Decoding the Membrane-Associated Assembly of Viral Replication Complex in Live Cells Using Nanotopography Engineering

Many detrimental RNA viruses, including SARS-COV-1 & -2, Dengue virus, and Zika virus, have been found in generating specialized membrane compartments in host cells to provide a protective microenvironment for viral genome replication. These membrane compartments only require a few non-structural proteins (nsPs) of viruses to form but exist in a variety of shapes with curvatures ranging from tens to hundreds of nanometers. However, the molecular mechanism of how nsPs bind and assemble around the membrane to induce or stabilize such highly curved structures in the host cells is largely unclear. It is mainly due to the technical challenges to manipulate the interaction between nsPs and nanoscale curved membranes in live cells that are too small to resolve by conventional fluorescence microscopy. In this proposal, we propose to introduce nanofabrication-enabled membrane topography engineering in cells to dissect the mechanism of how viral nsPs dynamically assemble on the curved membranes. Specifically, we will utilize the state-of-the-art high-resolution nanofabrication technologies (e.g. electron beam lithography, reactive ion etching, and super-resolution imaging) to engineer the host cell membrane into a variety of shape designs in nanoscale resolution and identify the key regulators for efficient assembly of functional viral RC in live cells. Specifically, we still focus on the following three research aims: 1) Identify viral nsPs and their domains that can sense and modulate their interaction with the curved membrane in cells; 2) Validate the key regulatory factors for curvature sensing and generation by reconstituting the membrane binding of viral nsPs in vitro; 3) Evaluate the impact of membrane topography on the formation and function of viral replication compartments with both nsPs and RNA template. Successful implementation of this project will provide new mechanistic insights on how membrane topography facilitates or interferes with the assembly of nsPs for functional viral replications in host cells. The evaluated nanochip designs will be critical to establishing a new platform for viral replication characterization in cells and in vitro, and the key regulators identified can serve as new targets for anti-viral therapeutics development.