FUNCTIONAL CHARACTERIZATION OF ALPHAVIRAL PROTEINS INVOLVED IN REPLICATION

Abstract

Alphavirus are positive sense RNA virus which belongs to Togaviridae family of virus classification. These viruses have wide geographical distribution naming New world and Old world. They have a wide range of hosts ranging from humans, equine to birds and main transmission in the enzootic and epizootic cycle is assisted by Culex mosquito species. Other arthropods like ticks, mites, lice uphold the alphaviral replication and also act as carrier vectors. Members of this genus are capable of causing various range of infections from arthritis to encephalitis with high rate of mortality. However the treatment in case of alphavirus is mostly symptomatic in absence of any effective drug or vaccine. In the initial years, early 20th century when the early cases of infection are reported the alphavirus were found to be confined to certain demographics only. But with time, members of this genus have evolved and now have spread and covered major parts of the seven continents. And in the light of this evolution along with the absence of any therapy against this, it has become imperative to discover inhibitory drug molecules. For this purpose different crucial mechanism vital for the survival of the virus are to be targeted. For these reasons, we have targeted alphavirus replication enzyme nsP2 (non structural protein 2) which plays important roles at the different steps of the virus replication. The Nterminal region of the protein is a helicase domain, and the C-terminal region possessing proteolytic activity is a papain-like cysteine protease. The C-terminal region is crucial for the processing of nsP1234 polyprotein, which on cleavage form individual proteins. These nsP’s are part of different replication complexes which virus forms during the course of replication. Also, this C-terminal region is important for the synthesis of 26S sub-genomic RNA as it is discovered to have interactions with the promoter region for sub-genomic RNA on the genomic RNA. The protein is also crucial for inactivating the host response like IFN’s. Based on these observations we have considered the C-terminal protease an ideal target for our study. By applying the biophysical and biochemical strategies we were able to decipher new features of this protein. From three different alphaviruses viz. AURA, CHIKV and SINV, the C-terminal region was cloned in an expression vector. In case of AURAV, we have cloned and purified the protease using Ion exchange and size exclusion chromatography. The protein purification was optimized to achieve better yields of the protein for crystallization purposes. However, we were not successful in crystallizing the protein. But we have developed a simple yet reliable assay based on the application of modified β-galactosidase protein as the substrate. The Chapter I 6 different sites at which nsP2 protease cleaves were inserted at optimum position inside the β- galactosidase gene. And then the proteolytic activity was checked on using this modified gene in presence of nsP2. SINV nsP2 was also cloned in pET series of expression vector and was purified using affinity and size exclusion chromatography. However in case of SINV, it was difficult during the initial stages to get a soluble protein. Various modifications in the inducer concentration to temperature did not yield any success in this case. But by optimizing media conditions we were able to get pure soluble protein. The protein was purified and crystallized using sitting drop method of vapor diffusion. But we were not able to further improve the crystal quality to get diffraction quality crystals. Protease domain from CHIKV was also cloned and purified and in this case was successfully crystallized to achieve diffraction quality crystals. The protein was crystallized with 5 mg/mL concentrations using sitting drop vapor diffusion method. The crystal was diffracted and structure was solved up to 2.6 Å. This protein is monomer in solution conditions but it was observed to be a tetramer in the crystal form. On analysis of the structure it was found to be bound to different glycerol molecules at certain positions. Also the active site region was found to be closed in comparison to the VEEV (PDB: 2HWK) and nsP23zbd SINV (PDB: 4GUA). Large amount of water molecules were found to be near the substrate binding regions of the protein. Different regions (especially active site) of high temperature factors were observed which showed that there is flexibility and chances of modifications in these regions. Also, TNBS based activity assay was also developed in case of nsP2CHIKV. The assay was used for understanding the kinetics of nsP2 for its different substrate sites. All these studies have shown that nsP2 protease is a unique and important protease. The protein cloned and expressed heterologously is stable and active. The protein shows preference for nsP3/4 site in comparison to other sites showing that other nsP’s might be necessary for cleavage at nsP1/2 and nsP2/3 site. The information from this study might help in the understanding of the mechanism of this preference of nsP2 as well as the structure information from nsP2CHIKV would help in structure based screening of inhibitory molecules. Not only this, structure information regarding the environment of the active site, conformation and properties of active and substrate site residues would help in designing the inhibitory molecules against nsP2 protease. This might contribute in the inhibition of the infection of the alphavirus members.