Key points are not available for this paper at this time.
Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication. Coronavirus (CoV) nucleocapsid (N) protein is a highly phosphorylated protein required for viral replication, but whether its phosphorylation and the related kinases are involved in the viral life cycle is unknown. We found the severe acute respiratory syndrome CoV N protein to be an appropriate system to address this issue. Using high resolution PAGE analysis, this protein could be separated into phosphorylated and unphosphorylated isoforms. Mass spectrometric analysis and deletion mapping showed that the major phosphorylation sites were located at the central serine-arginine (SR)-rich motif that contains several glycogen synthase kinase (GSK)-3 substrate consensus sequences. GSK-3-specific inhibitor treatment dephosphorylated the N protein, and this could be recovered by the constitutively active GSK-3 kinase. Immunoprecipitation brought down both N and GSK-3 proteins in the same complex, and the N protein could be phosphorylated directly at its SR-rich motif by GSK-3 using an in vitro kinase assay. Mutation of the two priming sites critical for GSK-3 phosphorylation in the SR-rich motif abolished N protein phosphorylation. Finally, GSK-3 inhibitor was found to reduce N phosphorylation in the severe acute respiratory syndrome CoV-infected VeroE6 cells and decrease the viral titer and cytopathic effects. The effect of GSK-3 inhibitor was reproduced in another coronavirus, the neurotropic JHM strain of mouse hepatitis virus. Our results indicate that GSK-3 is critical for CoV N protein phosphorylation and suggest that it plays a role in regulating the viral life cycle. This study, thus, provides new avenues to further investigate the specific role of N protein phosphorylation in CoV replication. The causative pathogen for the epidemic severe acute respiratory syndrome (SARS) 2The abbreviations used are: SARS, severe acute respiratory syndrome; SCoV, severe acute respiratory syndrome coronavirus; N protein, nucleocapsid protein; CPE, cytopathic effect; JHMV, JHM strain of mouse hepatitis virus; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; pI, isoelectric point; SR, serine-arginine; GSK, glycogen synthase kinase; GST, glutathione S-transferase; Bis-Tris, 2-bis(2-hydroxyethyl)amino-2-(hydroxymethyl)propane-1,3-diol; Ab, antibody; MS, mass spectrometry; MOPS, 4-morpholinepropanesulfonic acid; DIG, digoxigenin; CIP, calf intestinal alkaline phosphatase; TGEV, transmissible gastroenteritis coronavirus; IBV, infectious bronchitis virus. was identified as the SARS coronavirus (SCoV) in 2003 (1Drosten C. Gunther S. Preiser W. van der Werf S. Brodt H.R. Becker S. Rabenau H. Panning M. Kolesnikova L. Fouchier R.A. Berger A. Burguiere A.M. Cinatl J. Eickmann M. Escriou N. Grywna K. Kramme S. Manuguerra J.C. Muller S. Rickerts V. Sturmer M. Vieth S. Klenk H.D. Osterhaus A.D. Schmitz H. Doerr H.W. N. Engl. J. Med. 2003; 348: 1967-1976Crossref PubMed Scopus (3696) Google Scholar, 2Ksiazek T.G. Erdman D. Goldsmith C.S. Zaki S.R. Peret T. Emery S. Tong S. Urbani C. Comer J.A. Lim W. Rollin P.E. Dowell S.F. Ling A.E. Humphrey C.D. Shieh W.J. Guarner J. Paddock C.D. Rota P. Fields B. DeRisi J. Yang J.Y. Cox N. Hughes J.M. LeDuc J.W. Bellini W.J. Anderson L.J. N. Engl. J. Med. 2003; 348: 1953-1966Crossref PubMed Scopus (3571) Google Scholar). Its genome consists of a ∼30-kilobase positive-sense single-stranded RNA which encodes a 3′ co-terminal set of nine subgenomic mRNAs with a common leader sequence at their 5′ ends (3Snijder E.J. Bredenbeek P.J. Dobbe J.C. Thiel V. Ziebuhr J. Poon L.L. Guan Y. Rozanov M. Spaan W.J. Gorbalenya A.E. J. Mol. Biol. 2003; 331: 991-1004Crossref PubMed Scopus (1003) Google Scholar, 4Lai M.M. Cavanagh D. Adv. Virus Res. 1997; 48: 1-100Crossref PubMed Google Scholar). These subgenomic RNAs encode various structural and nonstructural proteins required to produce progeny virions, including the viral nucleocapsid (N) protein. The SCoV N protein is the most abundant viral structural