key: cord-255170-bp3irxlh
authors: Mark, John; Li, Xuguang; Cyr, Terry; Fournier, Sylvie; Jaentschke, Bozena; Hefford, Mary Alice
title: SARS coronavirus: Unusual lability of the nucleocapsid protein
date: 2008-12-12
journal: Biochemical and Biophysical Research Communications
DOI: 10.1016/j.bbrc.2008.09.153
sha: 
doc_id: 255170
cord_uid: bp3irxlh

Abstract The severe acute respiratory syndrome (SARS) is a contagious disease that killed hundreds and sickened thousands of people worldwide between November 2002 and July 2003. The nucleocapsid (N) protein of the coronavirus responsible for this disease plays a critical role in viral assembly and maturation and is of particular interest because of its potential as an antiviral target or vaccine candidate. Refolding of SARS N-protein during production and purification showed the presence of two additional protein bands by SDS–PAGE. Mass spectroscopy (MALDI, SELDI, and LC/MS) confirmed that the bands are proteolytic products of N-protein and the cleavage sites are four SR motifs in the serine–arginine-rich region—sites not suggestive of any known protease. Furthermore, results of subsequent testing for contaminating protease(s) were negative: cleavage appears to be due to inherent instability and/or autolysis. The importance of N-protein proteolysis to viral life cycle and thus to possible treatment directions are discussed.

Severe acute respi ra tory syn drome (SARS) is an atyp i cal pneu mo nia first described in Novem ber 2002 [1] . The caus a tive agent, the SARS coro na vi rus, belongs to the cor o na vi ra dae fam ily of envel oped, positive-sense RNA viruses and poss e ses four structural pro teins. The nucle o cap sid (N) pro tein has been found to bind to a spe cific pack ag ing-sig nal motif on the viral RNA; it is the interac tion of this pro tein-RNA com plex with the mem brane (M), envelope (E) and spike (S) pro teins that leads to bud ding through the cell mem brane and virus mat u ra tion. The N-pro tein is of par tic u lar inter est because of its potential as a vac cine can di date [2, 3] , as a diag nos tic marker for SARS [4, 5] , and because it appears to play a crit i cal role in the per tur ba tion of sev eral host cell pro cesses during infec tion [6] .

SARS N-pro tein is a 432 amino acid, 46 kDa pro tein with the high pI (10.1) and high con tent of basic amino acids char ac ter istic of many DNA-or RNA-bind ing pro teins. Some atyp i cal char acter is tics of the N-pro tein include a low per cent age of hydro pho bic amino acids and an absence of cys teine res i dues [7] . The peculiar com po si tion of the pro tein may be impor tant for the RNA-bind ing prop er ties, but the absence of strong intra mo lec u lar inter ac tions also sug gests that, in con trast to other viral struc tural pro teins, the struc ture of SARS N-pro tein is unsta ble [8] [9] [10] [11] [12] .

As with other viral N-pro teins, the SARS N-pro tein exhib its exten sive oli gi mer iza tion that is pre sum ably linked to SARS virus pack ag ing and mat u ra tion. Of the potential inter mo lec u lar binding sites that have been iden ti fied [13] [14] [15] , the first to be char acter ized was the highly hydro philic ser ine and argi nine rich region, 184 ssrsssrsrgnsr 196 [7, [16] [17] [18] . Dele tion of this sequence abro gates N-pro tein self-asso ci a tion and pre vents N-pro tein local i za tion around the nucleus and thus the RNA-bind ing and pack ag ing required for SARS virus mat u ra tion.

In the pro cess of puri fy ing the N-pro tein to study its potential in the pro duc tion of vac cines against the virus, the appear ance of two other pro tein/pro tein frag ment bands on SDS-PAGE was detected. This paper describes the work to iden tify these pro teins/ pro tein frag ments and to deter mine their source. A series of experi ments were designed to iso late and char ac ter ize these frag ments, pin point the cleav age site(s), and deter mine if the cleav age was due to a bac te rial pro te ase con tam i nant or to autol y sis. It is antic ipated that iden ti fi ca tion of the source/con di tions for cleav age and subsequent inac ti va tion of N-pro tein and con com i tant dis abling of the coro na vi rus' abil ity to pack age RNA and/or inter fere with host cell func tion, may lead to devel op ment of novel and effi ca cious treat ments for SARS.

