Molecular insight into the specific interactions of the SARS‐Coronavirus‐2 nucleocapsid with RNA and host protein

Abstract

The severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) nucleocapsid protein is the most abundantly expressed viral protein during infection where it targets both RNA and host proteins. However, identifying how a single viral protein interacts with so many different targets remains a challenge, providing the impetus here for identifying the interaction sites through multiple methods. Through a combination of nuclear magnetic resonance (NMR), electron microscopy, and biochemical methods, we have characterized nucleocapsid interactions with RNA and with three host proteins, which include human cyclophilin‐A, Pin1, and 14–3–3τ. Regarding RNA interactions, the nucleocapsid protein N‐terminal folded domain preferentially interacts with smaller RNA fragments relative to the C‐terminal region, suggesting an initial RNA engagement is largely dictated by this N‐terminal region followed by weaker interactions to the C‐terminal region. The nucleocapsid protein forms 10 nm ribonuclear complexes with larger RNA fragments that include 200 and 354 nucleic acids, revealing its potential diversity in sequestering different viral genomic regions during viral packaging. Regarding host protein interactions, while the nucleocapsid targets all three host proteins through its serine‐arginine‐rich region, unstructured termini of the nucleocapsid protein also engage host cyclophilin‐A and host 14–3–3τ. Considering these host proteins play roles in innate immunity, the SARS‐CoV‐2 nucleocapsid protein may block the host response by competing interactions. Finally, phosphorylation of the nucleocapsid protein quenches an inherent dynamic exchange process within its serine‐arginine‐rich region. Our studies identify many of the diverse interactions that may be important for SARS‐CoV‐2 pathology during infection.

A specific phosphorylation-dependent conformational switch of SARS-CoV-2 nucleoprotein inhibits RNA binding

“…In order to investigate possible changes in backbone conformation, 13 C α chemical shifts of pSR were compared to recently proposed random coil chemical shift values ( 64 , 65 ) for pN234(III), indicating that no significant induction of secondary structure results from hyperphosphorylation (supporting figure 1), as previously proposed for PKA-phosphorylated N (residues 1-209) ( 45 ).…”

“…The broadly phosphorylating protein kinase A (PKA) has been exploited to phosphorylate N in a single step, including via co-expression with N ( 45 ), and notably to investigate the impact on binding of the host factor 14-3-3 ( 66 ). Incubation of N with PKA in vitro results in phosphorylation of six sites in the SR region of N234 (supporting figure 4) whose backbone resonances were assigned, namely pS180, pS193, pS194, pS197, pS201 and pS202, although broader phosphorylation was observed by co-expression with PKA ( 45 , 66 ). This 6-fold phosphorylated form of N234 nevertheless maintains its binding to RNA (figure 5B).…”

“…Crucially however, phosphorylation by PKA has no measurable impact on RNA binding, suggesting that the pattern resulting from the physiologically relevant kinases is indeed specific for inhibition, either by binding tighter to the surface of N2, by occupying more RNA binding sites on N2, or by ensuring a uniform electrostatic screening of the RNA-binding surface. A recent study provided evidence that phosphorylation of N(1–209) with PKA resulted in chemical shifts of the β-hairpin (91–105) of N2 ( 45 ). Our measurements confirm this observation.…”

“…N4 forms a highly stable dimeric structure involving two domain-swapped b-hairpins. Although N2 is known to represent the main RNA binding domain, secondary RNA binding sites have been proposed to exist in both N3 (39,40,26,36) and N4 (39,(41)(42)(43)(44)(45), an observation supported by assembly of truncation mutants of N into large supramolecular complexes of the dimensions of RNPs (46). N5 has also been associated with assembly into higher order oligomeric states (47).…”

The immune-evasive proline-283 substitution in influenza nucleoprotein increases aggregation propensity without altering the native structure

“…Since HSF1 regulates the expression of a broad change of proteostasis factors ( 45 – 47 ), potential candidates include heat shock proteins that can bind to the partially folded conformation and prevent aggregation, and folding enzymes such as peptidyl prolyl isomerases that catalyze the often rate-limiting steps of protein folding. It has been observed that the NP of severe acute respiratory syndrome coronavirus 2 virus heavily interacts with peptidyl prolyl isomerases, such as cyclophilin A and peptidyl-prolyl cis - trans isomerase never-in-mitosis-gene-A-interacting 1 (Pin1), although it is not yet clearly known whether these enzymes play an important role in NP folding ( 48 ). The chaperones that can rescue Pro 283 NP may have homologous proteins in E. coli, because Pro 283 NP could be recombinantly expressed in E. coli without a noticeable yield decrease compared to Ser 283 NP.…”