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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Main Author: Tan, Yaw Bia
Other Authors: Luo Dahai
Format: Thesis-Master by Research
Language:English
Published: 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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spelling sg-ntu-dr.10356-903072023-02-28T18:51:29Z Structural and functional characterisation of chikungunya virus replication complex dynamics Tan, Yaw Bia Luo Dahai Wu Bin School of Biological Sciences LuoDahai@ntu.edu.sg Science::Biological sciences Science::Chemistry::Analytical chemistry::Proteins 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. Master of Science 2019-07-22T01:32:04Z 2019-12-06T17:45:22Z 2019-07-22T01:32:04Z 2019-12-06T17:45:22Z 2019 Thesis-Master by Research Tan, Y. B. (2019). Structural and functional characterisation of chikungunya virus replication complex dynamics. Master's thesis, Nanyang Technological University, Singapore. https://hdl.handle.net/10356/90307 http://hdl.handle.net/10220/49450 10.32657/10220/49450 en This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0). 60 p. application/pdf Nanyang Technological University
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
Science::Chemistry::Analytical chemistry::Proteins
spellingShingle Science::Biological sciences
Science::Chemistry::Analytical chemistry::Proteins
Tan, Yaw Bia
Structural and functional characterisation of chikungunya virus replication complex dynamics
description 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.
author2 Luo Dahai
author_facet Luo Dahai
Tan, Yaw Bia
format Thesis-Master by Research
author Tan, Yaw Bia
author_sort Tan, Yaw Bia
title Structural and functional characterisation of chikungunya virus replication complex dynamics
title_short Structural and functional characterisation of chikungunya virus replication complex dynamics
title_full Structural and functional characterisation of chikungunya virus replication complex dynamics
title_fullStr Structural and functional characterisation of chikungunya virus replication complex dynamics
title_full_unstemmed Structural and functional characterisation of chikungunya virus replication complex dynamics
title_sort structural and functional characterisation of chikungunya virus replication complex dynamics
publisher Nanyang Technological University
publishDate 2019
url https://hdl.handle.net/10356/90307
http://hdl.handle.net/10220/49450
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