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CORD-19:eaeb1d7ab877eef0ecb07e5e944b64baa724cbd7 JSONTXT 8 Projects

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Id Subject Object Predicate Lexical cue
T1 0-199 Sentence denotes Heat shock protein 70 in lung and kidney of specific-pathogen-free chickens is a receptor-associated protein that interacts with the binding domain of the spike protein of infectious bronchitis virus
T2 201-209 Sentence denotes Abstract
T3 210-290 Sentence denotes Avian infectious bronchitis virus (IBV) is a member of the family Coronaviridae.
T4 291-432 Sentence denotes A binding domain that mediates the attachment of the virus to its receptor has been identified in the S1 protein of prototype IBV strain M41.
T5 433-560 Sentence denotes In this study, we identified this binding domain in a different strain, as well as the cellular proteins that interact with it.
T6 561-745 Sentence denotes First, we expressed the S1 N proteins (residues 19-270) of M41 and another isolate, SCZJ3, and compared the binding capacities of recombinant S1 N -M41 and S1 N -SCZJ3 to host tissues.
T7 746-939 Sentence denotes Protein histochemistry showed that both S1 N -M41 and S1 N -SCZJ3 could bind to lung and kidney, and that recombinant S1 N -SCZJ3 displayed a distinctive staining pattern in the proventriculus.
T8 940-1056 Sentence denotes Recombinant S1 N -SCZJ3 was then employed to purify binding-associated proteins in lung, kidney, and proventriculus.
T9 1057-1186 Sentence denotes Using an affinity chromatography assay, two common bands of about 60 kDa and 70 kDa were obtained from the total tissue proteins.
T10 1187-1368 Sentence denotes These protein bands were identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) as protein disulfide isomerase (PDI) and heat shock protein 70 (HSP70).
T11 1369-1511 Sentence denotes Finally, infection of chicken embryo kidney (CEK) cells by SCZJ3 was found to be inhibited by anti-HSP70 but not anti-PDI polyclonal antibody.
T12 1512-1657 Sentence denotes These data indicate that HSP70 is part of the receptor complex of IBV and might help to understand the mechanism of S-mediated cell entry of IBV.
T13 1659-1735 Sentence denotes Infectious bronchitis virus (IBV) is a member of the genus Gammacoronavirus.
T14 1736-1821 Sentence denotes It is an enveloped virus with a positive-strand RNA genome, which is highly variable.
T15 1822-1884 Sentence denotes IBV variants of different genotypes have been found worldwide.
T16 1885-1995 Sentence denotes Mutations have been mainly found in the S1 protein that can result in poor cross-protection between genotypes.
T17 1996-2162 Sentence denotes The virus can replicate in many tissues [1] and causes various pathological changes, including respiratory symptoms and lesions of the kidney and proventriculus [2] .
T18 2163-2327 Sentence denotes The infection is initiated by the binding of the spike (S) protein to host tissues [3] , which has been shown to be a determinant of tropism and pathogenicity [4] .
T19 2328-2456 Sentence denotes A functional region has been identified on the S1 subunit of several coronavirus, called the receptor-binding domain (RBD) [5] .
T20 2457-2602 Sentence denotes Certain residues in the RBD are critical for binding activity, and single mutations may abolish or change the binding activity of the virus [6] .
T21 2603-2728 Sentence denotes In the case of IBV, the N-terminal portion of S1 (residues was mapped as the binding domain of the Mass type strain M41 [7] .
T22 2729-2837 Sentence denotes However, the S1 subunit is highly variable, and mutations have been found in S1 of different genotypes [8] .
T23 2838-2912 Sentence denotes These mutations may influence the binding activity of RBD to host tissues.
T24 2913-2996 Sentence denotes Thus, we attempted to identify the receptor-binding domain of a different genotype.
T25 2997-2999 Sentence denotes Z.
T26 3000-3012 Sentence denotes Zhang and X.
T27 3013-3054 Sentence denotes Yang contributed equally to the research.
