Minimal reconstitution of membranous web induced by a vesicle–peptide sol–gel transition

• Main Authors: Ho, James Chin Shing, Steininger, Christoph, Hiew, Shu Hui, Kim, Min Chul, Reimhult, Erik, Miserez, Ali, Cho, Nam-Joon, Parikh, Atul N., Liedberg, Bo
• Other Authors: School of Chemical and Biomedical Engineering
• Format: Article
• Language: English
• Published: 2020
• Subjects: Engineering::Chemical engineering; RNA; Vesicle-peptide Sol-gel Transition
• Online Access: https://hdl.handle.net/10356/143163
• Institution: Nanyang Technological University

Positive strand RNA viruses replicate in specialized niches called membranous web within the cytoplasm of host cells. These virus replication organelles sequester viral proteins, RNA, and a variety of host factors within a fluid, amorphous matrix of clusters of endoplasmic reticulum (ER) derived vesicles. They are thought to form by the actions of a nonstructural viral protein NS4B, which remodels the ER and produces dense lipid-protein condensates. Here, we used in vitro reconstitution to identify the minimal components and elucidate physical mechanisms driving the web formation. We found that the N-terminal amphipathic domain of NS4B (peptide 4BAH2) and phospholipid vesicles (∼100-200 nm in diameter) were sufficient to produce a gel-like, viscoelastic condensate. This condensate coexists with the surrounding aqueous phase and affords rapid exchange of molecules. Together, it recapitulates the essential properties of the virus-induced membranous web. Our data support a novel phase separation mechanism in which phospholipid vesicles provide a supramolecular template spatially organizing multiple self-associating peptides thereby generating programmable multivalency de novo and inducing macroscopic phase separation.