Structural and functional characterisation of chikungunya virus replication complex dynamics
Chikungunya virus (CHIKV) is a 12 kilobase (kb) positive sense single strand RNA (+ssRNA) mosquito-borne virus. Through mosquito bites, the infected victims develop Chikungunya Fever (CHIKF) associated with symptoms like acute fever and painful polyarthritis. Unfortunately, there are no current e...
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Format: | Thesis-Master by Research |
Language: | English |
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Nanyang Technological University
2019
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Online Access: | https://hdl.handle.net/10356/90307 http://hdl.handle.net/10220/49450 |
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Institution: | Nanyang Technological University |
Language: | English |
Summary: | Chikungunya virus (CHIKV) is a 12 kilobase (kb) positive sense single strand RNA (+ssRNA)
mosquito-borne virus. Through mosquito bites, the infected victims develop Chikungunya
Fever (CHIKF) associated with symptoms like acute fever and painful polyarthritis.
Unfortunately, there are no current effective antiviral drugs and vaccine treatments against
CHIKV and many other +ssRNA viruses due to the inadequacy of biological information of its
replication complex (RC) formations and the process of viral replications. Hence, this project
aims to provide the structural and functional characterisation of the replication dynamics of
CHIKV RC, made of 4 non-transmembrane non-structural proteins (nsPs; nsP1-4) contributing
their enzymatic activities essential to viral replication. Recombinant CHIKV RC bound to its
binding partners, such as 3’ untranslated region (UTR) region of viral RNA, was reconstituted
in vitro for its macromolecular 3D modelling and analysis through Electron Microscopy (EM).
A quick and convenient bacterial cell-based luciferase system to study the CHIKV replication
dynamics was established. The detection of the signal amplification of the Renilla Luciferase
(Rluc) luminescence indicated the presence of viral replication activity from the replicase
plasmid expressing CHIKV RC which acted on the 3’ UTR region coupled to Rluc reporter
gene in the reporter plasmid. This replicase-reporter system captured the importance of selfproteolytically
regulated non-structural polyprotein processing and polymerase activity of RC
required for the first event of viral replication to synthesise negative-strand RNA. Further
biochemical and structural determination of the enzymatic domains of CHIKV RC will reveal
the functional roles and spatial organisation of nsPs in the active assembly of RC. The
expansion of the experimental database will eventually build a structural-aided drug discovery
platform to identify the effective antiviral drug candidates to treat the infections of CHIKV and
+ssRNA group viruses. |
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