T28 3055-3228 Sentence denotes Electronic supplementary material The online version of this article (doi:10.1007/s00705-017-3280-x) contains supplementary material, which is available to authorized users.
T29 3229-3455 Sentence denotes Here, we have identified the binding domain of a prevalent strain SCZJ3, which has the characteristics of the QX-like genotype and shows only 75.9% amino acid sequence identity to M41 in the S1 N region (residues 19-270) [9] .
T30 3456-3545 Sentence denotes In vivo, M41 is a respiratory IBV strain that mainly affects the upper respiratory tract.
T31 3546-3653 Sentence denotes QX-like strains have been reported to be associated with distinct lesions of the proventriculus and kidney.
T32 3654-3729 Sentence denotes M41 was first isolated in 1996 and is now becoming epidemic in China [10] .
T33 3730-3908 Sentence denotes To investigate differences in the pathogenicity of QX-like and Mass-like strains [11] , the binding of S1 N -SCZJ3 and S1 N -M41 to lung, kidney and proventriculus were analyzed.
T34 3909-4065 Sentence denotes The virus-cell interaction of IBV is complicated; the binding of virus to host cells may initiate a chain of molecular interactions that enable viral entry.
T35 4066-4149 Sentence denotes Sialic acid and heparan sulfate have been reported to be involved in IBV infection.
T36 4150-4236 Sentence denotes Removal of sialic acid can abolish the binding of S1 to respiratory tissues [12, 13] .
T37 4237-4385 Sentence denotes However, sialic acid is present on cells that are not sensitive to IBV, indicating that sialic acid is not the only molecule that binds to IBV [4] .
T38 4386-4561 Sentence denotes Heparan sulfate was identified as a binding molecule only for the cell-adapted strain Beaudette, with the binding sites located within the S2 subunit (residues 686-691) [14] .
T39 4562-4655 Sentence denotes Up to now, there has been little study of the protein involved in the binding process of IBV.
T40 4656-4797 Sentence denotes In studies of SASR and 229E, RBDs showed specific binding to host receptors and showed higher binding activity than full-length S1 [15, 16] .
T41 4798-4880 Sentence denotes This binding activity has been utilized to define the receptor of virus [17, 18] .
T42 4881-5004 Sentence denotes In the present study, an affinity chromatography assay was used to isolate proteins that interacted with S1 N -SCZJ3 [19] .
T43 5005-5101 Sentence denotes Using an infection inhibition assay, we found that HSP70 is part of the receptor complex of IBV.
T44 5102-5212 Sentence denotes Infectious bronchitis virus (IBV) strains SCZJ3 (JF951370.1) and M41 (DQ834384.1) were kept in our laboratory.
T45 5213-5298 Sentence denotes Spodoptera frugiperda (Sf9) cells were maintained in Sf-900 TM SFM II medium (Gibco).
T46 5299-5470 Sentence denotes Chicken embryo kidney (CEK) cells were generated from fifteenday-old chicken embryos and maintained in DMEM with high glucose supplemented with 10% fetal calf serum (FCS).
T47 5471-5588 Sentence denotes Viral RNA was extracted from allantoic fluid and reverse transcribed using a PrimerScript TM RT Reagent Kit (Takara).
T48 5589-5789 Sentence denotes The sequences encoding S1 N -SCZJ3 and S1 N -M41 (nucleotides 55 to 810) were amplified and cloned into the Bac-to-Bac baculovirus expression system according to the manufacturer's instructions [20] .
T49 5790-5911 Sentence denotes Sf9 cell monolayers were infected with baculoviral stock at an MOI of 5 and incubated at 27°C for 72 h before harvesting.
T50 5912-6034 Sentence denotes Cells were frozen and thawed and then suspended in lysis buffer (10 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0).
T51 6035-6192 Sentence denotes The lysates were then centrifuged at 10,000 g for 10 min at 4°C, and the supernatants were purified using an Ni-NTA column (Ni Sepharose TM , GE Healthcare).
T52 6193-6425 Sentence denotes The purified proteins (S1 N -SCZJ3 and S1 N -M41) and PNGase F (NEB)-treated proteins were separated by 10% SDS-PAGE and detected using polyclonal IBV-M41 antiserum (diluted 1:100 in PBS, China Institute of Veterinary Drug Control).
