key: cord-0978233-8z9wf8vz
authors: Tugaeva, Kristina V.; Hawkins, Dorothy E. D. P.; Smith, Jake L. R.; Bayfield, Oliver W.; Ker, De-Sheng; Sysoev, Andrey A.; Klychnikov, Oleg I.; Antson, Alfred A.; Sluchanko, Nikolai N.
title: The mechanism of SARS-CoV-2 nucleocapsid protein recognition by the human 14-3-3 proteins
date: 2021-01-28
journal: bioRxiv
DOI: 10.1101/2020.12.26.424450
sha: d819c335833318b859cf79115170d1061691ebc3
doc_id: 978233
cord_uid: 8z9wf8vz

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains numerous phosphorylation sites located within unstructured regions. Binding of phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to ~1% of expressed proteins during infection. Although the association between 14-3-3 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, its molecular mechanism and the specific critical phosphosites are unknown. Here, we show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various efficiencies and deduce the apparent KD to selected isoforms, showing that these are in a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to Ser197, which is conserved among related zoonotic coronaviruses and located within the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the SR-rich region, while hijacking cellular pathways by 14-3-3 sequestration. As such, the assembly may represent a valuable target for therapeutic intervention. Highlights SARS-CoV-2 nucleocapsid protein (N) binds to all seven human 14-3-3 isoforms. This association with 14-3-3 strictly depends on phosphorylation of N. The two proteins interact in 2:2 stoichiometry and with the Kd in a μM range. Affinity of interaction depends on the specific 14-3-3 isoform. Conserved Ser197-phosphopeptide of N is critical for the interaction.

