SINGLE MOLECULE IMAGING AND MANIPULATION OF HOST-PATHOGEN INTERACTION

Virus are nature’s simplest replication machines, they undergo dynamic structural organization in their life cycle during host cell entry, genome release, replication and transcription of viral genome, assembly and budding from host cell to establish infection and escape host-immune system. We seek to understand the dynamic intricacies of these processes at molecular level, by following the dynamics of viral structural proteins or complex and their interactions with host macromolecules and translate these knowledge into the development of therapeutic interventions such as neutralizing antibodies and vaccine immunogens, by employing interdisciplinary approach with single molecule technologies including single molecule fluorescence resonance energy transfer (sm-FRET) imaging, high-resolution optical tweezers, molecular biology, virology, immunology, kinetic modeling and simulations.

• Conformational dynamics of enveloped viral glycoprotein during cellular entry.

Enveloped virus glycoprotein mediates entry into host cell via membrane fusion process between the virus membrane and endosomal membrane/cellular membrane. To facilitate this important fusion step, viral glycoprotein goes through large scale conformational change as evident from the X-ray, Cryo EM structural data, which provided the snapshots of the pre-fusion and post-fusion conformation of viral fusion proteins. Still, the information regarding key intermediates conformations on pathway to fusion, is currently lacking and hence our understanding of viral entry mechanism in not complete. Here, we aim to directly visualize the large conformational trajectories of the viral glycoprotein on the surface of infectious virion at a single molecule level by smFRET imaging, during receptor mediated triggering for membrane fusion reaction with supported bilayer and also with cellular membrane for entry. Current focuses are on understanding the entry mechanism of emerging infectious viruses such as Coronavirus (MERS-Cov), Nipah virus (NIV), Dengue (DENV), Influenza (IAV) and Ebola virus (EBOV).

• Mechanical basis of viral fusion machine mediated membrane fusion.

Membrane fusion is a fundamental step for enveloped virus entry process, occurs when two separate lipid bilayer membranes undergoes merging into a single continuous bilayer. The activation energy barrier for this fusion reaction is ~40KT. Viral fusion proteins catalyses this process to overcome high kinetic barrier for fusion, by undergoing through a series of conformational changes and ultimately mechanically merge the two membrane together. So, the force generation during conformational refolding step of fusion protein draws the two membranes (cellular and viral) together. Here, we aim to understand the force generation mechanism of viral fusion protein at a single molecule level during fusion reaction in reconstituted systems. This will facilitate us in delineating the steps and sub-steps involved in viral fusion protein mediated membrane fusion process.

• Antibody mediated regulation of neutralization dynamics and structure based immunogen design.

Broadly neutralizing antibody targets the viral fusion proteins epitopes and inhibit the conformational transition required for the membrane fusion for entry. Here, we aim to imaging the antibody mediated neutralization dynamics of viral enveloped fusion proteins. This will inform us, the conformational landscape of fusion protein favored by the antibody. Based on our single molecule imaging for structural dynamics data, we will design recombinant viral fusion protein (Immunogen) which will favorably remain in pre-fusion conformation and will test in mouse model to see its efficacy in terms of eliciting neutralizing antibody.

• Development of novel single molecule imaging and manipulation methods.

Single molecule biophysical techniques are extremely powerful, since they are not subject to the steady state averaging artifacts of conventional bulk methods. Our lab applies state of art smFRET imaging set up and high-resolution optical tweezer set up for single molecule imaging and manipulation respectively. We aim to focus on continuous developments of novel imaging and manipulation methods and combination of both techniques will deliver new insights of life at molecular level.