SARS N-pro tein expres sion and puri fi ca tion. Esch e richia coli M15 and BL21(DE3) cells, trans formed with pQE-2/NP (pQE-2 expressing the N-pro tein), were used to pro duce pro tein as pre vi ously described [17, 19] . Puri fi ca tion was per formed under dena tur ing con di tions using a His-trap HP metal affin ity col umn (GE Life sciences).

The puri fied N-pro tein was trans ferred to dial y sis tub ing (7500 MWCO) and dia lyzed into a urea-sup ple mented refold ing buffer (10 mM Tris, 100 mM sodium phos phate, 150 mM NaCl, and 8 M urea, pH 8.0). Urea was then grad u ally removed by a step wise replace ment of the buffer with Tris/phos phate buffer (10 mM Tris, 100 mM sodium phos phate, and 150 mM NaCl, pH 8.0) con tain ing decreas ing con cen tra tions of urea (8, 4, 2, 1, 0.5, and 0 M). In sam ples where the N-ter mi nal (His) 6 -tag was removed, the pH of the buffer was adjusted to 7.0 by dial y sis and Qiagen DA Pase (dipep ti dase) added to the pro tein solu tion.

N-pro tein solu tions were ana lyzed on SDS-poly-acryl am ide gel using the 12% cross-link method described by Lae mmli [20] or the 20% Tri cine method described by Sch ag ger [21] . Gels were stained using Coomassie blue or Sy pro Ruby Red.

Mass spec tro met ric anal y ses. SEL DI-TOF/MS data were gen er ated using a PBS-IIC instru ment (Ci pher gen, Fre mont, CA) that was cal i brated using All-in-One pep tide stan dards (Ci pher gen) adhered to a nor mal phase, NP20 pro tein array. One micro gram of the SARS N-pro tein was applied to each of the remain ing sam ple spots for anal y sis. SEL DI-TOF spec tra were gen er ated by laser desorp tion/ ion i za tion using an aver age 130 laser shots with an inten sity of 190-200 (arbi trary units) and detec tor sen si tiv ity of eight.

MALDI-TOF mass spec trom e try was per formed on pep tides after SDS-PAGE sep a ra tion and in-gel tryp tic diges tion of proteins/pep tide bands [22] . Pep tide frag ments were ana lyzed using a Mi cro mass MALDI-LR instru ment (Waters, Miss is sa u ga, ON) and ana lyzed using Mass LYNX 3.5 soft ware (Waters). Pep tide fin gerprint searches were per formed using MAS COT (Matrix Sci ence, Bos ton, MA) and the NCBI pro tein data base.

N-pro tein frag ments were puri fied on a Thermo Spec tra Sys tem HPLC using a Vy dac C8 reverse phase col umn. Pro teins were eluted using 0.01% tri flu o ro ace tic acid (TFA) and a 10-90% ace to ni trile gra di ent and pro tein-con tain ing frac tions ana lyzed by LC/MS as pre vi ously described [23] .

Pro te ase assess ment. Quan tic leave pro te ase assay using fluo res cein iso thi o cy a nate-(FITC)-con ju gated casein as described by man u fac turer (Pierce). Fluo res cence was detected using 485/538 nm exci ta tion/emis sion wave lengths in a Gen i os plate reader run ning XFlu or 4 soft ware (Tecan, Dur ham, NC).

Non-spe cific pro te ase activ ity was tested by mix ing a 10-fold excess (w/w rel a tive to SARS N-pro tein) of either oval bu min or RNAse A and co-refolded with the SARS N-pro tein. Oval bu min and RNase A were prepared by cleav ing and cap ping of disul fide bonds with iodoa cet a mide to pre vent disul fide bond for ma tion prior to refold ing. The test pro teins (N-pro tein and oval bu min or RNA ase) were then denatured by addi tion to 6 M gua ni dine buffer con tain ing SARS N-pro tein and pro teins simul ta neously refolded. The pres ence or absence of cleav age pep tides was deter mined using SDS-PAGE.

