BPS2026 – Multi-step folding of DNA by SARS-CoV-2 nucleocapsid protein

Folding of nucleic acids by the SARS-CoV-2 nucleocapsid protein (N protein) is an essential step in viral assembly and replication. To fold nucleic acids, N protein uses three interactions. First, N protein recognizes sequence-specific packaging signals on the genome. Second, N protein can use nonspecific electrostatic interactions to bind along the nucleic acid. Finally, N protein can oligomerize with other N proteins along the nucleic acid, further compacting it. We are interested in nonspecific interactions between the N protein and nucleic acid since these conserved interactions may be a promising target for broad-spectrum antivirals. To isolate these nonspecific interactions, we image N protein folding double-stranded DNA (dsDNA) instead of single-stranded RNA. This eliminates sequence-specific effects. Specifically, we image dsDNA of varying lengths (309 bp, 638 bp, and 5882 bp) with increasing concentrations (1–100 nM) of N protein using an atomic force microscope (AFM). Preliminary data show that N protein binds and folds dsDNA in a multi-step pathway by increasing dsDNA flexibility. Our work provides new mechanistic insight into nucleic acid compaction and genome packaging.