Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

Abstract

Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here we use biophysical tools to study N-protein interactions with oligonucleotides of different length, examining the size, composition, secondary structure, and energetics of the resulting states. We observe formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant change in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within macromolecular condensates.

Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding

“…Noteworthy, the hydrodynamic radius is close to the one recently reported for N protein purified under similar conditions [35]. Also, these results are in line with literature data showing that N protein readily oligomerizes into dimers [36,37]. The urea-treated N showed a circular dichroism profile comparable to that of the protein treated with RNase A (Figure 1E), indicating that the urea treatment did not substantially alter the protein secondary structure.…”

“…The urea-treated N showed a circular dichroism profile comparable to that of the protein treated with RNase A (Figure 1E), indicating that the urea treatment did not substantially alter the protein secondary structure. Both curves are close to previous reported N protein CD profiles [37]. On the other hand, CD profile of untreated N protein presents a positive ellipticity at 250-260 nm which represents the contribution of the nucleic acids present in this sample, most likely RNA, Negative stain images produced from the urea-treated N samples showed various rounded particles with dimensions smaller than 5 nm (Figure 2A).…”

“…As reported recently [36,37] and shown in Figure 1C, the N protein devoid of RNA is a dimer in solution, which was consistent with the notion that dimers are the fundamental oligomeric unit of full-length N. The toroid-like particles observed in Figure 3B had dimensions ranging from 7 to 10 nm, which were smaller than the expected N protein diameter estimated from Rh (2 x 8.0 nm in diameter) or Rg (2 x 6.6 nm in diameter) as described above. Thus, we reasoned that these particles could represent N protein dimers in a more compact conformation due to the presence of RNA.…”

“…However, such RNA-binding sites could be simultaneously occupied by multiple short RNA segments, as suggested by the GC models and confirmed experimentally with polyA15 and polyT10 [37]. Likewise, longer RNA segments could drive protein oligomerization, as observed with polyA50 and polyT20 [37].…”

SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA

“…In this study, we showed that the SARS-CoV-2 N protein phase separates with nonspecific RNA sequences in vitro and the viscosity and shape of these condensates depend on the structure of the RNA substrate. Concurrent studies have shown that the SARS-CoV-2 N protein expressed in bacteria forms biomolecular condensates with RNA in vitro [8,31,36,38,46,54,56,63,[71][72][73][74]. We purified N protein from mammalian cells to recapitulate the posttranslational modifications that occur in infected human cells [10].…”