play a key role in viral pathogenesis by facilitating glycan-host interaction during viral entry. Any protein or compound that binds to these glycans and blocks the interaction of virus with the host cell offers a potential approach to prevent viral infections. We have evaluated the antiviral potential of a chitinase (chi)-like lectin from Tamarindus indica (TCLL) that has specificity for N-acetylglucosamine (NAG). An enzyme linked immunosorbent assay (ELISA) confirmed the binding of TCLL to these alphaviruses glycans by utilizing an antibody against alphavirus glycoproteins. The dose-dependent reduction in virus titer in the presence of TCLL has been observed. The antiviral effects of TCLL against alphaviruses were investigated by plaque reduction assay, luciferase based assay and qPCR. Furthermore, the addition of NAG to TCLL abolished the antiviral activity confirming the antiviral effect of TCLL is due to its binding to the viral glycan moiety found on the surface viral glycoproteins. This was the first approach to inhibit virus by using a natural antiviral protein. In the second approach, nsP1 viral protein has been targeted for antiviral development. Alphaviruses encode four non-structural proteins namely nsP1, nsP2, nsP3 and nsP4. These nsPs are essential for the replication of viral RNA. nsP1 is a capping enzyme that helps in the formation of membrane-bound replication complex. It catalyzes the methyltransferase (MTase) and guanylyltransferase (GTase) enzymatic reactions that are required for the genomic (49S) and subgenomic (26S) RNA replication. To study the enzymatic activities of the nsP1 protein, CHIKV nsP1 was cloned, expressed and purified. The colorimetric assay was used to determine the MTase activity of nsP1. The GT activity of nsP1 was confirmed by western blotting which utilizes the anti-m7G cap antibody. We have developed a novel, non-radioactive ELISA based assay to detect the MTase and GT activities of CHIKV nsP1 and for screening inhibitors. The mutagenesis studies of S-adenosylmethionine (SAM) binding residue Asp63 to Ala further confirmed the nsP1 enzymatic activities. The presence of Mg2+ ions in the purified nsP1 was determined by inductively coupled plasma mass spectroscopy (ICP-MS) studies. The role of divalent Mg2+ ions ii for nsP1 GT activity has been explored. The inhibitory effects of sinefungin, aurintricarboxylic acid (ATA) and ribavirin against nsP1 have been investigated. In the third approach, the capsid protein has been targeted by small antiviral molecules. Alphavirus structural capsid protein (CP) is a multifunctional protein. It performs several functions including autoproteolysis, formation of nucleocapsid core, capsid-glycoprotein interactions and virus budding. The conserved hydrophobic pocket of CP is a potential drug target as it is involved in protein-protein interactions (PPIs). Various biophysical and structural studies have been performed using Aura virus CP (AVCP) and chikungunya virus CP (CVCP) in complex with piperazine. Piperazine is a small heterocyclic molecule that binds to the hydrophobic pocket of CP. The binding of piperazine to hydrophobic pocket of CP was analyzed by molecular docking and fluorescence studies. The piperazine molecule binds to the hydrophobic pocket of CVCP with more affinity as compared to AVCP. The antiviral studies of piperazine against CHIKV have been investigated. The thesis consists of four chapters. Identification and characterization of antiviral molecules against alphaviruses using different approaches have been carried out in the thesis objectives. Chapter 1 deals with the literature review. It describes the life cycle of alphaviruses and their mode of transmission. The structure of the virion and alphavirus genome organization has been described. The structural and non-structural polyprotein processing along with the functions of both structural and non-structural proteins have been explained. The alphavirus replication complex and viral replication and transcription have been described. The viral glycoproteins involvement in the course of infection has been depicted. Different inhibition strategies for alphaviruses have been explained. Chapter 2 describes the antiviral activity of chitinase (chi)-like lectin from tamarind against alphaviruses including chikungunya (CHIKV) and Sindbis virus (SINV). A chitinase (chi)-like lectin isolated from seeds of tamarind (Tamarindus indica) has specificity for N-acetylglucosamine (NAG) and binds