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CORD-19:f2230f91f1748d835721471e1618f9606d22b4da JSONTXT 7 Projects

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Id Subject Object Predicate Lexical cue
T1 0-150 Sentence denotes Suppression of innate antiviral response by severe acute respiratory syndrome coronavirus M protein is mediated through the first transmembrane domain
T2 152-160 Sentence denotes Abstract
T3 161-232 Sentence denotes Coronaviruses have developed various measures to evade innate immunity.
T4 233-437 Sentence denotes We have previously shown that severe acute respiratory syndrome (SARS) coronavirus M protein suppresses type I interferon (IFN) production by impeding the formation of functional TRAF3-containing complex.
T5 438-632 Sentence denotes In this study, we demonstrate that the IFN-antagonizing activity is specific to SARS coronavirus M protein and is mediated through its first transmembrane domain (TM1) located at the N terminus.
T6 633-703 Sentence denotes M protein from human coronavirus HKU1 does not inhibit IFN production.
T7 704-860 Sentence denotes Whereas N-linked glycosylation of SARS coronavirus M protein has no influence on IFN antagonism, TM1 is indispensable for the suppression of IFN production.
T8 861-1023 Sentence denotes TM1 targets SARS coronavirus M protein and heterologous proteins to the Golgi apparatus, yet Golgi localization is required but not sufficient for IFN antagonism.
T9 1024-1171 Sentence denotes Mechanistically, TM1 is capable of binding with RIG-I, TRAF3, TBK1 and IKKe, and preventing the interaction of TRAF3 with its downstream effectors.
T10 1172-1300 Sentence denotes Our work defines the molecular architecture of SARS coronavirus M protein required for suppression of innate antiviral response.
T11 1302-1374 Sentence denotes Host cells combat invading viruses by triggering innate immune response.
T12 1375-1472 Sentence denotes As a counter-defense, viruses encode various immunosuppressive proteins to evade innate immunity.
T13 1473-1561 Sentence denotes This interaction between host cells and viruses dictates the outcome of viral infection.
T14 1562-1656 Sentence denotes 1 Coronaviruses are enveloped and positive-stranded RNA viruses with a large genome of ,30 kb.
T15 1657-1708 Sentence denotes In the family Coronaviridae, there are four genera.
T16 1709-2060 Sentence denotes In addition to human coronaviruses 229E and NL63 in the genus Alphacoronavirus, four other viruses belonging to the genus Betacoronavirus, namely, human coronavirus OC43, human coronavirus HKU1, 2 severe acute respiratory syndrome (SARS) coronavirus 3, 4 and Middle East respiratory syndrome (MERS) coronavirus, 5 have also been found to infect human.
T17 2061-2268 Sentence denotes The concept that human coronaviruses are generally associated with mild respiratory diseases was overturned by the identification in 2003 of SARS coronavirus, which causes a severe and highly lethal disease.
T18 2269-2819 Sentence denotes 3, 4 Further investigation of the etiology of community-acquired pneumonia in an adult who was hospitalized in Hong Kong during the outbreak of SARS led to the identification of human coronavirus HKU1, 2 which circulates commonly in human population causing respiratory tract illnesses worldwide. [6] [7] [8] Although the mechanism by which SARS coronavirus and the newly identified MERS coronavirus cause severe respiratory diseases is not fully understood, their subversion of innate immunity is thought to contribute substantially to pathogenesis.
T19 2820-2909 Sentence denotes 4, 9 Type I interferons (IFNs) are major effector cytokines in innate antiviral response.
T20 2910-3119 Sentence denotes To induce IFN production, pathogen-associated molecular patterns such as viral double-stranded RNA are sensed by host pattern recognition receptors such as endosomal Toll-like receptor 3 and cytoplasmic RIG-I.
T21 3120-3260 Sentence denotes The activation of these receptors transmits a signal to downstream kinases TBK1 and IKKe that form a functional complex with TRAF3 and TANK.
T22 3261-3408 Sentence denotes Consequent phosphorylation of IRF3 and IRF7 transcription factors by these kinases leads ultimately to transcriptional activation of IFN promoters.
