Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/36444
Title: MECHANISTIC CHARACTERISATION OF DENGUE VIRUS RNA DEPENDENT RNA POLYMERASE NON-NUCLEOSIDE INHIBITOR BINDING POCKET THROUGH IN VITRO BIOCHEMICAL ASSAYS AND REVERSE GENETICS ANALYSES
Authors: DORCAS ADOBEA LARBI
Keywords: Dengue virus, RNA polymerase, Enzyme mechanism, Viral replication, Site-directed mutagenesis, Non-structural protein 5
Issue Date: 28-Nov-2012
Source: DORCAS ADOBEA LARBI (2012-11-28). MECHANISTIC CHARACTERISATION OF DENGUE VIRUS RNA DEPENDENT RNA POLYMERASE NON-NUCLEOSIDE INHIBITOR BINDING POCKET THROUGH IN VITRO BIOCHEMICAL ASSAYS AND REVERSE GENETICS ANALYSES. ScholarBank@NUS Repository.
Abstract: Dengue virus (DENV) is among the most important human arboviral pathogens. The virus infects about 50 million people worldwide leading to broad spectrum of outcome from a mild febrile illness to fatal haemorrhage and shock syndrome (Endy et al., 2010) and there are currently no clinically approved vaccines or antivirals for this disease. DENV has three structural and seven non-structural proteins (NS). NS5 has RNA-dependent RNA polymerase (RdRp) activity which plays a major role in viral replication and has also been associated with disease pathogenesis. DENV RdRp domain has been identified by X-ray crystallography to bind several non-nucleoside inhibitors. Thus, this research study was to assess the drug-ability and relevance of the RdRp binding pocket of two non-nucleoside inhibitor compounds from Novartis Institute for Tropical Diseases (NITD) that binds to the catalytic domain of the enzyme. Experiments were done to investigate the importance of the inhibitor binding pocket for in vitro polymerase activity and as well for replication fitness in context of the DENV 2 TSV01 infectious virus. For these studies, individual amino acids lining this pocket that interacted with the inhibitors were mutated to alanine. Biochemical enzymatic assays were used to measure the ability of the RdRp proteins to carry out de novo initiation and elongation activities. Results obtained showed decreased enzymatic activities for full length (FL) NS5 F399A, K402A, F486A, N493A, Y607A, N610A and D664A proteins whilst G605A and W796A proteins displayed an increase in de novo initiation activity but with no distinct change in elongation activity compared to the wild-type (WT) protein. The exception was Y607A which demonstrated significant increase in RdRp elongation activity whilst G605A also showed a slight decrease in activity. K402 residue showed to be required for both de novo initiation and elongation process whilst Y607 was determined to play an essential role in polymerase activity only during de novo initiation of viral RNA synthesis. Residues F486 and G605 generally demonstrated no effect in both de novo initiation and elongation steps of RNA replication suggesting that these residues are not crucial for RdRp enzyme activity. Similarly, engineering of five mutated residues into genomic RNA of infectious clone for viral infection studies showed that residues F399, N493, N610 and D664 are critical for viral replication. These residues have also demonstrated a significant role in functioning both at the step of de novo initiation and elongation during the synthesis of RNA. W796A exhibited ~50% decrease in IFA positive cells and was able to recover less than 25% of virus titres as compared to WT which was in contrast to its remarkable performance during in vitro enzyme activity studies. This work contributes to understanding the biological function of residues lining the RdRp catalytic domain in DENV NS5. Gaining insight into specific active site residues is essential for the development of anti-viral inhibitors.
URI: http://scholarbank.nus.edu.sg/handle/10635/36444
Appears in Collections:Master's Theses (Open)

Show full item record
Files in This Item:
File Description SizeFormatAccess SettingsVersion 
Larbi DA.pdf2.69 MBAdobe PDF

OPEN

NoneView/Download

Page view(s)

160
checked on Dec 11, 2017

Download(s)

33
checked on Dec 11, 2017

Google ScholarTM

Check


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.