The coronavirus nucleocapsid protein (N) controls viral genome packaging and contains 26 numerous phosphorylation sites located within unstructured regions. Binding of 27 phosphorylated SARS-CoV N to the host 14-3-3 protein in the cytoplasm was reported 28 to regulate nucleocytoplasmic N shuttling. All seven isoforms of the human 14-3-3 are 29 abundantly present in tissues vulnerable to SARS-CoV-2, where N can constitute up to 30 ~1% of expressed proteins during infection. Although the association between 14-3-3 31 and SARS-CoV-2 N proteins can represent one of the key host-pathogen interactions, 32 its molecular mechanism and the specific critical phosphosites are unknown. Here, we 33 show that phosphorylated SARS-CoV-2 N protein (pN) dimers, reconstituted via 34 bacterial co-expression with protein kinase A, directly associate, in a phosphorylation-35 dependent manner, with the dimeric 14-3-3 protein, but not with its monomeric mutant. 36 We demonstrate that pN is recognized by all seven human 14-3-3 isoforms with various 37 efficiencies and deduce the apparent K D to selected isoforms, showing that these are in 38 a low micromolar range. Serial truncations pinpointed a critical phosphorylation site to 39 Ser197, which is conserved among related zoonotic coronaviruses and located within 40 the functionally important, SR-rich region of N. The relatively tight 14-3-3/pN association 41 can regulate nucleocytoplasmic shuttling and other functions of N via occlusion of the Introduction 47 The new coronavirus-induced disease, COVID19, has caused a worldwide health 48 crisis with more than 90 million confirmed cases and 1.9 million deaths as of January 49 2021 [1] . The pathogen responsible, Severe Acute Respiratory Syndrome Coronavirus 2 uses angiotensin-converting enzyme 2 (ACE2) as entry receptor [5] . The ACE2 56 expression roughly correlates with the evidenced SARS-CoV-2 presence in different 57 tissue types, which explains the multi-organ character of the disease [6] (Fig. 1) . 58 In contrast to multiple promising COVID19 vaccine clinical trials [7-9], treatment of 59 the disease is currently limited by the absence of approved efficient drugs [10] . The 60 failure of several leading drug candidates in 2020 warrants the search for novel 61 therapeutic targets including not only viral enzymes, but also heterocomplexes involving 62 viral and host cell proteins. Unravelling mechanisms of interaction between the host and 63 pathogen proteins may provide the platform for such progress. 64 The positive-sense single-stranded RNA genome of SARS-CoV-2 coronavirus of human coronaviruses indicated that N might be the major factor conferring the 69 enhanced pathogenicity to SARS-CoV-2 [4] . N represents the most abundant viral 70 protein in the infected cell [12] [13] [14] , with each assembled virion containing approximately 71 one thousand molecules of N [15] . Given that each infected cell can contain up to 10 5 72 virions (infectious, defective and incomplete overall) [14] , the number of N molecules in 73 an infected cell can reach 10 8 , accounting for ~1% of a total number of cellular proteins 74 (~10 10 [16] ). 75 The N protein interacts with viral genomic RNA, the membrane (M) protein and 76 self-associates to provide for the efficient virion assembly [17] [18] [19] . It consists of two 77 structured domains and three unstructured regions, including a functionally important 78 central Ser/Arg-rich region ( Fig. 2A ) [20] [21] [22] . Such organization allows for a vast 79 conformational change, which in combination with positively charged surfaces [23], 80 facilitates nucleic acid binding [24] . Indeed, the crystal structure of the N-terminal 81 domain (NTD) reveals an RNA binding groove [25] [26] [27] , while crystal structures of the C- 82 terminal domain (CTD) show a highly interlaced dimer with additional nucleic acid 83 binding capacity [28, 29] . The N protein shows unusual properties in the presence of 84 RNA, displaying concentration-dependent liquid-liquid phase separation [ treatment with a kinase inhibitor cocktail eliminated the N/14-3-3 interaction, whereas 100 inhibition of 14-3-3θ expression by siRNA led to accumulation of N protein in the 101 nucleus [37] . These data suggest that 14-3-3 proteins directly shuttle SARS-CoV N 102 protein in a phosphorylation-dependent manner: a role which may be universal for N 103 proteins of all coronaviruses, including SARS-CoV-2. However, the molecular 104 mechanism of the 14-3-3/N interaction remains ill-defined. 105 14-3-3 proteins are amongst the top 1% of highest-expressed human proteins in 106 many tissues, with particular abundance in tissues vulnerable to SARS-CoV-2 infection 107 including the lungs, gastrointestinal system and brain [40, 41] (Fig. 1 ). 