Epistemic_Statements  

CORD-19:cf1631f486167031d3429ac3e81d2bb99f33f15e JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 238-353 Epistemic_statement denotes The SARS-CoV N protein has also been suggested to be involved in other important functions in the viral life cycle.
T2 794-892 Epistemic_statement denotes Bioinformatics reveal that other coronavirus N proteins could share the same modular organization.
T3 1416-1517 Epistemic_statement denotes In humans, coronaviruses are often associated with mild respiratory illnesses, including common cold.
T4 1518-1671 Epistemic_statement denotes However, a novel coronavirus has been identified as the etiology agent of severe acute respiratory syndrome (SARS), which has a case fatality rate of ca.
T5 2215-2326 Epistemic_statement denotes The M protein may also be involved in the formation of the nucleocapsid through interaction with the N protein.
T6 2774-2955 Epistemic_statement denotes From genetic and bioinformatics studies, the N protein can be divided into three putative regions: an Nterminal domain, a RNA-binding domain (RBD) and a C-terminal domain [12, 13] .
T7 2956-3047 Epistemic_statement denotes The N-and Cterminal domains are believed to play a role in interaction with other proteins.
T8 3212-3484 Epistemic_statement denotes Rather surprising, the mid-portion of the protein has been shown to interact with the M protein and hnRNP A1 [16, 17] , and structural studies have identified the region between amino acids 45-181 as the putative RNA-binding region, which is close to the N-terminus [18] .
T9 3637-3747 Epistemic_statement denotes However, the structural organization of coronavirus N proteins in general remains largely unknown to this day.
T10 3889-4062 Epistemic_statement denotes Through the power of nuclear magnetic resonance (NMR) spectroscopy, we present the first evidence that the SARS-CoV N protein consists of two independent structural domains.
T11 4538-4770 Epistemic_statement denotes The elucidation of the modular organization of the SARS-CoV N protein, particularly the boundary between disordered and structured regions, facilitates future studies of this class of proteins at the functional and structural level.
T12 4771-4839 Epistemic_statement denotes Sequence alignment, secondary structure and orderdisorder prediction
T13 4840-5053 Epistemic_statement denotes The full-length sequences of SARS and other coronavirus N proteins were aligned using CLU-STALW version 1.83 with the slow algorithm, an identity matrix, a window of 4 amino acids and standard gap penalties [19] .
T14 11678-11838 Epistemic_statement denotes We observed that the resonances from residues N45-181 have good chemical shift dispersion (Figure 2a) , indicating that the fragment has a structured character.
T15 12154-12290 Epistemic_statement denotes These results indicate that the N-terminal flanking region between amino acids 1-44 does not affect the structure of the N45-181 domain.
T16 12424-12551 Epistemic_statement denotes We found that the resonances from N248-365 are welldispersed (Figure 2c ), suggesting that N248-365 forms an ordered structure.
T17 12884-12988 Epistemic_statement denotes These results indicate that residues from 365 to the C-terminal do not affect the structure of N248-365.
T18 12989-13195 Epistemic_statement denotes Shortening the fragment to span amino acids 274-365 changes the 15 N-HSQC resonance pattern, which indicates that the 248-273 region is important for structure stabilization of this domain (data not shown).
T19 13196-13546 Epistemic_statement denotes To explore the structure of the region between residues 182-247 and their effect on the structure of N45-181 and N248-365, we constructed the fragment N45-365 which contains the two struc- The lack of resonance perturbation when the two domains are linked together suggests that interaction between these two domains is weak, if they interact at all.
T20 13672-13723 Epistemic_statement denotes These results are consistent with PONDR prediction.
T21 14066-14233 Epistemic_statement denotes To test whether the residues beyond the structural domains are truly disordered, we employed the CLEANEX-PM experiment to identify solvent-accessible resonances [27] .
T22 14357-14530 Epistemic_statement denotes When we compared the CLEA-NEX-PM spectrum of N1-181 (Figure 3b) with that of N45-181 (Figure 3a) , we observed 40 resonances that only appeared in N1-181 but not in N45-181.
T23 14531-14709 Epistemic_statement denotes This number agrees with that expected for the N-terminal region (5 prolines), indicating that all amide protons in the Nterminus of SARS-CoV N protein are exposed to the solvent.
T24 14710-14959 Epistemic_statement denotes We counted 39 additional peaks in the CLEANEX-PM spectrum of N248-422 (Figure 3d ) compared to that of N248-365 ( Figure 3c ) (51 expected since there are 6 prolines), suggesting that the majority of the C-terminal residues are also solvent-exposed.
