Structure and function of SARS-CoV-2 nucleocapsid protein measured using optical tweezers, confocal fluorescence, and AFM.

Abstract

The nucleocapsid (N) protein, one of four structural proteins expressed by SARS-CoV-2, primarily functions by binding and compacting the ∼30 kilobase viral RNA genome (>10 μm linear length) to enable packaging into viral particles (<100 nm diameter). We use optical tweezers to isolate a single ssDNA substrate and observe the binding and compacting function of N protein in real time. The compaction is a multistep process, with kinetics consistent with initial diffusion-limited binding of free protein to the substrate followed by a sudden stochastically triggered reorganization of the protein-DNA complex into a compacted form. Experiments utilizing truncated protein variants lacking either the ordered N or C terminal domains (NTD and CTD) and the intrinsically disordered linker demonstrate that initial binding is driven by the NTD while the formation of the compacted DNA structure requires the CTD. We further localize the binding of fluorescently labeled protein along the substrate using confocal imaging, showing N protein able to both uniformly coat the entire length of the substrate non specifically and subsequently form dense protein clusters through interprotein oligomerization. Finally, various DNA and RNA substrates of differing lengths and sequences are incubated with either full length or truncated N protein and imaged using AFM. Measurement of these nucleic acid-protein complexes reveal both the binding activity of N protein and its ability to remodel complex, folded nucleic acid structures. Our results help elucidate the biophysical mechanism through which N protein enables efficient packaging of the long vRNA under physiological conditions.