T23 3409-3570 Sentence denotes 10, 11 Among the structural proteins encoded by SARS coronavirus, M protein is a glycoprotein with three N-terminal transmembrane domains named TM1, TM2 and TM3.
T24 3571-3752 Sentence denotes 12 , 13 We have previously reported that SARS coronavirus M protein suppresses type I IFN production potently by preventing the formation of functional TRAF3-TANK-TBK1/IKKe complex.
T25 3753-3949 Sentence denotes 14 This suppression of innate antiviral response by SARS coronavirus M protein represents one novel viral countermeasure against host innate immunity, which could play a role in SARS pathogenesis.
T26 3950-4155 Sentence denotes 9 However, it remains to be seen whether M proteins of other coronaviruses, which share 28%-41% identity in aminoacid sequence with SARS coronavirus M protein, might also exhibit IFN-antagonizing activity.
T27 4156-4405 Sentence denotes Compared to SARS coronavirus, human coronavirus HKU1 is more frequently associated with less severe disease. [6] [7] [8] It will therefore be of interest to determine whether human coronavirus HKU1 M protein would be able to suppress IFN production.
T28 4406-4536 Sentence denotes On the other hand, the functional domain in SARS coronavirus M protein required and sufficient for IFN antagonism remains elusive.
T29 4537-4669 Sentence denotes In this study, we compared M proteins of SARS coronavirus and human coronavirus HKU1 for their ability to counteract IFN production.
T30 4670-4815 Sentence denotes Furthermore, we dissected the structural domains in SARS coronavirus M protein and mapped the region that mediates the IFN-antagonizing activity.
T31 4816-4966 Sentence denotes Plasmids and antibodies pIFNb-Luc reporter plasmid and RIG-I expression plasmid were obtained from Dr Takashi Fujita (Kyoto University, Kyoto, Japan).
T32 4967-5105 Sentence denotes 15 Expression vectors for TBK1, IRF3 and TRAF3 were generous gifts from Dr Genhong Cheng (University of California, Los Angeles, CA, USA).
T33 5106-5195 Sentence denotes 16, 17 pISRE-Luc reporter construct was purchased from Clontech (Mountain View, CA, USA).
T34 5196-5261 Sentence denotes MDA5, IKKe, MAVS and TANK expression vectors have been described.
T35 5262-5394 Sentence denotes 14 Monoclonal anti-FLAG (clone M2) and anti-a-tubulin (clone DM1A) antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA).
T36 5395-5449 Sentence denotes Anti-myc was bought from Santa Cruz (Dallas, TX, USA).
T37 5450-5509 Sentence denotes Anti-V5 was from Life Technologies (Grand Island, NY, USA).
T38 5510-5567 Sentence denotes Anti-GM130 was from BD Transduction (Lexington, KY, USA).
T39 5568-5629 Sentence denotes Anti-calnexin was from Affinity Bioreagent (Golden, CO, USA).
T40 5630-5870 Sentence denotes Cell culture and transfection HEK293 and HeLa cells were cultured in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and 1% penicillin/streptomycin at 37 uC in a humidified atmosphere of 5% CO 2 .
T41 5871-5972 Sentence denotes Cells were transfected with GeneJuice transfection reagent purchased from Novagen (Madison, WI, USA).
T42 5973-6112 Sentence denotes Protein analysis and reporter assay Western blotting, immunoprecipitation and dual luciferase assay were performed as previously described.
T43 6113-6244 Sentence denotes 18, 19 Relative luciferase activity (RLA) was derived by normalizing readings of firefly luciferase to those of Renilla luciferase.
T44 6245-6281 Sentence denotes It was expressed in arbitrary units.
T45 6282-6427 Sentence denotes All experiments were performed in triplicates and Student's t-test was used to assess statistically the differences between the indicated groups.
T46 6428-6529 Sentence denotes Confocal microscopy was performed on an LSM510 system (Carl Zeiss, Oberkochen, Germany) as described.
T47 6530-6572 Sentence denotes 20, 21 Cell fixation was achieved with 1 :
T48 6573-6598 Sentence denotes 1 (v/v) acetone-methanol.
T49 6599-6662 Sentence denotes Nuclei were counter-stained with 49,6-diamidino-2-phenylindole.
