Dengue virus (DENV) and Zika virus (ZIKV) are members of the flavivirus genus and infect hundreds of millions of people a year. Currently there are no approved drugs against ZIKV or highly effective vaccines against all DENV serotypes. A detailed understanding of the structural and dynamical aspects of the various components of DENV and ZIKV virus is being pursued in Singapore in an interdisciplinary effort that engages 14 different experimental and computational groups.
A Flavivirus is like an onion. The outermost layer is made up of proteins called the envelope (E) protein. The next layer of the onion is a lipid bilayer, within which parts of the E proteins along with membrane (M) proteins are embedded. Finally, the inner core of the onion contains capsid (C) proteins in complex with a mass of RNA; the RNA contains the genetic information of the virus.
The virus infects cells of the immune system. The mechanisms behind this process of infection are partly understood. First the virus is encapsulated in small compartments of cells called endosomes. It is within these compartments that a lowering of the pH occurs that triggers a mechanical/structural response within the outermost layer of the virus. The E proteins, which were initially lying parallel to the surface of the viral sphere in the form of dimers, undergo structural changes and transition to a trimeric form (Figure 1). This structural rearrangement is accompanied by exposure of the hydrophobic tips of the E protein, referred to as the fusion peptide (FP) regions. This exposure subsequently initiates fusion of the viral and the immune cell membranes, leading to release of the viral genome into the cytoplasm and hence infection. The pattern of amino acids that make up FP is conserved across all flaviviruses (these include dengue virus, west nile virus, yellow fever virus, Japanese encephalitis, tick-borne encephalitis) and naturally is a potential target for antiviral drugs that aim to block viral fusion and hence infection.
We know that the function of macromolecules in biology is intimately linked to their structure, mediated through dynamical changes. However, these changes occur over multiple time scales: thermal fluctuations in the range of femtoseconds (fs), picoseconds (ps) and nanoseconds (ns), and global rearrangements, folding, and assembly processes such as antibody binding in the range of microseconds (μs) to milliseconds (ms) (Figure 2). Each of these motions can be captured by different experimental and computational techniques. Wouldn't it be nice to integrate them seamlessly: to have a rheostat that can be tuned to any time window to obtain a movie of the associated molecular motions? The current project brings together the various practitioners who extend the timescales of their recording devices towards this aim. This will result in a detailed movie of the workings of the dengue virus (as shown in Figure 2) and hopefully guide the development of novel therapeutics. The methodological basis for the movie will rely primarily upon the use of molecular dynamics (MD) simulation - this represents a "computational microscope" that enables us to zoom in on the jiggling of atoms and molecules.
In the Bioinformatics Institute (BII) A*STAR, multiscale MD simulation approaches1 allow our scientists to probe biologically and experimentally relevant time and length scales, by application/development of both atomically-resolved and simplified coarse-grained models (Figure 2). Coarse-graining is achieved by representing all-atom systems with a reduced number of degrees of freedom, i.e. groups of atoms are represented as beads (Figure 2) and validated against all-atom simulations and experimental data. This powerful tool is illustrated by a simulation model of the entire dengue virus envelope, recently developed in our group (Figure 1).2 On the other end of the spectrum, we recently used fully atomistic simulation methods to study the conformational dynamics of the E protein FP region, a major epitope for potential antibodies against dengue, and found results in excellent agreement with data from fluorescence spectroscopy experiments.3 Likewise, the thermodynamics of the interaction of FP with lipid bilayers are being investigated via parallel computational and experimental approaches, providing novel insights into the mechanisms of viral/host membrane fusion.4 In addition atomistic approaches were employed in order to understand the autoinhibitory role of the virus C protein disordered N-Terminus mediating DENV C interaction with biological targets.5 At the same time, new computational approaches approaches for solvent mapping are being developed to reveal new druggable cryptic pockets.6 Recently we have also reported the genome sequences of ZIKV strains from two cases in Singapore and found through phylogenetic analyses that these strains form an earlier branch distinct from the recent large outbreak in the Americas.7
The current project combines experiments and molecular simulations for studying DENV (single proteins, complexes, whole virus), producing details of the virus at unparalleled spatial and temporal resolution. The emerging methodology will enable the application of new protocols we are developing at the Bioinformatics Institute (BII) A*STAR to probe the fluctuating surfaces of the virus in its various constituent states with a view to unravelling cryptic pockets/crevices that can subsequently be drugged with inhibitors of the viral motions and hence life cycle. Using dengue as an example and combining both experimental and computational results at different time and spatial scales will provide new insights into viral mechanisms and a platform for interrogating other complex biological systems, including related viruses such as ZIKV.
Principal investigators: Dr Chandra Verma and Dr Peter J. Bond (Bioinformatics Institute, BII, A*STAR); Prof Paul Matsudaira (Department of Biological Sciences, National University of Singapore).
Funding notes: The project is funded by a grant from the Ministry of Education in Singapore (MOE AcRF Tier 3 Grant Number MOE2012-T3-1-008).