Domain characterisation of CHIKV nsP3 protein
Chikungunya virus (CHIKV) is an Alphavirus and belongs to Togaviridae family of virus that infects the human while mosquitoes act as vector in its life cycle. Aedes aegypti and Aedes albopictus are the two mosquito species mediating the disease. Virus enters the host body as a result of bite during blood meal of infected female. In the host body, the virus is recognized by host immune cells at site of infection which results in influx of other immune cells which are responsible for activation of humoral immune response which controls and neutralises the virus. The disease results in fever, rashes, arthralgia and myalgia in human beings. The ssRNA genome encodes both non-structural proteins (nsP1, nsP2, nsP3 and nsP4) and structural proteins (C, E1, E2, E3 and 6K). The structural protein forms the virus capsid and envelope proteins. The non-structural proteins form the replication complex of the virus. The nsP1, nsP2, nsP3 and nsP4 possesses enzymatic activities which are crucial for viral replication and thus multiplication of the virus in the host body. This project focuses on the expression and characterisation of one of the domains of CHIKV. nsP3 protein plays a critical role in viral-host protein-protein interaction and consist of three domains namely the N-terminal Macrodomain, the central Alphavirus unique domain (AUD) and the C-terminal end Hypervariable domain (HVD). nsP3 macrodomain is known to have an ADP-ribosyl binding pocket and shows hydrolase activity. AUD contains a unique Zinc binding fold with four cysteine residues which coordinates with a zinc molecule which helps to bind RNA. HVD is highly disordered and interacts with host proteins with the help of conserved short peptide motif and utilise them for its growth. All the three domains help in viral replication and infection. In this work, domains will be cloned in pET vector and protein will expressed in E. coli bacterial strain. It is followed by a western-blot confirmation using nsp3 specific polyclonal antibody.
Keywords: Alphavirus, Macrodomain, Hypervariable domain, Cloning, Protein expression.