Fluo res cent res o nance energy trans fer-labelled pep tides (EDANS/DAB CYL-con ju gated 168 LPKGFYAEGSRGGSQ ASS 185 -and 181 SQASSRSSSRSRGNSRNSTP 200 -SARS N-pro tein pep tides) were pur chased from JPT Pep tide Tech nol o gies (Acton, MA). EDANS/DAB-CYL-con ju gated pep tides were designed to cor re spond to puta tive cleav age sites of the SARS N-pro tein. Cleav age of either pep tide would lead to sep a ra tion of the EDANS reagent from the DAB CYL and result in a 40-fold increase in fluo res cent sig nal. An ali quot (100 ll) of each EDANS/DAB CYL-con ju gated pep tide dis solved in PBS at pH 7.2 (0.5 mg/ml final con cen tra tion) was placed in a 96-well plate and 100 ll of either N-pro tein prep a ra tion (25 lg total pro tein) or tryp sin (positive con trol) was added. Fluo res cence (rel a tive fluo res cent units, RFU) was recorded using 360/465 nm exci ta tion/emis sion wave lengths over 60 min at 25 °C.

SARS N-pro tein pro duc tion, puri fi ca tion, and refold ing SARS N-pro tein was pro duced in both M15 and BL21(DE3) cells. Pro teins were expressed as insol u ble inclu sion bodies and purified using metal affin ity resin, then urea-dena tured pro teins were refolded by step wise dial y sis and the His-tag removed by DA Pase diges tion. SDS-PAGE con firmed that the recov ered N-pro tein was puri fied to near homo ge ne ity under dena tur ing con di tions (Fig. 1 ). How ever, fol low ing the refold ing, two addi tional bands (A and B bands) were observed at approx i mately 29 kDa (A band) and 25 kDa (B band). The masses were con sis tent with pro te ol yed N-pro tein (approx i mately 50 kDa) and sim i lar to the SARS N-pro tein pro te oly sis prod ucts reported in the pres ence of casp as es [24] .

To rule out the pos si bil ity that the A and B bands could be attrib uted either to N-pro tein cleav age by DA Pase or a con tam inant in DA Pase, the puri fi ca tion was repeated using BL21(DE3) cells with out DA Pase addi tion. BL21(DE3) cells lack the lon and ompT pro te ase genes and thus reduced recombinant pro tein pro teol y sis is expected. How ever, the A and B bands were still observed, con firm ing that the SARS N-pro tein cleav age was not due to the DA Pase prep a ra tion and was inde pen dent of the cell line used (data not shown).

To deter mine the source of the A and B bands, mass spec trom e try tech niques were used. The exper i ments used prep a ra tions of recombinant N-pro tein from which the His-tag had not been enzy mat i cally removed; thus, cal cu la tion of the resul tant pro tein/ pep tide masses must take into con sid er ation the pres ence of the His-tag (an addi tional 11 amino acid sequence). While SDS-PAGE showed two dis crete bands of approx i mately 29 and 25 kDa, sur face enhanced laser desorp tion ion i za tion (SEL DI) mass spec trom e try (data not shown) showed that the bands comprised of four or five pro teins/pep tides with sim i lar molec u lar masses: 24.0-26.0 (A band) and 21.5-23.0 kDa (B band). Each SEL DI peak was sep a rated from its near est neigh bor by approx i mately 200-250 Da (two amino acids). Thus, the A and B bands were tenta tively iden ti fied as sev eral site-spe cific hydro lyzed frag ments of the full-length N-pro tein (47 kDa).

Preliminary iden ti fi ca tions of the SARS N-pro tein frag ments were con firmed by in-gel diges tion of the A and B bands and MALDI-TOF/mass spec trom e try. Detected pep tide molec u lar weights were searched against the MAS COT data base and both the A and B bands were iden ti fied as SARS N-pro tein frag ments. MALDI-TOF/ MS data was then com pared to the expected molec u lar masses of tryp tic pep tides deter mined by an in sil ico digest of the N-pro tein sequence. Only pep tides cor re spond ing to the N-pro tein's C-termi nal region were detected in the A band, while only N-ter mi nal pep tides were detected in the B band. This result, com bined with SEL DI-TOF/MS data, indi cated that the recombinant N-pro tein under goes cleav age at multiple sites near the cen ter of its amino acid sequence.

To deter mine the exact loca tion of N-pro tein cleav age, N-pro tein sam ples were ana lyzed by LC/MS. Resul tant masses were com pared to the the o ret i cal masses of all pos si ble pep tides derived from the N-pro tein's amino acid sequence. The results showed that cleavage had occurred between res i dues 197/198, 201/202, 203/204, and 205/206 of the His-tagged N-pro tein (i.e., 184 ssr/sssr/sr/gnsr/ 196 in the untagged pro tein sequence). The decon vo luted mass spec trum of the cleaved C-ter mi nus pro tein frag ment is shown in Fig. 2 and is con sis tent with the result of SEL DI-TOF/MS that showed suc ces sive cleav age sites with gaps of approx i mately two amino acids.