T53 6426-6481 Sentence denotes Western blot analysis was performed as described [21] .
T54 6482-6592 Sentence denotes Specific-pathogen-free (SPF) eggs were purchased from Merial-Beijing and incubated in a 37°C humidity chamber.
T55 6593-6665 Sentence denotes Tissues were taken from 5-week-old chickens and fixed with 10% formalin.
T56 6666-6722 Sentence denotes Protein histochemistry was performed as described [13] .
T57 6723-6831 Sentence denotes S1 N -SCZJ3, S1 N -M41 (30 lg/ ml) and PBS as a control were applied to slides and incubated at 37°C for 2h.
T58 6832-6963 Sentence denotes Binding was then detected using a monoclonal antibody against the His-tag (HRP-conjugated, diluted 1:100 with PBS) at 37°C for 1 h.
T59 6964-7075 Sentence denotes Lung, kidney and proventriculus tissues were taken from 3-day-old SPF chickens and frozen with liquid nitrogen.
T60 7076-7180 Sentence denotes Tissues were ground and suspended in phosphate buffer (50 mg, 1 ml) containing 1% Triton X-100 and PMSF.
T61 7181-7278 Sentence denotes The lysates were subjected to ultrasonic treatment and centrifuged at 10,000 g for 10 min at 4°C.
T62 7279-7374 Sentence denotes The concentrations of the supernatants were determined using a NanoDrop 2000 spectrophotometer.
T63 7375-7542 Sentence denotes Ni-NTA resin coupled with S1 N -SCZJ3 or Ni-NTA resin alone as control (1 ml) was incubated with total tissue proteins (1 ml) and rotated end over end for 4 h at 16°C.
T64 7543-7597 Sentence denotes The tube was then centrifuged at 3000 g for 2 minutes.
T65 7598-7872 Sentence denotes Finally, the resins were washed four times with wash buffer (5 ml) with increasing concentrations of imidazole (20, 50, 100, 250 mM imidazole, 50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0), followed by 10 ml of elution buffer (50 mM NaH 2 PO 4 , 300 mM NaCl, 100 mM EDTA, pH 8.0).
T66 7873-8019 Sentence denotes The eluates were separated by 10% SDS-PAGE, and the bands were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS).
T67 8020-8229 Sentence denotes Binding blocking assay SCZJ3 virus (10 3 EID 50 ) diluted in PBS was pretreated with increasing concentrations (22, 45, 90 , 180 lg/ml) of chromatography eluates in equal volumes and incubated at 37°C for 1 h.
T68 8230-8389 Sentence denotes Chicken embryo kidney cells cultured in 6-well plates were infected with 1-ml incubation at 37°C for 1 h, and cells were harvested after three washes with PBS.
T69 8390-8457 Sentence denotes The virus loads were determined by real-time PCR as described [1] .
T70 8458-8662 Sentence denotes Chicken embryo kidney cells were cultured in 24-well plates, and the cells were incubated with increasing concentrations of anti-PDI or anti-HSP70 polyclonal antibody for 1 h at 37°C, or PBS as a control.
T71 8663-8763 Sentence denotes The cells were then infected with SCZJ3 virus (10 3 EID 50 , 200 ll) diluted in PBS for 1 h at 37°C.
T72 8764-8813 Sentence denotes Cells were harvested after three washes with PBS.
T73 8814-8862 Sentence denotes The virus loads were determined by realtime PCR.
T74 8863-9030 Sentence denotes Expression, purification and histochemistry of S1 N -SCZJ3 and S1 N -M41 SCZJ3 and M41 are indeed two separate IBV strains in terms of both genotype and pathogenicity.
T75 9031-9236 Sentence denotes The predicted amino acid sequences of the N-terminal peptides of S1 (S1 N , aa 19-270) were aligned by the ClustalW method, and the genetic distance between the S1 N -SCZJ3 S1 N and M41 peptides was 0.315.
