A Structural Domain in the genomic RNA of SARS-CoV-2 Folds into a Compact Granular Structure without the N protein: A Single-Molecule Fluorescence Spectroscopic Investigation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) packages its single-stranded genomic RNA (gRNA) having about 30,000 nucleotides into virions by forming 35–40 granular ribonucleoprotein (RNP) units. Each RNP unit has a diameter of ∼15 nm. While it is generally assumed that the assembly of these RNPs is driven by the binding of the nucleocapsid (N) protein to the gRNA in the cytoplasm, the precise molecular mechanism remains to be fully elucidated. In this study, we develop an experimental strategy based on single-molecule fluorescence and fluorescence correlation spectroscopies to examine the formation of long-range base pairing within a candidate structural domain corresponding to nt 12230-12686 of the gRNA (gRNA _12k ). Our results demonstrate that the 5’ and 3’ regions of gRNA _12k autonomously form long-range base pairing in near-physiological buffers containing mono- and divalent cations, independently of the N protein. This domain possesses an extensive secondary structure, is compact, and can unfold and refold reversibly upon heat treatment and cooling. Notably, the addition of the N protein melts the long-range base pairs, and causes the aggregation of multiple molecules of gRNA _12k . Based on these observations, we propose a refined mechanism for the genome assembly in SARS-CoV-2: gRNA initially forms autonomous granular structures, which are subsequently reorganized and condensed by the N protein to chaperone the assembly of the entire gRNA.