protein. During the viral life cycle multiple copies of the N protein interact with the viral genome to form the ribonucleoprotein complex, which is subsequently packaged by a lipid envelope during viral budding, possibly through its interaction with the viral structure membrane (M) protein (5Fang X. Ye L. Timani K.A. Li S. Zen Y. Zhao M. Zheng H. Wu Z. J. Biochem. Mol. Biol. 2005; 38: 381-385PubMed Google Scholar). In addition to its structural role, the N protein is also implicated in regulating the synthesis of viral RNA and protein (4Lai M.M. Cavanagh D. Adv. Virus Res. 1997; 48: 1-100Crossref PubMed Google Scholar, 6He R. Leeson A. Andonov A. Li Y. Bastien N. Cao J. Osiowy C. Dobie F. Cutts T. Ballantine M. Li X. Biochem. Biophys. Res. Commun. 2003; 311: 870-876Crossref PubMed Scopus (106) Google Scholar, 7Tahara S.M. Dietlin T.A. Nelson G.W. Stohlman S.A. Manno D.J. Adv. Exp. Med. Biol. 1998; 440: 313-318Crossref PubMed Scopus (40) Google Scholar). Using reverse genetics, the critical role of N protein in the replication of coronaviruses has been identified in HCoV-229E, TGEV (transmissible gastroenteritis coronavirus), and IBV (infectious bronchitis virus) (8Almazan F. Galan C. Enjuanes L. J. Virol. 2004; 78: 12683-12688Crossref PubMed Scopus (162) Google Scholar, 9Schelle B. Karl N. Ludewig B. Siddell S.G. Thiel V. J. Virol. 2005; 79: 6620-6630Crossref PubMed Scopus (114) Google Scholar, 10Spencer K.A. Dee M. Britton P. Hiscox J.A. Virology. 2008; 370: 373-381Crossref PubMed Scopus (35) Google Scholar). However, the molecular mechanisms in N protein participation in viral replication and the cellular gene(s) involved in regulating the unknown. The CoV N protein is highly and is of The to the protein and is to RNA H. A. S. S. J. 2005; PubMed Scopus Google Scholar, L. A.M. A. Z. N. P. J. 2004; PubMed Scopus Google Scholar). The to its H. H. A. J. J. J. Virol. PubMed Scopus Google Scholar, H. J. K. X. H. PubMed Scopus Google and the central contains a (SR)-rich motif with but which is possibly also involved in the of its H. Ye F. K. X. H. 2005; PubMed Scopus Google Scholar). N proteins are highly phosphorylated in cells D. J.A. J.M. A. Enjuanes L. J. Virol. 2005; PubMed Scopus Google Scholar, S.A. C.D. M.M. Virology. PubMed Scopus Google Scholar). M. R. J. Virol. 2005; 79: PubMed Scopus Google that SCoV N protein phosphorylation in at the The phosphorylation of the TGEV, IBV, and SCoV N proteins been identified as and D. J.A. J.M. A. Enjuanes L. J. Virol. 2005; PubMed Scopus Google Scholar, H. A. S.R. Dee M. Hiscox J.A. J. Virol. 2005; 79: PubMed Scopus Google Scholar, Z. Virus Res. PubMed Scopus Google Scholar, L. J. M. J. X. N. R. S. J. Mass PubMed Scopus Google Scholar). However, the for N protein phosphorylation and the of phosphorylation viral life cycle been The of N protein phosphorylation could its RNA and its S.A. C.D. M.M. Virology. PubMed Scopus Google Scholar, M. R. J. Virol. 2005; 79: PubMed Scopus Google Scholar, H. A. S.R. Dee M. Hiscox J.A. J. Virol. 2005; 79: PubMed Scopus Google Scholar, G.W. Stohlman S.A. S.M. J. Virol. PubMed Scopus Google Scholar). IBV, H. A. S.R. Dee M. Hiscox J.A. J. Virol. 2005; 79: PubMed Scopus Google that phosphorylated N protein was to viral RNA with a In the N protein was found in the the phosphorylated N protein was with the cellular membrane S.A. C.D. M.M. Virology. PubMed Scopus Google Scholar). and S. PubMed Scopus Google that of N protein by an cellular protein viral The phosphorylated SCoV N was to the to the by with the protein, as a for M. R. J. Virol. 