With the site of N-pro tein cleav age iden ti fied, exper i ments were per formed to deter mine whether this cleav age was the result of pro te ase con tam i na tion of the prep a ra tion, N-pro tein auto ca tal ysis, or an inher ent sus cep ti bil ity to spe cific bond cleav age. Ini tially, the com mer cially avail able Quan tic leave assay, which uses FITClabelled casein, was used to test for non-spe cific pro te ol y sis. When the FITC-labelled casein was added to prep a ra tions of the puri fied N-pro tein, no increase in fluo res cence was detected.

It was pos si ble that a con tam i nat ing pro te ase was pres ent but unde tect able by the Quan tic leave assay, as casein con tains sev eral R res i dues but no SR motif. To fur ther eval u ate the pos si bil ity that con tam i nat ing pro te ases were respon si ble for N-pro tein hydroly sis, dena tured SARS N-pro tein was refolded in the pres ence of excess dena tured oval bu min (pos sess ing an SR motif at a.a. 104-105) or dena tured RNAse A (pos sess ing an SR motif at a.a 36-37). After refold ing, the pro tein/pro tein frag ment mix tures were sepa rated by SDS-PAGE; while the SARS N-pro tein still showed the dis tinc tive cleav age pattern upon refold ing, nei ther oval bu min nor RNAse A showed any pro te ol y sis (Fig. 3) . If a con tam i nat ing pro te ase was respon si ble for cleav age of the SR motif, pref er ential diges tion of the more con cen trated (10-fold excess) oval bumin or RNAse A is expected. The absence of cleav age of these two pro teins sug gests the absence of pro te ase con tam i na tion in the N-pro tein sam ple.

A spe cific pro te ol y sis assay was then devel oped using pep tides con tain ing the SR-rich region cor re spond ing to the SARS N-pro tein cleav age site. Pep tides derived from the N-pro tein, were syn thes ised and con ju gated with EDANS and DAB CYL for FRET anal y sis. The FRETlabelled pep tide sub strates were incu bated with N-pro tein, tryp sin (positive con trol), or buffer (neg a tive con trol). In this assay pep tide cleav age would result in the lib er a tion of EDANS and DAB CYL and a con com i tant increase in fluo res cence [25] . In positive con trols, incuba tion of both FRET sub strates with tryp sin resulted in increased fluo res cence and dem on strated that the assay could detect pro te ases. How ever, in N-pro tein tri als, no sim i lar increase in fluo res cence was seen (Fig. 4) indi cat ing that con tam i nat ing pro te ases were unlikely unless in amounts below the detec tion limit of the assay (for trypsin, approx i mately 2.5 ng/ml). SARS N-pro tein pres ent in the same mix ture as these SR-con tain ing sub strates did, itself, undergo cleavage as evi denced by the appear ance of A and B bands on SDS-PAGE (data not shown), indi cat ing that if the N-pro tein is respon si ble for its own cleav age (autol y sis), it is unable to cleave FRET-labelled peptides con tain ing the cleav age site. These data can be explained if the sequences for pro te ase bind ing and subsequent cleav age are located in dif fer ent regions of the N-pro tein. Sep a ra tion of bind ing and cleavage sites on a pro te o lytic sub strate has been reported pre vi ously [26] but it is not com mon for a pro te ase to bind a sub strate at a sequence remote from the scis sile bond. Taken together, the results from the FITC-labelled casein, the refold ing, and the FRET (EDANS/DAB CYL) detec tion exper i ments strongly sug gest that the SR-spe cific cleav age of N-pro tein is not the result of a con tam i nat ing pro te ase activ ity.

