Characterization of SARS-CoV-2 N protein reveals multiple functional consequences of the C-terminal domain

Abstract

SummaryNucleocapsid protein (N) is the most abundant viral protein encoded by SARS-CoV-2, the causative agent of COVID-19. N plays key roles at different steps in the replication cycle and is used as a serological marker of infection. Here we characterize the biochemical properties of SARS-CoV-2 N. We define the N domains important for oligomerization and RNA binding that are associated with spherical droplet formation and suggest that N accessibility and assembly may be regulated by phosphorylation. We also map the RNA binding interface using hydrogen-deuterium exchange mass spectrometry. Finally, we find that the N protein C-terminal domain is the most immunogenic by sensitivity, based upon antibody binding to COVID-19 patient samples from the US and Hong Kong. Together, these findings uncover domain-specific insights into the significance of SARS-CoV-2 N and highlight the diagnostic value of using N domains as highly specific and sensitive markers of COVID-19.

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

“…With a 20nt NA ligand, we observe very high affinity binding, consistent with fluorescence polarization results by Wu et al (Wu et al, 2020). Our data show that cross-linking of N-protein dimers by a single 20mer can occur.…”

The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA

“…The solution environment and types of RNA used in our experiments are very different from the cytoplasm and viral RNA. However, similar results have been obtained in published and unpublished work by several other groups under a variety of conditions, including via in cell experiments [20][21][22][23][24][25][26][27] . Taken together, these results demonstrate that N protein can undergo bona fide phase separation, and that N protein condensates can form in cells.…”

“…Finally, given we observed phase separation with poly(rU), the behavior we are observing is likely driven by relatively nonspecific protein:RNA interactions. In agreement, work from a number of other groups has also established this phenomenon across a range of solution conditions and RNA types [20][21][22][23][24][25][26][27] .…”

“…Furthermore, the equivalent region was identified in SARS coronavirus as a nuclear export signal (NES), such that we suspect this too is a classical Crm1-binding leucine-rich NES 103 . Jack et al identified helix H4 as enriched for homotypic cross-links in the context of droplets, supporting a model in which this region promotes protein:protein interactions, an interpretation corroborated by hydrogen-deuterium exchange mass spectrometry on RBD-LINK in the dilute phase 20,26 .…”

Evaluation of “Caterina assay”: An Alternative Tool to the Commercialized Kits Used for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Identification

“…However, Chu et al found that the N gene assay was approximately 10 times more sensitive than the RdRp (ORF-1b) gene assay in detecting positive clinical specimens from two patients via rRT-PCR [ 30 ]. It has been reported that the N gene that encoded an internal N nucleoprotein protein was one of the most abundant proteins and was highly immunogenic and less prone to genetic changes [ 31 , 32 ]. However, recent study showed that some structural proteins, like the E protein, have low mutation rates across the residue sequence while other viral components, such as the Spike (S) [ 33 ] or the N protein showed higher degrees of variability [ 34 ].…”