T76 9237-9407 Sentence denotes Four main variable regions were found at aa positions 20-26, 53-80, 117-122, and 129-136, containing the amino acids critical for M41 (N38T, H43Q, P63S, T69V) (Fig. 1A) .
T77 9408-9497 Sentence denotes We then expressed S1 N (residues 19-270) of SCZJ3 and M41 in a baculovirus-insect system.
T78 9498-9725 Sentence denotes As shown in Fig. 1B , both proteins were highly glycosylated and could be detected by polyclonal IBV-M41 antiserum before and after treatment with PNGase F, indicating that S1 N -SCZJ3 and S1 N -M41 were expressed successfully.
T79 9726-9862 Sentence denotes To compare the biological activities of S1 N -SCZJ3 and S1 N -M41, we examined their ability to bind to lung, kidney and proventriculus.
T80 9863-10072 Sentence denotes Binding to peribronchial epithelial cells in the lung and renal tubular epithelial cells in the kidney was detected, indicating that the S1 N region of both M41 and SCZJ3 could play a role in receptor binding.
T81 10073-10238 Sentence denotes In addition, S1 N -SCZJ3 showed distinct binding to proventriculus mucous membrane epithelial cells (Fig 1C) , which may correlate the tissue tropism of SCZJ3 [10] .
T82 10239-10338 Sentence denotes Next, we decided to investigate the binding-associated proteins in lung, kidney and proventriculus.
T83 10339-10528 Sentence denotes Due to the lack of cultured cell lines for IBV, we attempted to isolate proteins that interact with S1 N -SCZJ3 in tissue lysates, which may increase the abundance of the obtained proteins.
T84 10529-10694 Sentence denotes Tissue lysates were incubated with Ni-NTA resins containing S1 N -SCZJ3 as ligand, and after extensive washing, chromatography eluates were analyzed by 10% SDS-PAGE.
T85 10695-10859 Sentence denotes As shown in Fig. 2A , two main common bands were detected (60 kDa and 70 kDa), and two distinctive bands were detected in proventriculus tissue (27 kDa and 45 kDa).
T86 10860-11054 Sentence denotes A similar result was obtained using a virus overlay protein binding assay (VOPBA), demonstrating that the molecules isolated using recombinant S1 N -SCZJ3 are specific (Supplementary Figure 1) .
T87 11055-11212 Sentence denotes In tissue lysates incubated with Ni-NTA resin alone, the nonspecific bands were weak and diverse under the same elution conditions (Supplementary Figure 2) .
T88 11213-11374 Sentence denotes The bands were then analyzed by trypsin digestion and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS), followed by Mascot search analysis.
T89 11375-11558 Sentence denotes The proteins were identified as 14-3-3 protein (27 kDa), tropomyosin (45 kDa), protein disulfide isomerase (PDI) (60 kDa) and heat shock protein 70 (70 kDa) (Supplementary Figure 3 ).
T90 11559-11819 Sentence denotes To confirm the presence of receptor-associated proteins in the chromatography eluates isolated by S1 N -SCZJ3, we attempted to block the binding of SCZJ3 to chicken embryo kidney cells (CEK) by pretreating virus with chromatography eluates, as shown in Fig 2A.
T91 11820-11948 Sentence denotes Before infection, SCZJ3 virus was incubated with purified chromatography eluates of lung (L), kidney (K) and proventriculus (P).
T92 11949-12053 Sentence denotes After infection, virus loads on CEK cells were determined by real-time PCR as described previously [1] .
T93 12054-12316 Sentence denotes As shown in Fig. 2B , virus binding was efficiently blocked by lung and kidney chromatography elutes in a dose-dependent manner, which is consistent with the hypothesis that certain molecules in lung and kidney eluates comprise the virus receptor complex of IBV.
T94 12317-12443 Sentence denotes However, the case of the proventriculus eluate is more complicated because certain molecules may enhance the binding of SCZJ3.
T95 12444-12663 Sentence denotes In order to determine whether the two proteins corresponding to the main bands (PDI and HSP70) in lung and kidney eluates are involved in the binding of SCZJ3, we examined whether these proteins could inhibit infection.