14-3-3 proteins 108 recognize hundreds of phosphorylated partner proteins involved in a magnitude of 109 cellular processes ranging from apoptosis to cytoskeleton rearrangements [42, 43] . 110 Human 14-3-3 proteins are present in most of the tissues as seven conserved 111 "isoforms" (β, Fig. 1) , with all-helical topology, forming dimers 112 possessing two identical antiparallel phosphopeptide-binding grooves, located at ~35 Å 113 distance from each other [44] . By recognizing phosphorylated Ser/Thr residues within 114 the structurally flexible (R/K)X 2-3 (pS/pT)X(P/G) consensus motif [44, 45], 14-3-3 binding 115 is known to regulate the stability of partner proteins, their intracellular localization and 116 interaction with other factors [46] . In addition to their high abundance in many tissues 117 susceptible to SARS-CoV-2 infection (Fig. 1 ), 14-3-3 proteins were reported as one of 118 nine key host proteins during SARS-CoV-2 infection [47], indicating their potential 119 association with viral proteins. 120 In this work, we dissected the molecular mechanism of the interaction between 121 SARS-CoV-2 N and human 14-3-3 proteins. SARS-CoV-2 N protein containing several 122 phosphosites reported to occur during infection, was produced using the efficient 123 Escherichia coli system that proved successful for the study of polyphosphorylated 124 proteins [48] . We have observed the direct phosphorylation-dependent association 125 between polyphosphorylated SARS-CoV-2 N and all seven human 14-3-3 isoforms and 126 determined the affinity and stoichiometry of the interaction. Series of truncated mutants 127 of N localized the key 14-3-3-binding site to a single phosphopeptide residing in the 128 functionally important SR-rich region of N. These findings suggest a topology model for 129 the heterotetrameric 14-3-3/pN assembly occluding the SR-region, which presents a 130 feasible target for further characterization and therapeutic intervention. phosphorylated locus is the SR-rich region ( Fig. 2A conservation between SARS-CoV and SARS-CoV-2 N proteins, many phosphosites 160 identified in the PKA-co-expressed N are likely shared by SARS-CoV N ( Fig. 2A) . 161 Importantly, many identified phosphosites lie within the regions predicted to be 162 disordered, and contribute to predicted 14-3-3-binding motifs, albeit deviating from the 163 optimal 14-3-3-binding sequence RXX(pS/pT)X(P/G) [44] ( Fig. 2A 214 We then questioned whether the interaction with pN is preserved for other human 215 14-3-3 isoforms. Analytical SEC clearly showed that the phosphorylated SARS-CoV-2 N 216 can be recognized by all seven human 14-3-3 isoforms, regardless of the presence of a 217 His-tag or disordered C-terminal tails on the corresponding 14-3-3 constructs (Fig. 4A ). 218 However, the efficiency of complex formation differed for each isoform. Judging by the 219 repartition of 14-3-3 between free and the pN-bound peaks, the apparent efficiency of 220 pN binding was higher for 14-3-3γ, 14-3-3η, 14-3-3ζ and 14-3-3ε, and much lower for 221 14-3-3β, 14-3-3τ and 14-3-3σ, in a roughly descending order (Fig. 4A ). The interaction 222 also appeared dependent on the oligomeric state of 14-3-3, since the monomeric 223 mutant form of 14-3-3ζ, 14-3-3ζm-S58E [56] (apparent Mw 29 kDa) showed virtually no 224 interaction relative to the wild-type dimeric 14-3-3ζ counterpart (apparent Mw 58 kDa), mechanism could be observed for 14-3-3ε, however in this case we could achieve pN. 419 saturation only at much higher 14-3-3ε concentrations ( Fig. 5B and C), and the 239 resulting apparent K D was ~7 times higher than for 14-3-3γ (Fig. 5C ). Nevertheless, 240 once again the stoichiometry was close to 2:2. These findings strongly disfavor the 241 earlier hypothesis that 14-3-3 binding affects dimerization status of N [57]. 242 We further asked what are the specific regions of SARS-CoV-2 N that are 243 responsible for interaction with human 14-3-3. 2:2 complex (one 14-3-3 dimer with two pN monomers) is not observed. The well-298 defined 2:2 stoichiometry of the 14-3-3γ complex with the full-length pN (Fig. 3 ) 299 suggests that the dimeric pN is anchored using two equivalent, key 14-3-3-binding sites, 300 each located in a separate subunit of N. It is tempting to speculate that, in the absence (Supplementary table 1) . 326 We conceived stepwise truncations to remove the most probable 14-3-3-binding 327 phosphosites, aiming to identify the iteration at which binding (observed for the pN.1-328 211 construct) ceased. 14-3-3 can bind incomplete consensus motifs at the extreme C- None of the truncated mutants interacted with 14-3-3γ in the unphosphorylated state 340 (Fig. 8B) . No binding to 14-3-3γ was detected with phosphorylated N.1-179 (Fig. 8C) and only very limited binding could be observed with phosphorylated N. (Fig. 8C) . 