2005; 79: PubMed Scopus Google Scholar). J. 2008; PubMed Scopus Google that phosphorylation of SCoV N at the SR-rich motif could its and also its the phosphorylation of N protein has been to in regulating viral replication, but is a of its critical We to this by of the cellular for phosphorylation of SCoV N protein. We mass spectrometric analysis and deletion mapping to the phosphorylation sites of SCoV N to the central SR-rich which contains several consensus substrate for the GSK-3 kinase. The role of GSK-3 in N protein phosphorylation was by both in vitro and in The of GSK-3 in N phosphorylation has also been in another coronavirus, Finally, found that of GSK-3 could the replication of both The results indicate that GSK-3 is critical for N phosphorylation but also suggest its in regulating viral replication. SCoV N and N were by of the The used for SCoV is strain Yang J.Y. H.W. S.F. P.J. S. A. 2004; PubMed Scopus Google and the neurotropic JHM strain of mouse hepatitis JHMV, was by M. C. S.A. M.M. J. Virol. PubMed Scopus Google Scholar). The for RNA and reverse was Yang J.Y. H.W. S.F. P.J. S. A. 2004; PubMed Scopus Google Scholar). The was into the and for and protein of specific into the SCoV N was by using the The SCoV N with deletion of SR-rich motif was in the of and by of of The for the active form of and were by of of and of of and VeroE6 and cells were in with and and The mouse were in with and and of cells were in with cells were to the were by using to the as P.J. P.J. Yang W.J. S. A. PubMed Scopus Google Scholar). and were with inhibitor and inhibitor The were separated by with of the protein to the of The was the membrane and at in at for with and and the was by the system The used in the N in Yang Yang J.Y. P.J. J. Med. Virol. 2004; PubMed Scopus Google N by J. and The specific the phosphorylated N proteins of SCoV and was by of into The sequence for the two is SCoV and of N cells were with and by of at for at the was with of at for The were with of and for the with of calf alkaline in of at for The were with of and by for Mass of SCoV N cells with was used for of the N protein of SCoV by with The proteins were with and were separated by with the protein to was for The was and in of at for and by a system of high and mass and several were used to the of with the effect of specific kinases N the cells with N with coronaviruses were with specific including and for for for kinase for for protein kinase and for The were into the with the cytopathic effect was the were for In used for the in vitro GSK-3 kinase are the proteins of SCoV N and the N proteins the The proteins were the as Yang Yang J.Y. P.J. J. Med. Virol. 2004; PubMed Scopus Google Scholar). The were in a kinase and of active of and of The were at for and by the addition of and separated by The were by and to also to the phosphorylated unphosphorylated N protein by the The protein were in and separated by the isoelectric using which were with the The isoelectric was at with of for for and for isoelectric the were with the separated by and for the of by and strain of SCoV was in cells in Yang J.Y. H.W. S.F. P.J. S. A. 2004; PubMed Scopus Google and the strain of mouse hepatitis was with the cells as S.A. M.M. J. Virol. PubMed Scopus Google Scholar). The viral titer of SCoV was as the of as with as PubMed Scopus Google and also by the The viral titer of was by in cells as S.A. M.M. J. Virol. PubMed Scopus Google and also by the viral RNA was into by the synthesis system Yang J.Y. H.W. S.F. P.J. S. A. 2004; PubMed Scopus Google Scholar). was with the The set for contains and and the set for SCoV contains and The was in a of of viral and reverse and of The was with as an at for by of at for at for and at for and the was at the of the for cycle. the of the a analysis was at the of by the at for and the to with the and for by at to with of the viral used the and to the The was and for the of the for as S.F. J. P.J. J. 2004; PubMed Scopus Google Scholar). cellular RNA was using the to the The RNA was and by with in MOPS, to and by The membrane was for the using the by the The used for was with during synthesis The used for of are as and were used for of SCoV N and were used for of N and and reverse were used for of the The were with which is The N of SCoV in VeroE6 and the phosphorylation of the SCoV N protein, used high resolution to the phosphorylated unphosphorylated VeroE6 cells were with protein and in high resolution identified a N protein the and with that the and most of the N protein in both was and this phosphorylation could the of this protein in The to in