Proteoforms of the SARS-CoV-2 nucleocapsid protein are primed to proliferate the virus and attenuate the antibody response

Abstract

The SARS-CoV-2 nucleocapsid (N) protein is the most immunogenic of the structural proteins and plays essential roles in several stages of the virus lifecycle. It is comprised of two major structural domains: the RNA binding domain, which interacts with viral and host RNA, and the oligomerization domain which assembles to form the viral core. Here, we investigate the assembly state and RNA binding properties of the full-length nucleocapsid protein using native mass spectrometry. We find that dimers, and not monomers, of full-length N protein bind RNA, implying that dimers are the functional unit of ribonucleoprotein assembly. In addition, we find that N protein binds RNA with a preference for GGG motifs which are known to form short stem loop structures. Unexpectedly, we found that N undergoes autoproteolytic processing within the linker region, separating the two major domains. This process results in the formation of at least five proteoforms that we sequenced using electron transfer dissociation, higher-energy collision induced dissociation and corroborated by peptide mapping. The cleavage sites identified are in highly conserved regions leading us to consider the potential roles of the resulting proteoforms. We found that monomers of N-terminal proteoforms bind RNA with the same preference for GGG motifs and that the oligomeric state of a C-terminal proteoform (N156-419) is sensitive to pH. We used mass spectrometry to show that N binds to a monoclonal antibody raised against full-length N. No antibody interactions were detected for N proteoforms without C-terminal residues, therefore locating antigenic regions towards the C-terminus. We then tested interactions of the proteoforms with the immunophilin cyclophilin A, a key component in coronavirus replication. We found that N1-209 and N1-273 bind directly to cyclophilin A, an interaction that is abolished by the approved immunosuppressant drug cyclosporin A. We propose that the proteoforms generated via autoproteolysis evade antibody detection through removal of the antigenic C-terminus and facilitate interactions with structured RNA or cyclophilin thereby enabling the virus to proliferate.

Characterisation of protease activity during SARS-CoV-2 infection identifies novel viral cleavage sites and cellular targets with therapeutic potential

“…17/19, 69/71), consistent with a degree of further exoproteolytic processing. Some of these cleavage sites have subsequently been identified as autolysis products following extended incubations with N in vitro (26).…”

Multivalent binding of the partially disordered SARS-CoV-2 nucleocapsid phosphoprotein dimer to RNA

“…Furthermore, the few biophysical studies that incorporate RNA binding, either on individual domains or FL-N, have been limited to short segments of RNA or to synthesized polynucleotide RNA models rather than the viral RNA (11,12,14,43). These limited and conflicting reports are not surprising considering that the FL-N is a large multidomain protein with three long disordered segments and is prone to autoproteolysis (46) and aggregation as well as nonspecific binding to bacterial proteins and RNA during recombinant expression and purification, as we find in our studies. Our work here, to our knowledge, offers the first thorough characterization of FL-N and its binding to a 1000-nt segment of the viral RNA and presents a model that explains the importance of FL-N bivalent structure to enhancing its binding affinity on multivalent sites of a long segment of the viral RNA.…”

1H, 13C and 15N Backbone chemical shift assignments of the n-terminal and central intrinsically disordered domains of SARS-CoV-2 nucleoprotein

“…N2 has been described using both X-ray crystallography (Kang et al 2020;Peng et al 2020) and NMR spectroscopy (Dinesh et al 2020). The dimerization domain N4 has been described using X-ray crystallography (Ye et al 2020;Peng et al 2020) and N has been shown to form higher order oligomers via N4 (Ye et al 2020;Zeng et al 2020;Lutomski et al 2020). The remaining 164 amino acids comprising N1, N3 and N5 are predicted to be intrinsically disordered, but despite evidence that these domains are essential for function in the related Mouse Hepatitis virus (Keane and Giedroc 2013), there is currently no atomic resolution information describing their behaviour in solution.…”

“…Peptides representing the SR region of N3 in its phosphorylated and non-phosphorylated forms were also described using NMR spectroscopy (Savastano et al 2020 ). Mass spectrometry has also revealed a number of auto-catalytic sites in N, two of which are present in N3 (Lu et al 2020 ; Lutomski et al 2020 ).…”