STUDIES OF SUBSTRATE-BASED INHIBITORS TARGETING NON-STRUCTURAL PROTEINS OF ALPHAVIRUSES

Abstract

Alphaviruses are members of Togaviridae family which are mainly arthropod-borne viruses having an envelope of icosahedral symmetry. This family contains around 30 kinds of viruses most of which are transmitted by mosquito vectors. Viruses of this genus can infect humans as well as animals causing diseases like encephalitis, which is caused by Venezuelan equine encephalitis virus (VEEV), Western equine encephalitis virus (WEEV) and Eastern equine encephalitis virus (EEEV) or fever, rash and arthritis caused by Chikungunya virus (CHIKV), Ross River virus (RRV) and Sindbis virus (SINV). Alphaviruses have emerged as a serious human health menace as serious epidemics have been caused by these viruses in the last two decades. Chikungunya virus became a well-known member of this family after outbreaks in 1999–2000 in the Democratic Republic of the Congo, and in 2007 in Gabon. In 2006 large outbreak was also reported in India and in several other South East Asian countries. Other member of the alphavirus genus is SINV whose outbreak was reported in Finland and Sweden. It is exclusively endemic to Northern Europe where large outbreaks have been reported. Study of several cases over a period of 2006-10 has reported the occurrence of SINV epidemics during late summer/early autumns. VEEV has been found in South America and North America with potency to cause an outbreak in that region. Recurrent alphaviral epidemics such as the one observed for CHIKV in 2006 and the unavailability of antiviral drug or vaccine against them makes it imperative to search for effective drug targets and antiviral molecules that can work against alphaviruses or alleviate the clinical symptoms associated with alphaviral infection. The four non-structural proteins of alphaviruses are actively involved as individual components in the intricate process of viral replication inside host cell as well as in combating host antiviral pathways. Various biochemical assays and conserved sequences of these proteins strongly support their major roles in the formations of viral replication complexes. To curb the spread of viral infection it is very essential to decipher the functions of viral non-structural proteins and their roles in the viral replication cycle so that potential antiviral drugs can be designed and used for alphaviral attenuation. In recent years, efforts for developing inhibitors against non-structural proteins of replicase complex, especially nsP1 and nsP2, have greatly increased. Further analyses of the viral replication inside host cell will facilitate a clear understanding of various non-structural proteins and will accentuate the quest for the development of inhibitors against them. x This thesis consists of four chapters. Chapter 1 reviews the published literature and gives a general overview of members of alphavirus genus, alphaviral genome architecture, re-emergence of chikungunya in different part of the globe and its epidemiology, description of alphavirus host and their transmission. Further, alphavirus replication and the role of various non-structural proteins involved in the formation of viral replication complex along with brief information of the research work carried out on non-structural proteins has been described. Additionally, inhibitors identified against non-structural proteins of alphaviruses have been reviewed. Self-oligomerization of polymerase in different RNA viruses, role of various domains in homo-oligomerization and proposed functional significance of this oligomerization has been discussed. A succinct overview into the functioning and use of capillary electrophoresis technique for enzyme characterization has been presented. Chapter 2 describes the development of a novel capillary electrophoresis (CE)-based methyltransferase assay for alphavirus capping enzyme, nsP1. nsP1 orchestrates the capping of viral RNA inside the host cell. This enzyme has a unique mechanism for the formation of cap0 structure on viral RNA. The capping of viral nascent RNA is carried out in two steps: Methyltransferase (MTase) and Guanyltransferase (GTase). The first step or the MTase reaction involves the transfer of methyl group to GTP molecule at m7 position. This m7GMP further binds itself to the protein making a covalent complex. This m7GMP is later transferred to 5’ cap of nascent viral RNA. This mechanism is unique for members of alphavirus genus. This makes nsP1 enzyme an attractive drug target. Till date, no non-radioactive assay is available for kinetic characterization and inhibitor screening for methyltransferase step of nsP1 enzyme. We have developed a CE-based assay to determine MTase activity of VEEV nsP1 and CHIKV. For this, cloning, expression and purification of VEEV nsP1 was done. Recombinant CHIKV nsP1 (C-terminal truncated) has been already cloned and purified in our lab. Expression and purification of VEEV nsP1 was optimised. Recombinant VEEV nsP1 and CHIKV nsP1 were purified to homogeneity using affinity chromatography and MTase reaction in the presence of the enzyme was carried out. Running conditions for Capillary electrophoresis including background electrolyte, injection parameters, internal standard, running voltage