This detec tion of SARS N-pro tein pro te ol y sis is not the first obser va tion of N-pro tein cleav age. Cleav age has also been reported in SARS coro na vi rus-infected Vero (mon key-derived) cells [24, 27] . Ying and co-work ers [24] observed three N-pro tein-related bands with appar ent molec u lar size of 27-31 kDa and four at 16-23 kDa in addi tion to bands for the full-length pro tein. This hydro ly sis attrib uted to the action of endog e nous caspase-3 as N-pro tein incu ba tion with exog e nous casp as es, resulted in sim i lar cleavage prod ucts. The authors did not note that the SARS N-pro tein sequence lacks the canon i cal-DXXD-caspase-3 cleav age site. More recently, Di emer et al. [27] showed that caspase-6 can cleave SARS N-pro tein fol low ing res i due 400 or 403, giv ing frag ments of 44 and 2 kDa. The cleav age was cell-type spe cific and only observed in Vero-E6 or human epi the lial and not Caco or N2a (murine neuro nal) lines. In the cur rent study, SDS-PAGE results using E. coliderived SARS N-pro tein showed sim i lar cleav age to that detected by Ying et al., but in the absence of any casp as es.

The SR-rich region thus appears to be read ily cleaved in both E. coli and Vero cells and our attempts to iden tify a con tam i nat ing pro te ase respon si ble for that cleav age, though not exhaus tive, were unsuc cess ful. Guru pra sad and co-work ers [28] report that cer tain dipep tide sequences are sta tis ti cally more likely to be found in pro teins with shorter in vivo half-lives than in those with longer in vivo half-lives. Both SR and RS (argi nine-ser ine) dipep tides (and the N-pro tein SR-rich region con tains both) are among those found more fre quently in "unsta ble" pro teins. In vivo insta bil ity is usu ally linked to diges tion by cir cu lat ing or cel lu lar pro te ases. There fore, a data base of pro te ases was searched to find an enzyme capa ble of the observed cleav ages. Not only is the SR motif cleav age site is not a rec og ni tion site for any known E. coli pro te ases (con sis tent with our inabil ity to detect pro te ase con tam i na tion in our N-protein prep a ra tions), it is not rec og nized by any other pro te ase in the data base [29] . Sev eral pro te ases do cleave fol low ing pos i tively charged res i dues, includ ing argi nine (Arg-C and clos tri pain) or any argi nine or lysine (tryp sin); how ever, this cleav age is otherwise non-spe cific and does not require S in the P2 position.

The puta tive bio log i cal role(s) of this SR-rich region is not com pletely known, but it has been pro posed that this region allows the SARS N-pro tein to form the dimers or tet ra mers [13, 17] thought Ovalbumin/N N-protein RNAse A/N protein Fig. 3 . SARS N-pro tein refold ing with excess oval bu min or RNAse A. SARS N-pro tein was refolded in the pres ence of a 10-fold excess of oval bu min or RNA ase. Sam ples were sep a rated by 20% Tri cine gel lane M, molec u lar size mark ers; lanes 1, 4, and 7, sam ples obtained after over night dial y sis into a final refold ing buffer (0 M urea); lanes 2, 5, and 8, obtained fol low ing addi tional 2 h incu ba tion at 4 °C; lanes 3, 6, and 9, obtained fol low ing 120 h incu ba tion at 4 °C. Lanes 1-3 con tained N-pro tein refolded in the absence of sec ond ary pro tein. Lanes 4-6 con tained SARS N-pro tein refolded with oval bu min, lanes 7-9 con tained SARS N-pro tein refolded with RNA ase. Arrows indi cate molec u lar weights of pre dicted oval bu min (32 and 12 kDa) and RNAse A (9 and 8 kDa) cleav age prod ucts.

to be essen tial in for ma tion of the mature viral nucle o cap sid and aid ing in com pact ing the RNA/nucle o cap sid com plex [13] [14] [15] 30] . Dele tion of the SR-rich region not only com pletely abol ished the oli gi mer iza tion of the N-pro tein but also resulted in a dis or dered dis tri bu tion of N-pro teins in mam ma lian cells [17] . Con sid er ing that the region respon si ble for self-asso ci a tion iden ti fied by these pre vi ous stud ies was iden ti cal to the cleav age region iden ti fied in this study, it is rea son able to spec u late that the cleav age of this sequence may aid in the unpack ing of viral RNA needed to allow this RNA to serve as a tem plate for viral genome rep li ca tion. If bind ing and subsequent lysis of the SR-rich amino acid region are inher ent prop er ties of the SARS N-pro tein, the SR-rich amino acid sequence presents a potential tar get for a SARS ther a peu tic and an agent that pre vents its cleav age (perhaps a com pet i tive inhibi tor such as a mimetic of the SR-region) may prove effec tive in inter rupt ing viral rep li ca tion in SARS-infected indi vid u als.

Thanks to Dr. Laura Stew art for crit i cal input into the man uscript.

Koles nik ova

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