T50 6663-6707 Sentence denotes Real-time RT-PCR was performed as described.
T51 6708-6891 Sentence denotes 22, 23 Briefly, total RNA was extracted using RNAiso Plus reagent (Takara, Shiga, Japan) and cDNA was synthesized by Transcriptor First Strand cDNA kit (Roche, Indianapolis, IN, USA).
T52 6892-6976 Sentence denotes Quantitation of target mRNA expression was achieved with the comparative C t method.
T53 6977-7167 Sentence denotes SARS coronavirus-specific inhibition of type I IFN production by M protein M proteins from SARS coronavirus and human coronavirus HKU1 share 35% identity in amino-acid sequence ( Figure 1 ).
T54 7168-7281 Sentence denotes We have previously demonstrated the capability of SARS coronavirus M protein to antagonize type I IFN production.
T55 7282-7454 Sentence denotes 14 To determine whether human coronavirus HKU1 M protein Figure 1 Amino-acid sequence alignment of M proteins from SARS coronavirus (SCV) and human coronavirus HKU1 (HKU1).
T56 7455-7553 Sentence denotes Alignment was generated by Clustal v (EMBL-EBI server (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
T57 7554-7601 Sentence denotes The identity between the two proteins is 34.8%.
T58 7602-7652 Sentence denotes Identical residues are indicated by asterisks (*).
T59 7653-7747 Sentence denotes Strongly and weakly similar residues are highlighted by colons (:) and dots (.), respectively.
T60 7748-7887 Sentence denotes SARS, severe acute respiratory syndrome. could have similar activity, we made a side-by-side comparison of the two M proteins ( Figure 2 ).
T61 7888-7991 Sentence denotes We used two luciferase reporter constructs to measure the activation of IFN response in cultured cells.
T62 7992-8196 Sentence denotes In plasmid pISRE-Luc, the expression of luciferase reporter was driven by IFN-stimulated response elements (ISRE), which are activated by type I IFNs and also bound to IRF3 and IRF7 transcription factors.
T63 8197-8312 Sentence denotes 24, 25 For plasmid pIFNb-Luc, reporter expression directly reflects the transcriptional activity of IFN-b promoter.
T64 8313-8397 Sentence denotes To activate IFN production, we expressed RIG-I, MDA5, MAVS and TBK1 in HEK293 cells.
T65 8398-8454 Sentence denotes RIG-I and MDA5 are sensors of viral double-stranded RNA.
T66 8455-8567 Sentence denotes 11, 15 MAVS is a mitochondrial adaptor protein and TBK1 is the protein kinase that phosphorylates IRF3 and IRF7.
T67 8568-8730 Sentence denotes 1, 11 These proteins represent three critical steps in the upstream, midstream and downstream of the intracellular signaling pathway that leads to IFN production.
T68 8731-8805 Sentence denotes All proteins stimulated transcriptional activity driven by ISRE (Figure 1a
T69 8807-8949 Sentence denotes We next sought to identify the molecular determinants in SARS coronavirus M protein that mediate virus-specific suppression of IFN production.
T70 8950-9099 Sentence denotes N-linked glycosylation of viral structural proteins might affect their folding, stability, sorting and sensing by innate and adaptive immune systems.
T71 9100-9316 Sentence denotes 26 Since SARS coronavirus M protein undergoes N-linked glycosylation at a single site at the N terminus, 12, 27 we first investigated the requirement of this type of posttranslational modification for IFN antagonism.
T72 9317-9469 Sentence denotes We constructed an N-linked glycosylation-defective mutant of SARS coronavirus M protein designated M-N4Q and interrogated its IFN-antagonizing activity.
T73 9470-9728 Sentence denotes Since SARS coronavirus M protein and its M-N4Q mutant displayed equally potent activity to suppress RIG-I-and TBK-1-induced activation of IFN production (Figure 3 ), N-linked glycosylation is not influential in the suppression of IFN production by M protein.
T74 9729-9840 Sentence denotes TM1 of SARS coronavirus M protein is required for IFN antagonism SARS coronavirus M protein has three TMs (TM1:
T75 9841-9863 Sentence denotes 1-38 amino acids; TM2:
T76 9864-9888 Sentence denotes 51-69 residues; and TM3:
T77 9889-9955 Sentence denotes 76-85 residues) and a cytoplasmic endodomain (86-221 amino acids).