the phosphorylated the N into cells and the N proteins by with Mass spectrometric analysis showed that of the protein sequence could be recovered We phosphorylation in analysis of this that was which to the with a of This is an of a phosphorylated the of and that the be the is the that be phosphorylated this that this be the is that for this that it is phosphorylated in The results analysis with for the phosphorylated form of the phosphorylation of this of N protein in cells phosphorylation sites including and whether phosphorylation was to be that with the of N protein in The of N at the SR-rich the of in the of N protein in a N with by was used for treatment and the The of this N that of N protein and that in addition to are major phosphorylation that major and by analysis and is located at the central SR-rich motif which are also for phosphorylation. by the J. 2008; PubMed Scopus Google this as the major phosphorylation for SCoV N in a N deletion of the protein, and it cells for the of N protein treatment that of by treatment the SR-rich motif is the major phosphorylation of N proteins in both and VeroE6 GSK-3 the to N at the SR-rich several kinases this motif the that the SR-rich motif is the substrate for cellular investigate which kinase to N protein cells N with specific of the including protein kinase MEK, and We that inhibitor for the kinase involved in N phosphorylation N phosphorylation and to a phosphorylation sites and the kinases by the for the SR-rich motif in the SCoV N for the consensus of GSK-3 in the N was by the but it the GSK-3 consensus for the consensus of GSK-3 in the N for the consensus of GSK-3 in the N for the consensus of GSK-3 in the N for the consensus of GSK-3 in the N for the consensus of GSK-3 in the N for the consensus of GSK-3 in the N for the consensus of GSK-3 in the N for the consensus of GSK-3 in the N The for the consensus of GSK-3 in the N was by the but it the GSK-3 consensus The for the consensus of GSK-3 in the N protein. in a new and of GSK-3 kinase in the of and in a kinase N protein phosphorylation GSK-3 to be the kinase for N protein phosphorylation. the for and N protein by with phosphorylated was that phosphorylation of this is also by also the consensus phosphorylation of further the of GSK-3 in regulating N protein used a of constitutively active was into cells with to whether it could the phosphorylation of N protein. a a constitutively active In the dephosphorylated N by both was by and but by and These thus, further that GSK-3 is the cellular kinase for N protein phosphorylation. SCoV N with and in and by and in analysis to whether GSK-3 with SCoV N protein in into the were to with and for analysis using in the same protein as the In an in vitro kinase using GSK-3 kinase to the protein. The protein of was used as a and could the N protein and but in vitro and could be phosphorylated by the results that the SR-rich motif of N protein is the major phosphorylation for of the by GSK-3 SCoV N is that GSK-3 separated by and the priming phosphorylation of a located to the phosphorylation A.M. Y. P.J. J. Biol. PubMed Google Scholar). the SR-rich of to the consensus GSK-3 substrate phosphorylation are in which be into two their and The contains the sequence with the as the priming phosphorylation The contains with as the priming phosphorylation L. J. M. J. X. N. R. S. J. Mass PubMed Scopus Google that both and of are phosphorylated in which was by their of the priming sites a phosphorylation at the by of the priming phosphorylation phosphorylation by GSK-3 is the kinase for phosphorylation of the SR-rich that of decrease phosphorylation of the N protein. N protein were used to this including and N were into cells with with The N protein showed to the that it was treatment with further the of this N protein and However, was identified in N proteins with the the effect of the two of GSK-3 consensus sites the N phosphorylation by a and N proteins cells were to the N was highly phosphorylated and in multiple same molecular but with were phosphorylated as the N protein were treatment