T25 15157-15342 Epistemic_statement denotes A total of 27 additional peaks can be resolved, compared to 64 expected (2 prolines), indicating that about half of the linker region between residues 182-247 is exposed to the solvent.
T26 15343-15498 Epistemic_statement denotes It should be noted here that due to resonance overlapping the numbers counted should be viewed as a lower limit for the number of solvent-exposed residues.
T27 15499-15717 Epistemic_statement denotes Nevertheless we can conclude that all N-terminal residues are solvent exposed whilst most of the residues in the Cterminus and in the linker region between the two structural domains are exposed to the solvent as well.
T28 15935-16089 Epistemic_statement denotes The long disordered linker between the two structural domains is consistent with the observation that there is little interaction between the two domains.
T29 16090-16343 Epistemic_statement denotes However, the number of counted peaks in the CLEANEX-PM spectra of the Cterminus and the linker region are less than that expected, so it is likely that parts of these regions are solvent-protected, possibly through the formation of transient structures.
T30 16560-16722 Epistemic_statement denotes The function of the N248-365 is not clear, but many reports have identified the C-terminal half of SARS-CoV N protein to be involved in oligomerization [14, 15] .
T31 16723-16931 Epistemic_statement denotes To test this possibility, we have applied analytical gel-filtration chromatography, chemical cross-linking and analytical ultracentrifugation to assay the self-association property of the N protein fragments.
T32 16932-17128 Epistemic_statement denotes As shown in Figure 4a , N45-181 elutes out at a molecular weight of 18 kDa and N248-365 elutes out as a 28-kDa molecule, suggesting that N45-181 exists as a monomer and N248-365 exists as a dimer.
T33 17129-17249 Epistemic_statement denotes The self-association between the two N248-365 monomers is very strong, since we could not detect any monomeric fraction.
T34 17250-17359 Epistemic_statement denotes Similarly, N45-365 eluted out at molecular weight of $70 kDa, suggesting that N45-365 also exists as a dimer.
T35 17786-17924 Epistemic_statement denotes Only one major peak was detected for each of these three protein fragments, indicating that they are structurally homogeneous in solution.
T36 18359-18459 Epistemic_statement denotes Taking together all three results indicate that N45-181 exists as a monomer and N248-365 as a dimer.
T37 18460-18586 Epistemic_statement denotes The fact that dimerization occurs through a structural domain strongly suggest that the process is dependent on the structure.
T38 18686-18883 Epistemic_statement denotes It is interesting to note that we did not observe the formation of higher-order multimer in our studies, which may be important for the formation of the ribonucleoprotein complex within the virion.
T39 18884-19063 Epistemic_statement denotes A possible explanation is that multimer formation may require additional factors, such as the presence of RNA or other parts of the N protein that were not present in our samples.
T40 19064-19201 Epistemic_statement denotes Also we can not exclude the possibility that multimers do form at much higher protein concentrations than the ones used in these studies.
T41 19202-19301 Epistemic_statement denotes We suggest that the dimeric form represents a basic building block of the nucleocapsid of SARS-CoV.
T42 19302-19425 Epistemic_statement denotes Since coronavirus N proteins belong to the same protein family, it is probable that they share similar structural features.
T43 19963-20163 Epistemic_statement denotes The C-terminus of MHV N protein, which is disordered, has been shown to interact with hnRNP A1 [31] , whereas the disordered region in the middle is responsible for its RNA-binding activity [13, 32] .
T44 20164-20353 Epistemic_statement denotes In SARS-CoV, the disordered region in the middle of the N protein has been implicated in N-protein selfinteraction [33] , interaction with the M protein [16] and hnRNP A1 interaction [17] .
T45 20354-20504 Epistemic_statement denotes These experimental observations suggest that disordered regions of coronavirus N proteins are probable interaction sites with functional implications.
T46 21012-21190 Epistemic_statement denotes The extensive secondary structure and high similarity suggests that the two structural domains observed in SARS-CoV N protein also exist in the N proteins of other coronaviruses.
T47 21191-21381 Epistemic_statement denotes The results from the order-disorder prediction and secondary structure prediction coupled with sequence alignment suggest that coronavirus N proteins all share the same modular organization.
T48 21694-21779 Epistemic_statement denotes The ability of the N-terminal domain to bind RNA is closely related to its structure.
T49 21780-21910 Epistemic_statement denotes Although the structure of the C-terminal domain has not been determined, we suggest that dimerization is also structure-dependent.
T50 21911-22228 Epistemic_statement denotes A number of experimental observations support our hypothesis: First, it has been found that oligomer dissociation and protein unfolding of SARS-CoV N protein occur simultaneously [34] ; second, most self-interaction studies have mapped the oligomerization domain to regions containing the structural domain [14, 15] .
