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

Abstract 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 (gRNA12k). Our results demonstrate that the 5’ and 3’ regions of gRNA12k 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 gRNA12k. 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. Significance SARS-CoV-2 organizes its exceptionally long genomic RNA (gRNA) having about 30,000 nt into 35–40 granular ribonucleoprotein (RNP) units for viral packaging. It has been assumed that the nucleocapsid (N) protein drives the formation of the RNP granules. In this study, we challenge this prevailing view by demonstrating that a specific region of the gRNA sequence inherently encodes the information to fold into a compact, granular architecture independently of any proteins. Unexpectedly, we found that the N protein partially melts the autonomous structures, suggesting that it acts as an RNA chaperone to facilitate flexible genome assembly. Our findings redefine the interplay between viral proteins and gRNA, offering a new perspective on the mechanism of coronavirus replication. Competing Interest Statement The authors have declared no competing interest. Footnotes The authors declare no competing interest.