MOLECULAR CHARACTERIZATION OF ALPHAVIRUS RNA POLYMERASE

Abstract

Alphaviruses are member of Togaviridae family and transmitted through mosquitoes. Alphaviruses have evolved as a major threat to humans and causes rashes, fever, headache, encephalitis, and arthritis. It contains ~30 members including chikungunya virus (CHIKV), Sindbis virus (SINV), Aura virus (AURAV), Venezuelan equine encephalitis virus (VEEV). Alphaviruses are enveloped viruses that contain a positive-sense single-stranded RNA genome. It contains a 49S RNA genome that is ~12 kb long comprising of non-structural proteins (nsPs) and structural proteins. The nsPs are translated as two types of polyproteins P123 and P1234 that is cleaved into matured nsPs by proteolytic activity of nsP2 protein. nsP1 works as the RNA capping enzyme, nsP3 holds as ADP-ribose phosphate phosphatase activity, and nsP4 works as RNA dependent RNA polymerase (RdRp). Structural proteins are also translated as polyprotein in which capsid protein contains autoproteolytic activity and forms nucleocapsid. The envelope protein forms by the host lipid bilayer and contains spikes, assembled from E2 and E3 proteins. 6K functions as signaling polypeptide. The RdRp protein is synthesized by the polyprotein P1234, which is translated by the read-through of an opal codon present between the coding region of nsP3 and nsP4. The nsP4 is 611 amino acid long protein and the molecular weight is ~70 kDa. The N-terminal of this protein doesn’t show any sequence and structural similarity with other viral members. It has been important to interact with the other nsPs for an efficient replication process. C-terminal of RdRp has conserved structural motifs. It contains the distinctive ‘GDD’ motif that is characteristic feature of RdRp enzymes. The nsP4 protein has been an important antiviral target due to its prime role in the replication process of the virus life cycle. To date, there is no efficient antiviral drug or vaccine is commercially available against the alphavirus infection. Therefore, RdRp has been a potential antiviral target for drug development. Chapter 1 is about the review of literature on alphavirus. It explains the life cycle of alphavirus, the structure of alphavirus, genomic organization of alphavirus, detailed roles of alphavirus non-structural and structural proteins. The structure of nsP4 protein, two-metal ion mechanism, different channels, various motifs, and polymerization mechanism has been discussed in detail. It has also included the antiviral studies performed targeting the RdRp protein and other alphaviral proteins. Chapter 2 shows the expression, purification, and biophysical studies performed with the alphavirus RdRp protein. The SINV Δ97nsP4 protein was expressed and purified using Ni+2-NTA affinity chromatography. The protein was studied for its secondary structural features, by circular dichroism (CD) experiments. The interaction of metals with RdRp protein was also studied using the CD. Furthermore, the study of structural change upon rNTP interaction was studied by fluorescence spectroscopy. The cloning of CHIKV Δ118nsP4 was done in the pET28c vector. Additionally, cloning of CHIKV nsP4 1-611 residues and -7-611 residues, as well as the crystallization of SINV Δ97nsP4 protein, were also attempted. Chapter 3 reports the in silico approach for the model building of alphavirus RdRp protein. DELTA-BLAST and multiple sequence alignment of CHIKV RdRp domain (151-611 amino acids) were done. The three-dimensional models of CHIKV and SINV RdRp were predicted using the online MODELLER and Phyre2 servers. Developed models were analyzed for their structural features and model stability. The chimeric model of CHIKV RdRp and human rhinovirus (RV-A16) was used for molecular docking studies of rNTPs and small molecules. Chapter 4 deals with the surface plasmon resonance (SPR) based interaction analysis of various analytes (divalent metal ions, rNTPs, oligonucleotides, and small molecules) with the SINV Δ97nsP4 protein. The purified and homogenized protein was immobilized over the sensor CM5 surface. The analytes were passed over the immobilized protein and sensograms were obtained. The association constant (Ka), dissociation constant (Kd), and affinity constant (KD) were calculated for all analytes. Chapter 5 describes the in vitro cell-based antiviral analysis of small molecules. The anti-SINV experiments were performed in BHK-21 cells. Further, anti-CHIKV studies were performed in Vero cell line using plaque reduction assay and EC50 was calculated. The EC50 against CHIKV was 6.68 ± 1.34 µM for piperine (PIP), 27.88 ± 13.34 µM for 2-thiouridine (2TU), 36.26 ± 17.53 µM for pyrazinamide (PZA), and 53.62 ± 21.68 µM for chlorogenic acid (CGA). qRT-PCR and immunofluorescence assay have further validated the antiviral property of selected small molecules. The early stage CHIKV inhibition was confirmed by reverse transcriptase PCR.Chapter 6 concludes the thesis topic. The SINV Δ97nsP4 protein was successfully purified and studied using different biophysical techniques. The predicted alphavirus RdRp model was used for molecular docking and the selected small molecules were showing efficient interactions with the nucleotide-binding site. The binding of different analytes with SINV Δ97nsP4 protein was analyzed by the SPR technique. Results of in vitro experiments have validated the antiviral role of the PIP, 2TU, PZA, and CGA.