T96 12664-12785 Sentence denotes Before infection, CEK cells were pretreated with anti-PDI and anti-HSP70 polyclonal antibody at different concentrations.
T97 12786-12899 Sentence denotes As shown in Fig. 2C , reduction of virus load in CEK cells was observed after treatment with anti-HSP70 antibody.
T98 12900-12990 Sentence denotes However, no significant reduction was detected in cells pretreated with anti-PDI antibody.
T99 12991-13072 Sentence denotes These results indicate that HSP70 is essential for the binding of SCZJ3 to cells.
T100 13073-13169 Sentence denotes The presence of HSP70 in chromatography eluates was confirmed by Western blot analysis (Fig. 2D)
T101 13171-13239 Sentence denotes The spike (S) protein is a determinant of coronavirus tropism [13] .
T102 13240-13316 Sentence denotes Retargeting the ectodomain of the protein can change the host tropism [22] .
T103 13317-13476 Sentence denotes IBV isolates have been reported in recent years that not only affect the upper respiratory tract but also show broader tissue tropism and higher pathogenicity.
T104 13477-13609 Sentence denotes The QX-like genotype is one of the epidemic IBV strains in China, according our epidemiology study based on analysis of the S1 gene.
T105 13610-13710 Sentence denotes Distinctive pathological changes to the proventriculus by QX-like strains have been described [23] .
T106 13711-13824 Sentence denotes Here, we demonstrate that the binding characteristics of recombinant S1 N -SCZJ3 is accordant with virus tropism.
T107 13825-13933 Sentence denotes In a recent study, a Y43H change in the ArkDPI S1 protein was shown to enhance binding to the trachea [24] .
T108 13934-14137 Sentence denotes In comparison to S1 N -M41, four main divergent regions were found in the S1 N -SCZJ3 sequence (Fig 1A) , and certain mutations may correlate with the binding properties of S1 N -SCZJ3 and virus tropism.
T109 14138-14329 Sentence denotes A galactose-binding-like domain was found in the S1 protein between aa 137 and 230 by structure prediction Fig. 1 (A) Amino acid sequence alignment of S1 N (residues 19-270) of M41 and SCZJ3.
T110 14330-14407 Sentence denotes Four main divergent regions are located at aa 20-26, 53-80, 117-122, 129-136.
T111 14408-14502 Sentence denotes The critical amino acids for M41 (N38T, H43Q, P63S, T69V) are shown in bold. (B) Western blot.
T112 14503-14595 Sentence denotes S1 N -SCZJ3 and S1 N -M41 were purified using an Ni-NTA column and analyzed by Western blot.
T113 14596-14839 Sentence denotes The samples were both recognized by polyclonal IBV-M41 antiserum before and after treatment with PNGase F. (C) Histochemistry was performed by incubating S1 N -M41 and S1 N -SCZJ3 with lung, kidney and proventriculus samples of PBS as control.
T114 14840-14963 Sentence denotes Binding to peribronchial epithelial cells in lung tissue and to the renal tubular epithelial cells in kidney were detected.
T115 14964-15079 Sentence denotes Distinctive binding of S1 N -SCZJ3 to mucous membrane epithelial cells was detected in the proventriculus (Phyre2).
T116 15080-15242 Sentence denotes As shown in Fig. 1A , this domain is relatively conserved in S1 N -SCZJ3 and S1 N -M41 and may be important for the virus to maintain its ability to infect birds.
T117 15243-15390 Sentence denotes However, the amino acid sequence from aa 19 to 36 is highly variable, and mutations may lead to conformational changes in S1 N -SCZJ3 to S1 N -M41.
T118 15391-15529 Sentence denotes Two amino acid changes at the N-terminal spike protein of transmissible gastroenteritis virus result in the loss of enteric tropism [25] .
T119 15530-15606 Sentence denotes In the case of SARS coronavirus, the structure of the RBD has been analyzed.
