Structural and functional characterization of MERS coronavirus papain-like protease
© Lin et al.; licensee BioMed Central Ltd. 2014
Received: 23 April 2014
Accepted: 19 May 2014
Published: 4 June 2014
A new highly pathogenic human coronavirus (CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries since September 2012. Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally. Studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent. In the present study, MERS-CoV papain-like protease (PLpro) is expressed, and its structural and functional consequences are elucidated.
Circular dichroism and Tyr/Trp fluorescence analyses indicated that the secondary and tertiary structure of MERS-CoV PLpro is well organized and folded. Analytical ultracentrifugation analyses demonstrated that MERS-CoV PLpro is a monomer in solution. The steady-state kinetic and deubiquitination activity assays indicated that MERS-CoV PLpro exhibits potent deubiquitination activity but lower proteolytic activity, compared with SARS-CoV PLpro. A natural mutation, Leu105, is the major reason for this difference.
Overall, MERS-CoV PLpro bound by an endogenous metal ion shows a folded structure and potent proteolytic and deubiquitination activity. These findings provide important insights into the structural and functional properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro against highly pathogenic coronaviruses.
KeywordsMERS coronavirus Papain-like protease Deubiquitination Antiviral target
In September 2012, a new highly pathogenic human coronavirus (CoV)1, Middle East respiratory syndrome coronavirus (MERS-CoV), has emerged in Jeddah and Saudi Arabia and quickly spread to some European countries [1–3]. The virus causes symptoms similar to Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), yet involving an additional component of acute renal failure . Until 15 May 2014, it has infected at least 572 people with a fatality rate of about 30% globally (World Health Organization, global alert and response, http://www.who.int/csr/don/2014_05_15_mers/en/). Recently, human-to-human transmission of MERS-CoV has been confirmed; albeit, a serological study of major livestock suggested dromedary camels also to be a possible host [5, 6]. Nevertheless, these findings indicate that the virus have the opportunity to spread globally and pose a significant threat to world health and the economy. Therefore, studies to understand the virus and to develop antiviral drugs or therapy are necessary and urgent.
Like other CoVs, the MERS-CoV nonstructural polyproteins (pp1a and pp1ab) are cleaved by two types of viral cysteine proteases, a main protease (EC 126.96.36.199) and a papain-like protease (PLpro) (EC 188.8.131.52) . This processing is considered to be a suitable antiviral target because it is required for viral maturation. Unfortunately, initial screening of the existing SARS-CoV PLpro inhibitor, a benzodioxolane derivative against MERS-CoV PLpro, revealed no significant inhibition . The difference represents the requirement of further understanding the MERS-CoV PLpro. In addition to proteolytic activity, similar to those of SARS-CoV, NL63-CoV and murine hepatitis virus, MERS-CoV PLpro acts on both deubiquitination and ISG15-linked ISGylation [8–11]. As a viral deubiquitinating protease (DUB), MERS-CoV PLpro is able to deubiquitinate interferon regulatory factor 3 (IRF3), which can prevent its nuclear translocation and suppress production of interferon β . These studies support the multifunctional nature of coronaviral PLpro. Recently, with the crystal structure of SARS-CoV PLpro C112S mutant in complex with ubiquitin (Ub), we have demonstrated that Ub core (residue 1–72) makes mostly hydrophilic interactions with PLpro, while the Leu-Arg-Gly-Gly C-terminus of Ub is located in the catalytic cleft of PLpro, mimicking the P4-P1 residues . This bound pattern is similar to that of the ubiquitin-specific proteases (USPs), one of the five distinct DUB families [13, 14].
The MERS-CoV PLpro domain in nsp3 of the pp1a proteins (residue 1484–1800) has been identified [7, 10, 15]. Like other PLpro, there is a catalytic triad consisting of the residues Cys1592, His1759 and Asp1774. Homology modeling suggests that MERS-CoV PLpro, similar to other known PLpro, may have a right-hand-like architecture constituted by palm, thumb, and fingers domains, although their sequence identity are only about 30% . Furthermore, MERS-CoV PLpro is able to recognize and cleave at the LXGG consensus cleavage site, which is essential for most CoV PLpro-mediated processing . Despite this large body of knowledge on MERS-CoV PLpro, in the absence of detailed structural and functional characterization, the molecular basis for its catalytic mechanism remains poorly unknown.