and time were optimised for the separation of reaction products in minimal time. Reaction conditions viz. pH, salt, metal ion concentration and temperature were also optimised. Active site mutants (D63A) were generated to be used as a negative control to confirm that the observed activity was due directly to the purified enzyme. Additionally, IC50s of two known inhibitors (Aurintricarboxylic acid and sinefungin) were calculated and compared with published data (calculated using radiolabel-based assay) to validate the assay. This assay was xi also used to check the inhibition efficiency of a few substrate analogs against VEEV nsP1 and CHIKV nsP1 enzymes. H37A mutant of VEEV nsP1 was generated and used for carrying out MTase reaction for examining the formation of m 7GTP moiety during the capping reaction. Another CE-based method using neutral a coated capillary was developed for the positive identification of m7 Chapter 3 focusses on characterization of two newly identified inhibitors of chikungunya in a cell-culture based study. We have isolated a CHIKV strain from patients' sera from 2016 Indian epidemic and identified it as a recently sequenced strain 119067. Nucleotide and amino acid sequence of the strain was used for sequence analysis of the strain prevalent during the 2016 epidemic and non-synonymous mutations were identified on comparison with E1 sequences of other CHIKV strains. Sequence of the strain was used for phylogenetic tree construction, which demonstrated that the 119067 strain belonged to East Central South African (ECSA) clade of CHIKV. Two peptidomimetic compounds, Pep-I and Pep-II were identified previously by our group against nsp2 protease (nsP2pro) of CHIKV using structure-based drug design approach. The antiviral potential of these two compounds was examined with viral plaque reduction assays against this clinical strain. Both these compounds showed substantial reduction of viral titer in BHK-21 cells infected with the clinical isolate of CHIKV in a dose-dependent manner at concentrations well below their respective maximum non-toxic doses. To further validate the anti-CHIKV activity of these compounds, qRT-PCR was used for quantification of intracellular viral RNA. The data from qRT-PCR clearly demonstrated that the treatment of infected cells with Pep-I and Pep-II reduced viral RNA levels considerably. Additionally, time-of-addition studies were also performed to determine the stage of CHIKV infection cycle where these compounds exert their antiviral effect, and obtained results were in coherence with the anti-CHIKV nsP2pro activity exhibited by both the compounds. SINV was also propagated in BHK-21 cell line using RNA transcripts generated from a cDNA clone of SINV (pTOTO64). Antiviral activity of the two compounds was also assessed against SINV using plaque-reduction assay. Interestingly, no reduction in viral title or viral-induced cytopathic effects was observed in case of SINV. From this study we inferred that the two newly identified peptidomimetic inhibitors Pep-I and Pep-II specifically inhibit CHIKV replication. This also lead us to believe that this new approach of viral inhibition using peptidomimetic inhibitors of nsP2pro can be undertaken for the development of effective antiviral therapy against CHIKV. GTP generated by H37A mutant of VEEV nsP1. Taken together, this chapter explains the development of a novel MTase assay for kinetic characterization and inhibitor screening against alphaviral capping enzyme, nsP1. xii Chapter 4 deals with N-truncated (98-610aa) nsP4 of SINV which makes up the core domain of this RNA dependent RNA polymerase (RdRp). The N-terminal region of nsP4 is predicted to be unstructured and previously N-terminal truncated SINV nsP4 has been purified for molecular and biochemical analysis of the protein. This truncated form of nsP4 does not show de novo polymerase activity when incubated with a RNA template but was able to add poly A nucleotide chain in a non-templated fashion in the presence of divalent metal ion and showed terminal adenyltransferase (TATase) activity. Truncated nsP4 SINV was purified with the help of affinity chromatography and size exclusion chromatography. Crystallisation trials of the protein were done. Size exclusion chromatography revealed that this protein exists in oligomeric form along with its monomers in the solution. The oligomeric form of the protein was shown to bind nucleic acid present in the lysate during purification. To investigate the role of nucleic acid binding of the protein in its oligomerisation electrophoretic mobility shift assay (EMSA) was performed, For EMSA study, RNA corresponding to four different promoter regions of SINV genome were in-vitro transcribed. Two mutants (Double mutant D465A, D466A; Triple mutant: R545A, R 546A, R547A) thought to disrupt metal/RNA binding of the protein were generated using site-directed mutagenesis. Unfortunately, both these mutant proteins expressed as insoluble aggregates in E.Coli. EMSA of protein with RNA were run on non-denaturing gel. But, the correlation between nucleic acid binding and oligomerisation of the protein could not be established. Sequence analysis and homology modelling of the protein was done to identify various characteristic RdRp sequence motifs.