342 This strongly indicated that all phosphosites of the 1-196 segment (including at least 343 three phosphosites within the SR-rich region, i.e., Ser180, Ser188 and Ser194, see Fig.   344 8A) are dispensable for 14-3-3 binding and at most could contribute only as auxiliary 345 sites (as suggested by the scheme in Fig. 7B ). This narrowed the 14-3-3-binding region 346 within SARS-CoV-2 N down to 15 residues from 196 to 211, leaving only two possible 347 sites centered at Ser197 and Thr205 (Fig. 8A) . 348 By contrast, pN.1-204 showed a similar interaction with 14-3-3γ to that of pN.1-211 349 (Fig. 8C ). Although we cannot fully exclude that Thr205 phosphosite contributes to 14-3-350 3 binding in the context of the full-length pN, it is clearly not critical for 14-3-3 351 recruitment, unlike Ser197. Moreover, in contrast to Thr205, Ser197 is preserved in 352 most related coronavirus N proteins (see Fig. 2A, Fig. 9 ). binding sites ( Fig. 2A) . 375 Biochemical analysis confirmed that polyphosphorylated N is competent for 376 binding to all seven human 14-3-3 isoforms ( Fig. 3 and 4) , but revealed remarkable 377 variation in binding efficiency between them (Fig. 4) . This was supported by the 378 quantified affinities to two selected isoforms, 14-3-3γ (K D of 1.5 µM) and 14-3-3ε (K D of 379 10.7 µM) (Fig. 5 ). Our observations are in line with the recent finding that 14-3-3γ and 380 14-3-3η systematically bind phosphopeptides with higher affinities than 14-3-3ε and 14- Supplementary Fig. 3) , which could potentially lower the binding affinity to 401 14-3-3 dimers in light of the 2:2 stoichiometry. Nonetheless, sufficient binding was 402 clearly observed between the dimeric 14-3-3γ and the monomeric N-terminal constructs 403 ( Fig. 6 and 7) . 404 Truncation of the SR-rich region streamlined the search for the 14-3-3-binding 405 site to the 15-residue stretch of amino acids 196-211. This sequence hosts two 406 principally similar potential 14-3-3-binding sites, RNpS 197 TP and RGpT 205 SP ( Fig. 2A   407 and C). Importantly, the proximity of these sites rules out bidentate binding to the 14-3-3 408 dimer: 14-3-3 binds in an antiparallel manner requiring a minimum of 13-15 residues between phosphosites on a single peptide [44, 68] . Thus, binding to Ser197 and Thr205 410 sites must be mutually exclusive. 411 The markedly different binding between pN.1-204 and pN.1-196 to 14-3-3 (Fig. 8 ) 412 prompted us to propose Ser197 as the critical phosphosite. This finding aided the 413 design of a topology model for the complex (Fig. 9A) , in which the 14-3-3 dimer is 414 anchored by two identical Ser197 phosphosites from the SR-rich region in the two 1-419, N.1-211, N.1-238, N.212-419 versions of human 14-3-3η (Uniprot ID Q04917), 14-3-3β (Uniprot ID P31946), 14-3-3ε 498 (Uniprot ID P62258), 14-3-3τ (Uniprot ID P27348), 14-3-3γ (Uniprot ID P61981) and 14-499 3-3σ (Uniprot ID P31947) devoid of the short disordered segment at the C terminus, cloned into pProExHtb vector and carrying TEV-cleavable His 6 -tags on their N-terminal 501 ends were obtained as described previously [66, 85] . The monomeric mutant form of 502 untagged full-length human 14-3-3ζ carrying monomerizing amino acid substitutions 503 and pseudophosphorylation in the subunit interface (14-3-3ζm-S58E) was obtained as 504 before [56] . 505 14-3-3γ, 14-3-3ζ and 14-3-3ζm-S58E were expressed and purified using 506 ammonium sulfate fractionation and column chromatography as previously described presence (**) in various tissues of COVID19 patients based on the data from [6] , shown 893 with abundances (indicated in ppm, part per million, i.e., one molecule of a given protein 894 per 1 million of all proteins from a given tissue) of the seven human 14-3-3 isoforms, 895 extracted from the PAXdb database [40] . Different tissues are shown in the order 896 corresponding to the SARS-CoV-2 presence, starting, at the top, from the highest virus 897 presence [6] . The shown relative scale of ACE2 expression is also taken from [6] . The 898 total abundance of the seven human 14-3-3 isoforms in a given tissue and the average 899 abundance of an isoform in 12 selected tissues are also indicated. The latter values 900 were used for ordering the data for 14-3-3 isoforms, left to right, from the highest 901 average abundance (14-3-3ζ; 2423 ppm, or ~0.24%) to the lowest average abundance 902 (14-3-3η; 575 ppm, or ~0.06%). Note that the average abundance of all seven 14-3-3 903 proteins in three tissues with the highest SARS-CoV-2 presence (oral cavity, 904 gastrointestinal tract, lungs) reaches 1.21% of all proteins. 

