Download The Molecular Basis of the Flavivirus Replication Process

Research Theme
Infectious and Immunity
Research Project Title
The Molecular Basis of the Flavivirus Replication Process
Principal Investigator
Assistant Professor Luo Dahai, LKCMedicine
Co-supervisor (if any)
TBA
Collaborator(s)
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).
Fig. 1 (A) DENV life cycle. The intracellular stages of virus replication (yellow box). (B)
The DENV polyprotein and its predicted membrane topology and known 3D structures.
Contact Us
If you have questions regarding this project, please email the Principal Investigator.
Assistant Professor Luo Dahai - [email protected]