T78 9956-10180 Sentence denotes 27 To determine which part of this protein is required for IFN antagonism, we made and expressed in HEK293 cells two truncated mutants designated M1 and M2 containing the TMs and the endodomain separately (Figure 4a and b) .
T79 10181-10290 Sentence denotes We next compared M and its mutants M1 and M2 for their ability to circumvent IFN induction by RIG-I and TBK1.
T80 10291-10432 Sentence denotes Whereas M and M1 exhibited IFNantagonizing activity of similar potency, M2 was unable to suppress IFN-b promoter activity (Figure 4b and c) .
T81 10433-10543 Sentence denotes Hence, the TMs are required for the suppressive effect of SARS coronavirus M protein on type I IFN production.
T82 10544-10622 Sentence denotes We further investigated which of the three TMs is required for IFN antagonism.
T83 10623-10840 Sentence denotes Because the single TM was unstable and secreted out when expressed alone in cultured HEK293 cells (data not shown), we conjugated it to the endodomain, which was shown to be dispensable for IFN antagonism (Figure 4 ).
T84 10841-11055 Sentence denotes The mutants named TM19, TM29 and TM39, which respectively contain TM1 (amino acids 1-38), TM2 (amino acids 51-69) and TM3 (amino acids 76-85) fused to the endodomain, were constructed and expressed in HEK293 cells.
T85 11056-11144 Sentence denotes All mutant proteins were stable and abundantly detected in the cell lysate (Figure 5a) .
T86 11145-11248 Sentence denotes Interestingly, only TM19 was capable of inhibiting IFN-b promoter activity activated by RIG-I and TBK1.
T87 11249-11326 Sentence denotes Neither TM29 nor TM39 exhibited IFN-antagonizing activity (Figure 5b and c) .
T88 11327-11750 Sentence denotes In further support of the importance of TM1, a chimeric M protein containing TM1 (residues 1-38) of SARS coronavirus and the cytoplasmic domain (87-223 residues) derived from human coronavirus HKU1 was equally active as SARS coronavirus M protein in the suppression of IFN production induced by RIG-I or TBK1, whereas another chimera carrying TM1 of human coronavirus HKU1 was totally inactive in this assay (Figure 6a-c) .
T89 11751-11918 Sentence denotes Taken together, because TM2, TM3 and endodomain are dispensable for IFN antagonism, only TM1 is required and probably sufficient for the suppression of IFN production.
T90 11919-12153 Sentence denotes Mechanism of TM1 suppression of IFN production SARS coronavirus M protein suppresses IFN production by associating with and sequestering transducer proteins, thereby preventing the formation of functional TRAF3-TANK-TBK1/IKKe complex.
T91 12154-12318 Sentence denotes 14 If TM1 mediates IFN antagonism of SARS coronavirus M protein, it should be able to associate with transducer proteins and impede TRAF3 interaction with partners.
T92 12319-12478 Sentence denotes In addition, because Golgi targeting would be the cause of transducer protein sequestration, 14 TM1 should also be expected to be found in the Golgi apparatus.
T93 12479-12588 Sentence denotes Both SARS coronavirus M protein and the M1 mutant are known to localize predominantly to the Golgi apparatus.
T94 12589-12730 Sentence denotes 14, 27 In agreement with this, SARS coronavirus M protein colocalized substantially with GM130 (Figure 7a-c) , a marker of the Golgi complex.
T95 12731-12816 Sentence denotes 28 Interestingly, Golgi localization was also evident for TM19 mutant (Figure 7g-i) .
T96 12817-12938 Sentence denotes Neither M nor TM19 colocalized significantly with calnexin (Figure 7d-f and j-l) , a marker of the endoplasmic reticulum.
T97 12939-13024 Sentence denotes 29 In contrast, TM29 and TM39 were largely concentrated in the endoplasmic reticulum.
T98 13025-13133 Sentence denotes Their localization patterns were similar to that of calnexin, but distinct from that of GM130 (Figure 7mx) .