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Some phosphosites (red spheres and labeled) are predicted as 953 suboptimal 14-3-3 binding sites (green arrows). B. A Phos-tag gel showing that bacterial 954 co-expression of the full-length N with PKA yields polyphosphorylated protein. C. A 955 fragmentation spectrum of the representative phosphopeptide carrying phosphorylation 956 at Ser197 and Thr205

Absorbance spectra show that both recombinant 958 unphosphorylated and PKA-phosphorylated N proteins elute from the Ni-affinity column 959 bound to random E.coli nucleic acid

with multi-angle light scattering detection (SEC-MALS) and SDS-PAGE of the

Binding affinity. pN at a fixed concentration (~10

Titration of pN with 14-3-3γ. B. Titration of pN with 14-3-3ε. Changes of the 1056 elution profiles associated with the increasing 14-3-3 concentration are shown by 1057 arrows. C. Binding curves used for apparent K D determination

Interaction of human 14-3-3γ with individual SARS-CoV-2 N protein 1092 domains. A. Schematic representation of the N protein sequence with the main 1093 domains/regions highlighted. Yellow indicates NES, small red circles designate 1094 phosphosites within the two corresponding phosphorylatable loci. B. SEC profiles of 1095 individual 14-3-3γ (black traces)

unphosphorylated N fragments, the bottom row corresponds to their phosphorylated 1099 counterparts, as indicated on each panel. In all cases, 50 the case of N.247-364 and 1101 pN

Note that the well-defined complexes with 14-3-3 1102 were observed only for pN.1-211 and pN.1-238 fragments. The experiment was 1103 repeated twice and the most typical results are presented

Interaction of human 14-3-3γ with the phosphorylated monomeric SARS

SEC-MALS analysis of individual pN, 14-3-3γ and their 1124 complex formed at the indicated molar excess of pN. Protein concentration, MALS-1125 derived Mw distributions across the peaks and the respective average Mw values are 1126 indicated. Note that the difference shown by a two-headed arrow roughly corresponds 1127 to a pN.1-238 monomer mass

Localization of the 14-3-3-binding sites within the SR-rich region of the 1156 SARS-CoV-2 N protein. A. Sequence of the SR-rich region showing potential 14-3-3-1157 binding sites (green and blue font) and the truncations designed to exclude one or two 1158 main 14-3-3-binding sites (blue font). # denotes the designed C-terminus

Each graph 1161 contains SEC profiles for the individual 14-3-3 (black line), individual N or pN construct 1162

Note that only the phosphorylated mutants interacted with 14-3-3γ 1166 and that only pN.1-211 and pN.1-204 formed a defined complex with 14-3-3γ. Column: 1167 Superdex 200 Increase 5/150, flow rate: 0.45 ml/min. The experiment was repeated 1168 twice and the most typical results are presented

A topology model for the 1200 complex of SARS-CoV-2 N protein dimer with the dimer of human 14-3-3 illustrating the 1201 occlusion of the SR-rich region. B. Local alignment of SR-rich regions of the most 1202 similar coronavirus N proteins in order of descending sequence identity (s.id.) 1203 determined using entire N protein sequences. Alignment was performed using Clustal 1204 omega and visualized using Mview