N proteins with at at to the decrease in phosphorylation and the N proteins with at the was to that of the with The results further that both of GSK-3 consensus to N protein phosphorylation and that of and the SR-rich motif the phosphorylation of N protein by The of two of GSK-3 substrate phosphorylation sites has been further by the that but N could be by and Finally, the in vitro kinase using the N protein as the The phosphorylation of N was abolished in the N with that of N protein The results that two are critical for GSK-3 to the protein. GSK-3 the of further investigate whether GSK-3 could N phosphorylation in the infectious system and the viral life the effect of SCoV in VeroE6 We its N CPE, viral and viral RNA We that most N protein in the was as it could be by and also showed to that of the N protein in VeroE6 cells However, in the N protein phosphorylation with the of phosphorylated N protein in the cells that this GSK-3 inhibitor could also reduce N phosphorylation in the infectious effect was for the treatment of We further found that treatment could reduce with the effect of GSK-3 the cells with and was for and for the of showed that viral titer was to with treatment with that as the further showed an decrease for the infectious viral titer treatment with that of the analysis that the viral RNA was to be by GSK-3 in the of N was that at of consensus sequence for was also at the central SR-rich of TGEV, and IBV G.W. Stohlman S.A. S.M. J. Virol. PubMed Scopus Google the of GSK-3 in regulating the phosphorylation of N in this the effect of GSK-3 the phosphorylation of N another coronavirus, strain of The GSK-3 substrate phosphorylation sites SR-rich of with as the priming phosphorylation GSK-3 is also involved in regulating the phosphorylation of sites in a with that of the the phosphorylated of N for the of this of was by analysis to investigate as the priming for this of the N was but it be by The and N thus, to the priming N be by and that the priming at The GSK-3 substrate phosphorylation sites in thus, of The phosphorylation of this of was further to be by GSK-3 and and but by several kinase and in this of phosphorylation sites the phosphorylated N with the dephosphorylated N in high resolution The thus, used for of the effect of N phosphorylation. of of N at were in the cells with a The with were by by treatment that could of to a to that by GSK-3 is also the major kinase involved in phosphorylation of as as that of The effect of GSK-3 phosphorylation at has been further in the infectious and treatment decrease the phosphorylation of N at and We that the N in be by The of GSK-3 phosphorylation of N at has been in the infectious system by including kinase for analysis and Finally, also the effect of GSK-3 the replication of and the viral titer could be by and treatment to that for the of the GSK-3 the and the viral titer has been by including the kinase in the and the analysis also that viral RNA synthesis in Our GSK-3 kinase is involved in the phosphorylation of the N protein of at for SCoV and the of consensus GSK-3 at the SR-rich of N protein in most the of GSK-3 for regulating N protein phosphorylation in coronaviruses is highly with this that the phosphorylation by analysis in TGEV was also a consensus sequence for GSK-3 a to in the SCoV N two the central are the phosphorylation identified the phosphorylated the for analysis by of N protein with is which was to the phosphorylation at and L. J. M. J. X. N. R. S. J. Mass PubMed Scopus Google Scholar). the of the priming N the for the priming is unknown. kinases been to be with N phosphorylation using in vitro phosphorylation including mitogen-activated protein kinase M. R. J. Virol. 