T99 13134-13211 Sentence denotes In other words, TM2, TM3 or endodomain was not essential for Golgi targeting.
T100 13212-13316 Sentence denotes Thus, TM1 is both required and probably sufficient for Golgi localization of SARS coronavirus M protein.
T101 13317-13490 Sentence denotes On the other hand, reciprocal immunoprecipitation and immunoblotting experiments indicated the association of TM19 with RIG-I, TRAF3, TBK1 and IKKe (Figure 8a, lanes 1-4) .
T102 13491-13560 Sentence denotes Neither TM29 nor TM39 had the same property (Figure 8a, lanes 5-12) .
T103 13561-13733 Sentence denotes In addition, the interaction of TRAF3 with TBK1, IKKe and TANK was inhibited in the presence of TM19 (Figure 8b, lanes 1-6) , but not TM29 or TM39 (Figure 8b, lanes 7-10) .
T104 13734-13938 Sentence denotes In light of this, TM1 is both required and probably sufficient for interacting with TRAF3 and other transducer proteins and thereby preventing TRAF3 from engaging its partners such as TBK1, IKKe and TANK.
T105 13939-14037 Sentence denotes Indeed, both SARS coronavirus M protein and its TM19 mutant colocalized with TRAF3 (Figure 9a-f ).
T106 14038-14263 Sentence denotes However, human coronavirus HKU1 M protein also localized to the Golgi apparatus (data not shown) and colocalized with TRAF3 (Figure 9g-i) , but did not impede the interaction of TRAF3 with TANK, TBK1 or IKKe (data not shown).
T107 14264-14388 Sentence denotes Plausibly, Golgi localization and interaction with TRAF3 are required but not sufficient for suppression of IRF3 activation.
T108 14389-14602 Sentence denotes In this study, we showed that SARS coronavirus M protein suppressed the production of type I IFNs in a virus-specific manner and it did not share IFN-antagonizing property with M protein of human coronavirus HKU1.
T109 14603-14826 Sentence denotes IFN antagonism of SARS coronavirus M protein was mediated by N-terminal TM1 (amino acids 1-38), which targets M protein to the Golgi complex and associates with TRAF3 to prevent it from interacting with TANK, TBK1 and IKKe.
T110 14827-14948 Sentence denotes Our findings provide additional molecular details for suppression of type I IFN production by SARS coronavirus M protein.
T111 14949-15049 Sentence denotes The inability of human coronavirus HKU1 M protein to suppress IFN production was not too surprising.
T112 15050-15238 Sentence denotes Although the two full M proteins of SARS coronavirus and human coronavirus HKU1 share 35% identity, only 26% of the amino-acid residues in the TM1 region of the two proteins are identical.
T113 15239-15328 Sentence denotes This difference in the TM1 region might be critical in governing the inhibition of TRAF3.
T114 15329-15440 Sentence denotes SARS coronavirus is known to suppress innate antiviral response at multiple levels leading to a severe disease.
T115 15441-15501 Sentence denotes 4, 9 This might be similar to the emerging MERS coronavirus.
T116 15502-15640 Sentence denotes 30, 31 In contrast, human coronavirus HKU1 is commonly found in human population and is associated with less severe respiratory illnesses.
T117 15641-15718 Sentence denotes 6-8 Accordingly, it might not have the same ability to evade innate immunity.
T118 15719-15842 Sentence denotes Viral proteins are not well characterized and IFN-antagonizing proteins have not been identified in human coronavirus HKU1.
T119 15843-16063 Sentence denotes 32 It remains to be seen whether the distinct properties of M proteins in the suppression of IFN production could account at least partly for the severity of disease caused by SARS coronavirus and human coronavirus HKU1.
T120 16064-16374 Sentence denotes In one model to explain this difference, SARS coronavirus and MERS coronavirus are not yet adapted to the new environment in human, 4, 9, 33 whereas the well-adapted human coronavirus HKU1 and the other human coronaviruses such as OC43 and 229E have lost some of their immunosuppressive tools during evolution.
T121 16375-16542 Sentence denotes To test this idea, the coronaviruses in the two groups should be compared more thoroughly for their abilities to induce IFNs and to suppress IFN production and action.