T51 22229-22288 Epistemic_statement denotes The structural domains may also serve additional functions.
T52 22289-22415 Epistemic_statement denotes For example, a putative loop between W302 and P310 in the C-terminal domain has been suggested to bind to cyclophilin A [35] .
T53 22416-22497 Epistemic_statement denotes These additional functions may also be dependent on the structure of the protein.
T54 22498-22688 Epistemic_statement denotes Although the two structural domains do not interact with each other, we cannot discount the possibility that the two domains could act in concert to carry out important biological functions.
T55 22689-22793 Epistemic_statement denotes The long flexible linker between the two domains provides enough freedom to make this scenario possible.
T56 22794-22909 Epistemic_statement denotes Previously, the lack of information on structural organization precluded the study of multiple-domain interactions.
T57 23061-23162 Epistemic_statement denotes This disordered region may enable transient interactions with several structurally distinct partners.
T58 23163-23260 Epistemic_statement denotes It has been shown that the M protein of SARS-CoV binds to this region between a.a. 168-208 [16] .
T59 23261-23381 Epistemic_statement denotes Interestingly, human cellular hnRNP A1 has also been shown to bind to almost the same region between a.a. 161-210 [17] .
T60 23382-23502 Epistemic_statement denotes The disordered state of this region potentially allows it to interact with different partners depending on context, e.g.
T61 23590-23775 Epistemic_statement denotes The exact mechanism by which this occurs is not known, but it could involve different induced folding pathways, which has been shown to occur in other disordered proteins [23, 36, 37] .
T62 23976-24060 Epistemic_statement denotes The same region has also been shown to bind murine hnRNP A1 in infected cells [31] .
T63 24061-24294 Epistemic_statement denotes It seems that the coronavirus N proteins share the common theme of using the flexible linker as an interaction ''hotspot'', and use characteristics of disordered regions to achieve multiple functions within a limited sequence length.
T64 24396-24593 Epistemic_statement denotes SARS-CoV N protein has been shown to get serine-phosphorylated by multiple kinases and phosphorylation is proposed to be a possible mechanism for nucleocytoplasmic shuttling of the N protein [38] .
T65 24594-24659 Epistemic_statement denotes Disordered regions represent potential sites for phosphorylation.
T66 24777-24839 Epistemic_statement denotes In fact, this region can be phosphorylated in vitro (Dr. W.-Y.
T67 24871-25078 Epistemic_statement denotes Recent in silico prediction suggested that most of the potential phosphorylation sites fall in the disordered regions, although the exact phosphorylations sites have not been identified experimentally [38] .
T68 25079-25246 Epistemic_statement denotes Although the exact role of phosphorylation has not been elucidated, it could be related to regulate functions such as RNAbinding and localization within the host cell.
T69 25921-26007 Epistemic_statement denotes Coronavirus N proteins seem to employ a widespread property to allow for modification.
T70 26008-26170 Epistemic_statement denotes Whether or not such modification affects the folding or structural properties of the protein and how these properties affect its function remain to be determined.
T71 26424-26572 Epistemic_statement denotes However, structure determination of the full-length protein is hindered by the disordered regions, which often interfere with crystallization [43] .
T72 26616-26720 Epistemic_statement denotes 90 kDa) also makes full-length structure determination through NMR extremely difficult due to T2 issues.
T73 26721-26818 Epistemic_statement denotes The fact that the two structural domains do not interact provides a handle to solve this problem.
T74 26819-26939 Epistemic_statement denotes The two structural domains can be solved independently and still provide fair representation of the full-length protein.
T75 27193-27396 Epistemic_statement denotes The structures of the N-terminal domains would be of special interest since in SARS-CoV it has been identified as an RNAbinding domain, whereas in other coronaviruses the exact function is not yet known.
T76 27544-27709 Epistemic_statement denotes At present the molecular mechanism involving N protein/RNA interaction is still not fully understood and the RNA binding site(s) have not been unequivocally defined.
T77 27710-27865 Epistemic_statement denotes It is possible that the Nterminal structural domain folds into different tertiary structures and plays different roles in different coronavirus N proteins.
T78 27866-27945 Epistemic_statement denotes It is also possible that the linker region may also be involved in RNA binding.
T79 27946-28045 Epistemic_statement denotes Another interesting point that needs further study is the role of the C-terminal structural domain.
T80 28046-28196 Epistemic_statement denotes It is not yet known whether it plays the same dimerization role in other coronavirus as in SARS-CoV, although there are hints in the literature [44] .