2005; 79: PubMed Scopus Google and protein kinase J. 2008; PubMed Scopus Google Scholar). However, kinases are for the phosphorylation of the priming by the cells with specific for of a in the N protein, which a to for cellular kinases for the priming The effect of GSK-3 the phosphorylation of N protein was both in VeroE6 cells and in cells in which and treatment to a decrease in phosphorylation. and treatment could also and viral The that the of N phosphorylation by and be critical for coronavirus replication, the that phosphorylation of a a viral N for the effect of N phosphorylation viral life cycle be to reverse by the phosphorylation priming and for SCoV and for JHMV, in the viral which the N protein to be as the unphosphorylated The viral life cycle could be including RNA and replication viral and cytopathic effects. K.A. Dee M. Britton P. Hiscox J.A. Virology. 2008; 370: 373-381Crossref PubMed Scopus (35) Google used a reverse analysis and showed that the N protein a to the of IBV the These results the that N protein phosphorylation plays a role in viral replication. that a reverse the role of N phosphorylation in the viral life this another that the effect is directly by the of N by in the are required to the of N phosphorylation for the viral life cycle. to this the effect of GSK-3 the of and proteins regulating viral life cycle. the of N phosphorylation in regulating its RNA its S.A. C.D. M.M. Virology. PubMed Scopus Google Scholar, M. R. J. Virol. 2005; 79: PubMed Scopus Google Scholar, H. A. S.R. Dee M. Hiscox J.A. J. Virol. 2005; 79: PubMed Scopus Google Scholar, G.W. Stohlman S.A. S.M. J. Virol. PubMed Scopus Google Scholar, J. 2008; PubMed Scopus Google Scholar). These are critical for various of the life including and RNA However, is of the of to the phosphorylation of N protein. this results that using the GSK-3 the priming sites of GSK-3 the phosphorylation of N protein and the effect of N phosphorylation be several that the N protein also several cellular including the of cellular R. Leeson A. Andonov A. Li Y. Bastien N. Cao J. Osiowy C. Dobie F. Cutts T. Ballantine M. Li X. Biochem. Biophys. Res. Commun. 2003; 311: 870-876Crossref PubMed Scopus (106) Google Scholar, T. S. M. S. Biochem. Biophys. Res. Commun. 2004; PubMed Scopus Google the cycle M. B. J. Biol. PubMed Scopus Google and as a S.A. L. M. R.A. P. J. Virol. PubMed Scopus Google Scholar, Y. K. J. Virol. PubMed Scopus Google Scholar). These also in regulating viral replication thus, are for the effect of N phosphorylation in the viral life cycle. The of the for of phosphorylated N proteins the of N phosphorylation in the viral life cycle. found that the N proteins both in the SCoV and as the However, N proteins are phosphorylated cells and This two for the of the role of N protein phosphorylation in the viral life cycle. it of viral nucleocapsid In the of L.J. C.S. M. J. Virol. PubMed Scopus Google Scholar, J.C. J. Virol. 2003; PubMed Scopus Google that phosphorylation of protein the of during which the of viral for viral replication. However, at the and the proteins to be which the of with the viral RNA and for L.J. C.S. M. J. Virol. PubMed Scopus Google Scholar, J.C. J. Virol. 2003; PubMed Scopus Google Scholar). the effect of N phosphorylation with viral RNA and regulating the nucleocapsid is of as analysis showed that treatment the synthesis of viral phosphorylated N thus, in viral RNA In JHMV, the phosphorylated N protein is with the membrane S.A. C.D. M.M. Virology. PubMed Scopus Google the viral RNA E.J. van der Y. J. van der J. A.M. J. Virol. PubMed Scopus Google Scholar). thus, the that the phosphorylated form of N protein to the viral replication in membrane and in viral RNA In of N protein has been to with coronavirus replication in the during viral Spaan W.J. van der Y. J. Virol. PubMed Google Scholar, der Y. E.J. Dobbe J.C. S. Spaan W.J. J. Virol. PubMed Google Scholar). The of phosphorylated N with the membrane and with the replication be by using the the phosphorylated N In that a cellular is to both SCoV and N This a role for the GSK-3 kinase in regulating the viral life cycle. Our thus, a for the treatment of coronavirus and new avenues to investigate the specific role of N protein phosphorylation in coronavirus life We the of N J. of The
Wu et al. (Wed,) studied this question.
Synapse has enriched 5 closely related papers on similar clinical questions. Consider them for comparative context: