PhD by Research Programme (Funded Research Programme - Infectious and Immunity)

Research Project Title: The Molecular Basis of the Flavivirus Replication Process 

Principal Investigator: Assistant Professor Luo Dahai, LKCMedicine

Collaborators: Associate Professor Julien Lescar, School of Biological Sciences, NTU
Professor Subhash Vasudevan, Duke-NUS Graduate Medical School

Project Description:

Dengue is a major public-health problem: hundreds of thousands cases of the severe hemorrhagic form occur every year and a large and increasing proportion of the world population is at risk to contract the disease. In the absence of a vaccine conferring true and lasting cross-protection against the four - and possibly five - serotypes of DENV, outbreak control and patient care has to rely on symptomatic treatment and specific antiviral molecules. Plus-strand RNA virus replication occurs in association with cytoplasmic host-cell membranes, where both viral and cellular host factors cooperate within an organelle-like replication factory called replication complex (RC). Several non-structural proteins of the RC constitute validated drug targets because of their crucial functions during viral replication. However, a major impediment in developing drugs targeting the dengue virus (DENV) RC is that both its morphology and composition, the interplay between its molecular constituents as well as the precise molecular mechanisms for viral RNA replication are still elusive. Over the last decade, individual protein components of the RC were characterized both at the functional and structural level. However, a main challenge lying ahead is to gain a better understanding of the entire flavivirus RC. The study proposed here aims at revealing key protein-protein and protein-RNA interactions within the DENV RC to guide and accelerate drug discovery. 

Methods: Protein/RNA/Lipid Biochemistry, Biophysics, Structural biology, cell biology and Virology. The project will elucidate how the NS2B-NS3 protease-helicase and NS5 methyltransferase-polymerase interact with each other and with RNA. Their interactions with the membrane-bound NS4B protein at various stages of virus replication will also be elucidated. Conceptually, two main (and complementary) approaches will be followed targeting non-structural proteins from DENV and building on previous work on NS2B-NS3, NS4B and NS5:

A) Reconstitution of the DENV RC via the expression and assembly of selected individual components. 
As a first step towards the study of the entire DENV RC, we will characterize the structure and dynamic properties of subassemblies of the RC and study in situ the various enzymatic activities responsible for RNA replication. Recombinant viral non-structural proteins will be expressed to develop tools and reagents including monoclonal antibodies (mAbs) and small molecule inhibitors to probe the structure of the flavivirus RC. Crystal structures for NS2B-NS3, NS4B and NS5 as multi-proteins assemblies and bound to viral RNA will be determined, to capture the various stages of viral replication. 

B) The study and isolation of the RC from DENV infected cells.
In this complementary strategy, the DENV RC from infected cell lines will be extracted using pull-down with specific mAbs and its composition analyzed using biochemical and proteomics methods. Genetic complementation analysis will help identify protein-protein interactions crucial for the formation and function of the RC.  

In summary, this project will shed light on the structure and dynamics of the RC from a major human viral pathogen and on important viral proteins. It will offer novel opportunities in terms of therapeutic interventions to treat dengue and other flavivirus infections. Several results obtained by studying the DENV RC will provide a useful comparison with the evolutionary-related Hepatitis C virus (HCV).

If you have questions regarding this project, please email the Principal Investigator, Assistant Professor Luo Dahai - LuoDahai@ntu.edu.sg