Here, we expressed and purified the MERS-CoV PLpro by E. coli with high yield and high purity. The secondary, tertiary and quaternary structure of MERS-CoV PLpro was then investigated by circular dichroism (CD) spectroscopy, Tyr/Trp fluoresecence and analytical ultracentrifugation (AUC), respectively. The kinetic and DUB activity assays indicated that MERS-CoV PLpro exhibits potent DUB activity but lower proteolytic activity, compared with SARS-CoV PLpro. The present study provides a foundation for understanding the structural and biochemical properties of coronaviral PLpro family, which is applicable to develop strategies inhibiting PLpro for the effective control of highly pathogenic coronaviral infection.
Expression plasmid construction
The sequence of MERS-CoV PLpro (GenBank accession number NC_019843.2; polyprotein residues 1484–1800) was synthesized (MDBio Inc.), digested by Nco I-Xho I and then inserted into the pET-28a(+) vector (Novagen). In the construct, the 6 x His tag was retained at the C-terminus. The reading frame was confirmed by sequencing.
Expression and purification of MERS-CoV PLpro
The expression vector was transformed into E. coli BL21 (DE3) cells (Novagen). For large scaled protein expression, cultures were grown in LB medium of 0.8 liter at 37°C for 4 h, induced with 0.4 mM isopropyl-β-D-thiogalactopyranoside, and incubated overnight at 20°C. After centrifuging at 6,000 x g at 4°C for 15 min, the cell pellets were resuspended in lysis buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 5% glycerol, 0.2% Triton X-100, and 2 mM β-mercaptoethanol) and then lysed by sonication. The crude extract was then centrifuged at 12,000 x g at 4°C for 25 min to remove the insoluble pellet. The supernatant was incubated with 1-ml Ni-NTA beads at 4°C for 1 h and then loaded into an empty column. After allowing the supernatant to flow through, the beads were washed with washing buffer (20 mM Tris, pH 8.5, 250 mM NaCl, 8 mM imidazole, and 2 mM β-mercaptoethanol), and the protein was eluted with elution buffer (20 mM Tris, pH 8.5, 30 mM NaCl, 150 mM imidazole, and 2 mM β-mercaptoethanol). The protein was then loaded onto a S-100 gel-filtration column (GE Healthcare) equilibrated with running buffer (20 mM Tris, pH 8.5, 100 mM NaCl, and 2 mM dithiothreitol). The purity of the fractions collected was analyzed by SDS-PAGE and the protein was concentrated to 30 mg/ml by Amicon Ultra-4 10-kDa centrifugal filter (Millipore).
Circular dichroism spectroscopy
CD spectra of the recombinant MERS-CoV PLpro using a JASCO J-810 spectropolarimeter showed measurements from 250 to 190 nm at 20°C in 50 mM phosphate pH 6.5. The protein concentration was 1.0 mg/ml. In wavelength scanning, the width of the cuvette was 0.1 mm. The far-UV CD spectrum data were analyzed with the CDSSTR program [16, 17]. In this analysis, the α-helix, β-sheet, and random coil were split. To estimate the goodness-of-fit, the normalized root mean square deviation was calculated.
The fluorescence spectra of the enzyme at 1 μM were monitored in a Perkin-Elmer LS50B luminescence spectrometer at 25°C. The excitation wavelength was set at 280 nm, and the fluorescence emission spectrum was scanned from 300 to 400 nm. Measurement in the maximal peak, intensity, and average emission wavelength were used to confirm the protein folding [18, 19].
Analytical ultracentrifugation analysis
The AUC experiments were performed on a XL-A analytical ultracentrifuge (Beckman Coulter) using an An-50 Ti rotor [12, 19–22]. The sedimentation velocity experiments were performed using a double-sector epon charcoal-filled centerpiece at 20°C with a rotor speed of 42,000 rpm. Protein solutions of MERS-CoV PLpro (1.0 mg/ml) (330 μl) and reference (370 μl) solutions were loaded into the centerpiece, respectively. The absorbance at 280 nm was monitored in a continuous mode with a time interval of 300 s and a step size of 0.003 cm. Multiple scans at different time intervals were then fitted to a continuous c(s) distribution model using the SEDFIT program . All size-and-shape distributions were analyzed at a confidence level of p = 0.95 by maximal entropy regularization and a resolution N of 200 with sedimentation coefficients between 0 and 20 S or molar mass between 0 and 1000 kDa.
Steady-state kinetic analysis
in which kcat is the rate constant, [E] and [S] denote the enzyme and substrate concentration, and Km is the Michaelis-Menten constant for the interaction between the peptide substrate and the enzyme.
The fluorogenic substrate Ub-7-amino-4-trifluoro-methylcoumarin (Ub-AFC) (Boston Biochem) added at 0.5 or 1.0 μM to 50 mM phosphate pH 6.5 was used for deubiquitination assays as described . The enzymatic activity at 30°C was determined by continuously monitoring the fluorescence emission and excitation wavelength of 350 and 485 nm, respectively.
Results and discussion
Recombinant MERS-CoV PLpro preparation
Purification of MERS-CoV PL pro from E. coli
Total protein (mg)
Total activity (Ua)
Specific activity (U/mg protein)
Ni affinity chromatography
Gel-filtration by S-100 column
Furthermore, the recombinant MERS-CoV PLpro was digested by trypsin and then analyzed by MALDI mass spectrometry to confirm the amino acid sequence (Additional file 1: Figure S1). The molecular weight of fifteen peptides, which covered 60% amino acid sequence, was observed and confirmed (Figure 1B and Figure 1C). It indicated that our expression and purification of MERS-CoV PLpro by E. coli is successful. For convenience, in the present studies, the MERS-CoV PLpro domain (polyprotein 1a 1484–1800) is numbered to residue 2 to 317, while the first residue is a methionine.
Secondary, tertiary and quaternary structure analysis of MERS-CoV PLpro
Proteolytic activity of MERS-CoV PLpro
The kinetic parameters and DUB activity of MERS-CoV PL pro
(10-3 s-1 μM-1)
19.2 ± 2.6
0.4 ± 0.02
0.2 ± 0.03
0.11 ± 0.02
35.7 ± 3.8
16.5 ± 0.9
4.6 ± 0.6
0.11 ± 0.01
30.8 ± 8.0
0.01 ± 0.001
0.003 ± 0.001
0.004 ± 0.001
25.2 ± 5.1c
11 ± 2c
4.4 ± 1.2c
0.12 ± 0.02
To verify this, we produced the L105W and P162L mutants of MERS-CoV PLpro, and our kinetic data showed that the L105W mutant has a 23-fold increase in activity measured based on kcat/Km, as a result of a 41-fold increase in kcat and 1.9-fold increase in Km (Figure 5A and Table 2). The results conform to our prediction. However, in contrast, the P162L mutant has a 67-fold loss in kcat/Km, as a result of a 40-fold loss in kcat and 1.6-fold increase in Km (Figure 5A and Table 2). It suggests the requirement of the Proline residue in this site, although the reason is still not known. Nevertheless, the significant activity recovery by L105W mutation confirms the essential role of this residue on coronaviral PLpro catalysis. Theoretically, PLpro with lower proteolytic activity may result in late maturation of viral nsp1, nsp2, and nsp3 proteins; nonetheless, its influence on MERS-CoV remains unknown.
DUB activity of MERS-CoV PLpro
To characterize the DUB activity of MERS-CoV PLpro, the fluorogenic substrate Ub-AFC was used. Interestingly, in contrast with its rather low proteolytic activity, MERS-CoV PLpro shows comparable DUB activity to SARS-CoV PLpro (Table 2 and Figure 5B). It suggests that the two PLpro may show similar binding ability to the Ub core domain (residue 1–72). However, it is inconsistent with our previous observation on the structure of SARS-CoV PLpro in complex with Ub . As mimicking the equivalent residue of MERS-CoV PLpro, the arginine mutation of a key residue for Ub core domain binding, Glu168, can result in unstable binding of SARS-CoV PLpro and Ub and significant loss of DUB activity . To verify this inconsistency, a structure of MERS-CoV PLpro in complex with Ub is quite necessary.
Structural characterization of type 1 and type 2 PLpro have revealed that there are four cysteine residues coordinating to a zinc ion within the fingertips region in the finger domain [25, 28]. Remove of zinc from SARS-CoV PLpro will cause the tertiary structure more unstable and lead to less active . Based on sequence alignment, MERS-CoV PLpro also has four cysteine residues (Cys190, Cys193, Cys225 and Cys227) on the corresponding position. Here the DUB activity of MERS-CoV PLpro in various EDTA was examined to delineate the possible metal ion effect. The activity was 79% in 10 mM, and 72% left in 50 mM EDTA (Figure 5B). These results suggest the existence of endogenous metal ion, which is beneficial for its DUB activity. By the way, it has been clarified that exogenous zinc ion can efficiently inhibit SARS-CoV PLpro with the IC50 value of 1.3 μM [24, 29]. Here we also confirmed the potent inhibitory effect of zinc ion on MERS-CoV PLpro (Figure 5B); whereas the mechanism of this inhibition by zinc is not yet understood.
In summary, following our protocol, active MERS-CoV PLpro can be expressed by E. coli and purified with high yield and high purity. The secondary, tertiary and quaternary structural studies concluded that MERS-CoV PLpro has a similar scaffold to other coronaviral PLpro, as a right-hand-like architecture consisting of palm, thumb and fingers domains. The result of functional assay indicated that MERS-CoV PLpro exhibits potent DUB activity but rather low proteolytic activity. A natural mutation, Leu105, is the major reason for this difference. The present study not only demonstrates the structural and functional characterization of MERS-CoV PLpro, but provides a foundation for further understanding the coronaviral PLpro family, which is an ideal antiviral target. Next, with pure protein and effective proteolytic activity assay, potent inhibitors of MERS-CoV PLpro can be high throughput screened and identified.
interferon regulatory factor 3
Middle East respiratory syndrome coronavirus
polymerase chain reaction
severe acute respiratory syndrome coronavirus
This research was supported by grants from National Science Council, Taiwan (98-2320-B-010-026-MY3 and 101-2320-B-010-061) to CYC and CGMH-NYMU Joint Research Grant (CMRPG2D0211) to CYC and CYS. We also thank NYMU for its financial support (Aim for Top University Plan from Ministry of Education).
- Anderson LJ, Baric RS: Emerging human coronaviruses–disease potential and preparedness. N Engl J Med. 2012, 367 (19): 1850-1852. 10.1056/NEJMe1212300.View ArticlePubMedGoogle Scholar
- Chan JF, Li KS, To KK, Cheng VC, Chen H, Yuen KY: Is the discovery of the novel human betacoronavirus 2c EMC/2012 (HCoV-EMC) the beginning of another SARS-like pandemic?. J Infect. 2012, 65 (6): 477-489. 10.1016/j.jinf.2012.10.002.View ArticlePubMedGoogle Scholar
- Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus AD, Fouchier RA: Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med. 2012, 367 (19): 1814-1820. 10.1056/NEJMoa1211721.View ArticlePubMedGoogle Scholar
- Eckerle I, Muller MA, Kallies S, Gotthardt DN, Drosten C: In-vitro renal epithelial cell infection reveals a viral kidney tropism as a potential mechanism for acute renal failure during Middle East Respiratory Syndrome (MERS) Coronavirus infection. Virol J. 2013, 10: 359-10.1186/1743-422X-10-359.PubMed CentralView ArticlePubMedGoogle Scholar
- Assiri A, McGeer A, Perl TM, Price CS, Al Rabeeah AA, Cummings DA, Alabdullatif ZN, Assad M, Almulhim A, Makhdoom H, Madani H, Alhakeem R, Al-Tawfiq JA, Cotten M, Watson SJ, Kellam P, Zumla AI, Memish ZA, Team KM-CI: Hospital outbreak of Middle East respiratory syndrome coronavirus. N Engl J Med. 2013, 369 (5): 407-416. 10.1056/NEJMoa1306742.PubMed CentralView ArticlePubMedGoogle Scholar
- Reusken CB, Haagmans BL, Muller MA, Gutierrez C, Godeke GJ, Meyer B, Muth D, Raj VS, Smits-De VL, Corman VM, Drexler JF, Smits SL, El Tahir YE, De Sousa R, van Beek J, Nowotny N, van Maanen K, Hidalgo-Hermoso E, Bosch BJ, Rottier P, Osterhaus A, Gortazar-Schmidt C, Drosten C, Koopmans MP: Middle East respiratory syndrome coronavirus neutralising serum antibodies in dromedary camels: a comparative serological study. Lancet Infect Dis. 2013, 13 (10): 859-866. 10.1016/S1473-3099(13)70164-6.View ArticlePubMedGoogle Scholar
- Kilianski A, Mielech AM, Deng X, Baker SC: Assessing activity and inhibition of Middle East respiratory syndrome coronavirus papain-like and 3C-like proteases using luciferase-based biosensors. J Virol. 2013, 87 (21): 11955-11962. 10.1128/JVI.02105-13.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen Z, Wang Y, Ratia K, Mesecar AD, Wilkinson KD, Baker SC: Proteolytic processing and deubiquitinating activity of papain-like proteases of human coronavirus NL63. J Virol. 2007, 81 (11): 6007-6018. 10.1128/JVI.02747-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Clementz MA, Chen Z, Banach BS, Wang Y, Sun L, Ratia K, Baez-Santos YM, Wang J, Takayama J, Ghosh AK, Li K, Mesecar AD, Baker SC: Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases. J Virol. 2010, 84 (9): 4619-4629. 10.1128/JVI.02406-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Yang X, Chen X, Bian G, Tu J, Xing Y, Wang Y, Chen Z: Proteolytic processing, deubiquitinase and interferon antagonist activities of Middle East respiratory syndrome coronavirus papain-like protease. J Gen Virol. 2014, 95 (Pt 3): 614-626.View ArticlePubMedGoogle Scholar
- Zheng D, Chen G, Guo B, Cheng G, Tang H: PLP2, a potent deubiquitinase from murine hepatitis virus, strongly inhibits cellular type I interferon production. Cell Res. 2008, 18 (11): 1105-1113.PubMedGoogle Scholar
- Chou CY, Lai HY, Chen HY, Cheng SC, Cheng KW, Chou YW: Structural basis for catalysis and ubiquitin recognition by the Severe acute respiratory syndrome coronavirus papain-like protease. Acta Crystallogr D Biol Crystallogr. 2014, 70 (Pt 2): 572-581.View ArticlePubMedGoogle Scholar
- Hu M, Li P, Song L, Jeffrey PD, Chenova TA, Wilkinson KD, Cohen RE, Shi Y: Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14. EMBO J. 2005, 24 (21): 3747-3756. 10.1038/sj.emboj.7600832.PubMed CentralView ArticlePubMedGoogle Scholar
- Renatus M, Parrado SG, D'Arcy A, Eidhoff U, Gerhartz B, Hassiepen U, Pierrat B, Riedl R, Vinzenz D, Worpenberg S, Kroemer M: Structural basis of ubiquitin recognition by the deubiquitinating protease USP2. Structure. 2006, 14 (8): 1293-1302. 10.1016/j.str.2006.06.012.View ArticlePubMedGoogle Scholar
