UC Riverside

The SARS-CoV-2 structural proteins, membrane (M), envelope (E), nucleocapsid (N), and spike (S), orchestrate all stages of viral assembly, budding, and release, yet the molecular mechanisms by which they remodel host membranes and drive particle formation remain incompletely understood. This dissertation presents a series of biophysical and biochemical investigations into the structure, assembly, and membrane interactions of SARS-CoV-2 structural proteins, with the goal of elucidating the physical principles underlying coronavirus particle formation. In the first study, a SUMO-tag-based Escherichia coli expression system was developed to produce large quantities of full-length, native M protein. Recombinant M protein dimers were inserted into lipid bilayers and characterized by atomic force microscopy (AFM), cryo-electron microscopy (cryo-EM), and molecular dynamics (MD) simulations. AFM and cryo-EM imaging revealed M protein dimers exclusively in the compact "short" conformation, while MD simulations confirmed localized membrane thinning and curvature induction in the vicinity of the dimers. Coarse-grained simulations further demonstrated that endodomain interactions among M protein dimers, combined with line tension from membrane thinning, can drive viral assembly and budding. The expression platform developed for M protein was extended to the SARS-CoV-2 envelope (E) protein, yielding recombinant E protein in quantities sufficient for membrane reconstitution. Collaborative experiments using this purified E protein demonstrated that E forms multiple coexisting oligomeric species in ERGIC-mimetic lipid bilayers, induces membrane thinning and curvature, and drives concentration-dependent morphological transitions in giant vesicles. Real-time confocal microscopy further demonstrated that introduction of N protein–RNA ribonucleoprotein complexes to giant vesicles containing both M and E triggers rapid inward vesicle budding, establishing a minimal reconstituted system for SARS-CoV-2 particle assembly in vitro.The second study examined the production and characterization of SARS-CoV-2 virus-like particles (VLPs) in mammalian cells. Expression of all four structural proteins at ratios reflecting viral mRNA abundance in infected cells yielded VLPs validated by Western blotting, AFM, and dynamic light scattering. RNA extracted from purified VLP fractions showed a size distribution dominated by small RNAs, consistent with selective packaging rather than nonspecific cellular RNA incorporation.Finally, a study of p38 MAPK signal transduction demonstrated that the central human stress response kinase encodes information about experienced stresses as frequency oscillations of its activation state, and that downstream specificity is achieved through bioresonance between p38 and its target substrates.