T120 15607-15786 Sentence denotes The receptor-binding motif (RBM) that makes contacts with ACE2 has been identified at residues 424 to 494, and mutations in the RBM are critical for affinity or infectivity [26] .
T121 15787-15999 Sentence denotes In IBV, a study of the conformation of the binding domains of different genotypes may help to identify the main binding sites that contact cellular receptors and investigate their correlation with tissue tropism.
T122 16000-16080 Sentence denotes Affinity chromatography is an efficient method to isolate virus receptors [19] .
T123 16081-16226 Sentence denotes However, inhibition of binding and infection provides only indirect evidence for identifying virus receptors, and more-direct evidence is needed.
T124 16227-16472 Sentence denotes However, BHK-21 cells transfected with one of the proteins described above were not sensitive to IBV infection (not shown), suggesting that the virus-receptor interaction of IBV is a multi-step process or that the infection is celltype-specific.
T125 16473-16548 Sentence denotes Certain molecules may be critical for virus reproduction in the cell [27] .
T126 16549-16665 Sentence denotes Thus, reconstruction of the infection pathway of IBV will be more convenient if chicken-derived cell lines are used.
T127 16666-16811 Sentence denotes The presence of the S1 N -SCZJ3 protein and other proteins in chromatography eluates complicates the analysis of the functions of these proteins.
T128 16812-16890 Sentence denotes The functions of 14-3-3 and tropomyosin in virus infection need further study.
T129 16891-16968 Sentence denotes These proteins may be important for infection of the proventriculus by SCZJ3.
T130 16969-17112 Sentence denotes Surface-displayed HSP70 has been reported to be part of receptor complex in the case of dengue virus and Japanese encephalitis virus [17, 28] .
T131 17113-17234 Sentence denotes HSP70 is a kind of stress protein that mainly induce ATP binding, and hydrolysis regulates its affinity for polypeptides.
T132 17235-17384 Sentence denotes HSPs are important for folding of viral proteins [29] . b Fig. 2 (A) Isolation of S1 N -SCZJ3 binding-associated proteins by affinity chromatography.
T133 17385-17502 Sentence denotes Total proteins of lung (L), kidney (K), and proventriculus (P) were incubated with S1 N -SCZJ3-coupled Ni-NTA resins.
T134 17503-17560 Sentence denotes The chromatography eluates were analyzed by 10% SDS-PAGE.
T135 17561-17803 Sentence denotes Four bands were obtained (as indicated) and identified as 14-3-3 protein (27 kDa), tropomyosin (45 kDa), protein-disulfide isomerase (60 kDa), and heat shock protein 70 (70 kDa) by LC-MS/ MS. (B) Blocking of binding by chromatography eluates.
T136 17804-17954 Sentence denotes SCZJ3 (10 3 EID 50 ) was pre-incubated with affinity chromatography eluates of lung (L), kidney (K), proventriculus (P) and PBS as virus control (VC).
T137 17955-18078 Sentence denotes Subsequently, CEK cells were infected with 1 ml of these preparations and the viral loads were determined by real-time PCR.
T138 18079-18145 Sentence denotes (C) Inhibition of infection by anti-HSP70 and anti-PDI antibodies.
T139 18146-18257 Sentence denotes CEK cells were pretreated with increasing concentrations of anti-HSP70 and anti-PDI antibodies for 1 h at 37°C.
T140 18258-18530 Sentence denotes Cells were infected with SCZJ3 (10 3 EID 50 , 200 ll), and the viral loads were determined by real-time PCR. (D) Affinity chromatography eluates were incubated with anti-HSP70 antibody HSP70 is probably upregulated by infection with a virus like avirulent NDV or by fever.
T141 18531-18605 Sentence denotes This might help to explain the co-infections with IBV and other pathogens.
T142 18606-18745 Sentence denotes In conclusion, this study revealed that S1 N -SCZJ3 is the receptor binding domain of SCZJ3 and that HSP70 is part of the receptor complex.
T143 18746-18899 Sentence denotes Studying the interaction between S1 N peptides and the receptor complex will allow us to better understand the infection process and pathogenesis of IBV.