- Mielech AM, Kilianski A, Baez-Santos YM, Mesecar AD, Baker SC: MERS-CoV papain-like protease has deISGylating and deubiquitinating activities. Virology. 2014, 450–451: 64-70.View ArticlePubMedGoogle Scholar
- Sreerama N, Woody RW: Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem. 2000, 287 (2): 252-260. 10.1006/abio.2000.4880.View ArticlePubMedGoogle Scholar
- Whitmore L, Wallace BA: DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res. 2004, 32 (Web Server issue): W668-673.PubMed CentralView ArticlePubMedGoogle Scholar
- Chang HP, Chou CY, Chang GG: Reversible unfolding of the severe acute respiratory syndrome coronavirus main protease in guanidinium chloride. Biophys J. 2007, 92 (4): 1374-1383. 10.1529/biophysj.106.091736.PubMed CentralView ArticlePubMedGoogle Scholar
- Chou YW, Cheng SC, Lai HY, Chou CY: Differential domain structure stability of the severe acute respiratory syndrome coronavirus papain-like protease. Arch Biochem Biophys. 2012, 520 (2): 74-80. 10.1016/j.abb.2012.02.015.View ArticlePubMedGoogle Scholar
- Cheng SC, Chang GG, Chou CY: Mutation of Glu-166 blocks the substrate-induced dimerization of SARS coronavirus main protease. Biophys J. 2010, 98 (7): 1327-1336. 10.1016/j.bpj.2009.12.4272.PubMed CentralView ArticlePubMedGoogle Scholar
- Hsieh YH, Chou CY: Structural and functional characterization of human apolipoprotein E 72–166 peptides in both aqueous and lipid environments. J Biomed Sci. 2011, 18: 4-10.1186/1423-0127-18-4.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu CG, Cheng SC, Chen SC, Li JY, Fang YH, Chen YH, Chou CY: Mechanism for controlling the monomer-dimer conversion of SARS coronavirus main protease. Acta Crystallogr D Biol Crystallogr. 2013, 69 (Pt 5): 747-755.View ArticlePubMedGoogle Scholar
- Schuck P: Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys J. 2000, 78: 1606-1619. 10.1016/S0006-3495(00)76713-0.PubMed CentralView ArticlePubMedGoogle Scholar
- Chou CY, Chien CH, Han YS, Prebanda MT, Hsieh HP, Turk B, Chang GG, Chen X: Thiopurine analogues inhibit papain-like protease of severe acute respiratory syndrome coronavirus. Biochem Pharmacol. 2008, 75 (8): 1601-1609. 10.1016/j.bcp.2008.01.005.View ArticlePubMedGoogle Scholar
- Wojdyla JA, Manolaridis I, van Kasteren PB, Kikkert M, Snijder EJ, Gorbalenya AE, Tucker PA: Papain-like protease 1 from transmissible gastroenteritis virus: crystal structure and enzymatic activity toward viral and cellular substrates. J Virol. 2010, 84 (19): 10063-10073. 10.1128/JVI.00898-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Brown PH, Schuck P: Macromolecular size-and-shape distributions by sedimentation velocity analytical ultracentrifugation. Biophys J. 2006, 90 (12): 4651-4661. 10.1529/biophysj.106.081372.PubMed CentralView ArticlePubMedGoogle Scholar
- Benkert P, Biasini M, Schwede T: Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics. 2011, 27 (3): 343-350. 10.1093/bioinformatics/btq662.PubMed CentralView ArticlePubMedGoogle Scholar
- Ratia K, Saikatendu KS, Santarsiero BD, Barretto N, Baker SC, Stevens RC, Mesecar AD: Severe acute respiratory syndrome coronavirus papain-like protease: structure of a viral deubiquitinating enzyme. Proc Natl Acad Sci U S A. 2006, 103 (15): 5717-5722. 10.1073/pnas.0510851103.PubMed CentralView ArticlePubMedGoogle Scholar
- Han YS, Chang GG, Juo CG, Lee HJ, Yeh SH, Hsu JT, Chen X: Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, purification, characterization, and inhibition. Biochemistry. 2005, 44 (30): 10349-10359. 10.1021/bi0504761.View ArticlePubMedGoogle Scholar
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