T122 16543-16656 Sentence denotes Particularly, the IFN-antagonizing property of M proteins from more coronaviruses should be assessed in parallel.
T123 16657-16900 Sentence denotes Although attempts have been made to culture the virus in primary human ciliated airway epithelial cells and type II alveolar epithelial cells, [34] [35] [36] clinical isolates of human coronavirus HKU1 remain unculturable in most laboratories.
T124 16901-16958 Sentence denotes This hampers further comparative study in cultured cells.
T125 16959-17089 Sentence denotes To remedy this, the dynamics of IFN induction should be analyzed in human subjects naturally infected with human coronavirus HKU1.
T126 17090-17269 Sentence denotes We not only defined a minimal TM1 domain in SARS coronavirus M protein that mediates suppression of IFN production, but also provided new mechanistic details for this suppression.
T127 17270-17469 Sentence denotes Although TM1 alone is unstable and secreted out, it is stable and functional when conjugated to TM2-TM3 or endodomain of SARS coronavirus M protein or of human coronavirus M protein (data not shown).
T128 17470-17653 Sentence denotes TM1 contains the critical determinants for Golgi localization, for association with RIG-I, TRAF3, TBK1 and IKKe, and for prevention of TRAF3 from interacting with TANK, TBK1 and IKKe.
T129 17654-17801 Sentence denotes Our results are compatible with the notion that TM1 targets SARS coronavirus M protein and its associated cellular proteins to the Golgi apparatus.
T130 17802-17910 Sentence denotes It will be of importance to clarify exactly how this might contribute to the suppression of IRF3 activation.
T131 17911-18044 Sentence denotes Notably, human coronavirus HKU1 M protein also targets the Golgi complex, interacts with TRAF3, but does not suppress IFN production.
T132 18045-18149 Sentence denotes Hence, Golgi localization and interaction with TRAF3 are required but not sufficient for IFN antagonism.
T133 18150-18329 Sentence denotes Mechanistically, the perturbation of the interaction of TRAF3 with TANK, TBK1 and IKKe by TM1 of SARS coronavirus M protein is more critical in the suppression of IRF3 activation.
T134 18330-18486 Sentence denotes This could plausibly be mediated by preoccupation and steric hindrance, but further investigations are required to provide additional support to this model.
T135 18487-18660 Sentence denotes For example, it will be of interest to see whether artificial fusion of TM1 to TRAF3 might impede its binding with TBK1 and IKKe or inhibit its activation of IFN production.
T136 18661-18818 Sentence denotes Mapping and comparing the TM1-and TBK1-interacting domains in TRAF3 would also shed light on how the binding of TRAF3 with TM1 affects the binding with TBK1.
T137 18819-19028 Sentence denotes More importantly, a thorough comparison of the TM1 regions of SARS coronavirus and human coronavirus HKU1 will define the critical residues for the suppression of TRAF3 interaction with downstream transducers.
T138 19029-19389 Sentence denotes Nevertheless, this model of suppression by sequestration and pre-occupation represents a new mechanism for immune evasion, which might also be used by other viral and cellular proteins such as microtubule-associated TRAF3-binding protein MIP-T3, which localizes to the centrosome and cilia and also impedes the formation of functional TRAF3-containing complex.
T139 19390-19591 Sentence denotes 37 Furthermore, our definition of a small TM1 domain that mediates immune evasion will pave the way for rational design and development of new immunosuppressive Cells were then stained for M and TRAF3.
T140 19592-19643 Sentence denotes Nuclear morphology (blue) was visualized with DAPI.
T141 19644-19708 Sentence denotes Specific fluorescent signals from V5 and TRAF3 were then merged.
T142 19709-19774 Sentence denotes Transfected cells in the merged panels are highlighted by arrows.
T143 19775-19806 Sentence denotes Colocalization appeared yellow.
T144 19807-19884 Sentence denotes All panels shown are representative of the results of triplicate experiments.
T145 19885-19895 Sentence denotes Bar520 mm.
T146 19896-19990 Sentence denotes DAPI, 49,6-diamidino-2-phenylindole; IFN, interferon; SARS, severe acute respiratory syndrome.