to the glycans/sugar molecules having NAG. Thus, it is hypothesized that tamarind chi-like lectin (TCLL) might bind to the N-glycan rich surface of alphavirus and block the entry step of the virus. An ELISA based assay confirmed the binding of TCLL to these alphaviruses by utilizing mouse anti-alphavirus glycoprotein antibody. The antiviral activity of iii TCLL against alphaviruses has been investigated by plaque reduction assay, luciferase based assay and qPCR. Interestingly, the direct treatment of TCLL with the virus reduced the virus infection. Further, the addition of NAG to TCLL abolished antiviral activity confirming that the NAG binding property of TCLL is accountable for its antiviral activity. Taken together, this study will prove to be beneficial in developing lectin therapeutics targeting alphavirus glycans. Chapter 3 reports antivirals that target the methyltransferase (MTase) and guanylyltransferase (GT) activity of CHIKV nsP1. A novel non-radioactive ELISA based assay that was developed to identify nsP1 inhibitors has been described in this chapter. Alphaviruses have a distinct molecular mechanism of capping their genome. The first step involves the transfer of a methyl group from S-adenosylmethionine (SAM) to a guanosine triphosphate (GTP) molecule at its N7 position with the help of nsP1 methyltransferase (MTase). The second step involves the hydrolysis of methylated GTP to guanosine monophosphate (GMP) with the help of nsP1 guanylyltransferase (GTase) and further, a covalent complex is formed between nsP1 and methylated GMP (m7GMP). In the next steps, m7GMP moiety is added to the 5ʹ end of the viral ppRNA by nsP1 GTase resulting in the formation of cap0 structure. Thus, nsP1 is a potential target for antiviral drug development. For activity analysis, the cloning, expression and purification of CHIKV nsP1 were done. The MTase activity of CHIKV nsP1 protein has been firstly confirmed by using an indirect non-radioactive colorimetric assay. The GT activity of the CHIKV nsP1 protein has been confirmed by western blotting analysis using anti-m7G cap antibody. A non-radioactive plate based ELISA assay has been developed which determines the MTase as well as GT activity of CHIKV nsP1 for screening inhibitors. ICP-MS experiments revealed the presence of Mg2+ ions in the purified nsP1. The enzymatic activity was further confirmed by mutagenesis analysis. The inhibitory effects of sinefungin, ATA and ribavirin were assessed using the developed ELISA assay and the IC50 values were estimated to be 2.69 μM, 5.72 μM and 1.18 mM, respectively. In conclusion, this chapter explains the development of an ELISA assay for screening of inhibitors against alphavirus capping enzyme, nsP1. Chapter 4 focuses on the studies of antiviral molecule piperazine that was studied by analyzing the AVCP and CVCP complexes. Structural analysis of CP from different alphavirus members has revealed the conserved hydrophobic pocket that is involved in protein-protein interactions and it may serve as a potential drug target. The binding of small heterocyclic molecules like piperazine to iv the hydrophobic pocket of CP offers a new perspective for therapeutic intervention. Piperazine based drugs against human immunodeficiency virus (HIV) are already present in the market. Therefore, piperazine was chosen as a potential molecule for targeting the hydrophobic pocket of AVCP and CVCP. The structural and biophysical studies of AVCP in complex with piperazine have been investigated. The piperazine was found to be present in the hydrophobic pocket of CP of Aura virus. This piperazine-bound structure was compared with the already known dioxane-bound structure of AVCP. The piperazine binds at the same place where the dioxane molecule binds in the hydrophobic pocket. The molecular interactions of both piperazine and dioxane with the hydrophobic pocket residues were compared. Also, the piperazine-bound structure was compared with active AVCP and Sindbis virus CP (SCP). The docking analysis of piperazine into the CVCP structure shows that piperazine binds to the same position in CVCP hydrophobic pocket. The binding free energy values of piperazine to AVCP and CVCP were compared. The antiviral activity of piperazine against CHIKV was determined by immunofluorescence assay and plaque reduction assay. Therefore, piperazine derivative drugs can be designed as these might offer a broad spectrum antiviral therapy against various members of the alphavirus genus.