title: Inhibition of the human respiratory syncytial virus small hydrophobic protein and structural variations in a bicelle environment.
abstract: The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by the human respiratory syncytial virus (hRSV). SH protein has a single α-helical transmembrane (TM) domain that forms pentameric ion channels. Herein, we report the first inhibitor of the SH protein channel, pyronin B, and we have mapped its binding site to a conserved surface of the RSV SH pentamer, at the C-terminal end of the transmembrane domain. The validity of the SH protein structural model used has been confirmed by using a bicellar membrane-mimicking environment. However, in bicelles the α-helical stretch of the TM domain extends up to His-51, and by comparison with previous models both His-22 and His-51 adopt an interhelical/lumenal orientation relative to the channel pore. Neither His residue was found to be essential for channel activity although His-51 protonation reduced channel activity at low pH, with His-22 adopting a more structural role. The latter results are in contrast with previous patch clamp data showing channel activation at low pH, which could not be reproduced in the present work. Overall, these results establish a solid ground for future drug development targeting this important viroporin. Importance: The human respiratory syncytial virus (hRSV) is responsible for 64 million reported cases of infection and 160,000 deaths each year. Lack of adequate antivirals fuels the search for new targets for treatment. The small hydrophobic (SH) protein is a 64-amino-acid polypeptide encoded by hRSV and other paramyxoviruses, and its absence leads to viral attenuation in vivo and early apoptosis in infected cells. SH protein forms pentameric ion channels that may constitute novel drug targets, but no inhibitor for this channel activity has been reported so far. A small-molecule inhibitor, pyronin B, can reduce SH channel activity, and its likely binding site on the SH protein channel has been identified. Black lipid membrane (BLM) experiments confirm that protonation of both histidine residues reduces stability and channel activity. These results contrast with previous patch clamp data that showed low-pH activation, which we have not been able to reproduce.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Janet
authors.initials: J
authors.lastname: To
authors.affiliation: Department of Physics, Universitat Jaume I, Castellon, Spain.
authors.forename: Carmina
authors.initials: C
authors.lastname: Verdià-Baguena
authors.affiliation: Institute of Pharmacology and Toxicology, Paracelsus Medical University, Salzburg, Austria.
authors.forename: Silvia
authors.initials: S
authors.lastname: Dossena
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Wahyu
authors.initials: W
authors.lastname: Surya
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Mei
authors.initials: M
authors.lastname: Huang
authors.affiliation: Institute of Pharmacology and Toxicology, Paracelsus Medical University, Salzburg, Austria.
authors.forename: Markus
authors.initials: M
authors.lastname: Paulmichl
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore.
authors.forename: Ding Xiang
authors.initials: DX
authors.lastname: Liu
authors.affiliation: Department of Physics, Universitat Jaume I, Castellon, Spain.
authors.forename: Vicente M
authors.initials: VM
authors.lastname: Aguilella
authors.affiliation: School of Biological Sciences, Nanyang Technological University, Singapore jtorres@ntu.edu.sg.
authors.forename: Jaume
authors.initials: J
authors.lastname: Torres
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005814:Genes, Viral; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D018408:Patch-Clamp Techniques; D011754:Pyronine / Q000031:analogs & derivatives / Q000494:pharmacology; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D014709:Vero Cells; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics
chemical_list: D008051:Lipid Bilayers; D014764:Viral Proteins; C012020:pyronine B; D011754:Pyronine
references.citation: J Biomol NMR. 1999 Mar;13(3):289-302
references.pmid: 10212987
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references.pmid: 9757107
references.citation: Anal Biochem. 2011 Sep 1;416(1):100-6
references.pmid: 21624344
references.citation: Bioinformatics. 2012 Aug 1;28(15):2074-5
references.pmid: 22628523
references.citation: J Infect Dis. 2000 Oct;182(4):1228-33
references.pmid: 10979923
references.citation: J Am Chem Soc. 2003 Jul 30;125(30):9179-91
references.pmid: 15369375
references.citation: Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13961-6
references.pmid: 9391135
references.citation: Proc Natl Acad Sci U S A. 2008 Aug 5;105(31):10967-72
references.pmid: 18669647
references.citation: Nature. 2008 Jan 31;451(7178):591-5
references.pmid: 18235503
references.citation: Proc Natl Acad Sci U S A. 1972 Dec;69(12):3561-6
references.pmid: 4509315
references.citation: Nat Protoc. 2007;2(11):2728-33
references.pmid: 18007608
references.citation: Nature. 2013 Jun 27;498(7455):521-5
references.pmid: 23739335
references.citation: J Biol Chem. 2014 May 2;289(18):12535-49
references.pmid: 24668816
references.citation: PLoS Pathog. 2014 May 01;10(5):e1004077
references.pmid: 24788150
references.citation: Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1450-5
references.pmid: 10677482
references.citation: J Am Chem Soc. 2003 Nov 19;125(46):13932-3
references.pmid: 14611209
references.citation: J Am Chem Soc. 2002 Mar 20;124(11):2450-1
references.pmid: 11890789
references.citation: J Virol. 2006 Feb;80(4):1700-9
references.pmid: 16439527
references.citation: Eur J Endocrinol. 2005 Nov;153(5):693-9
references.pmid: 16260428
references.citation: Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7892-5
references.pmid: 1715584
references.citation: EMBO J. 2009 May 6;28(9):1351-61
references.pmid: 19279667
references.citation: PLoS One. 2013 Sep 12;8(9):e74281
references.pmid: 24069290
references.citation: Vaccine. 1996 Dec;14(17-18):1637-46
references.pmid: 9032893
references.citation: J Gen Virol. 2004 May;85(Pt 5):1153-1165
references.pmid: 15105532
references.citation: Arch Virol. 1997;142(6):1247-54
references.pmid: 9229012
references.citation: J Mol Biol. 1997 Oct 17;273(1):283-98
references.pmid: 9367762
references.citation: Nat Rev Mol Cell Biol. 2002 Aug;3(8):600-14
references.pmid: 12154371
references.citation: Proc Soc Exp Biol Med. 1956 Jul;92(3):544-9
references.pmid: 13359460
references.citation: Virology. 1993 Nov;197(1):1-11
references.pmid: 8212546
references.citation: Biophys J. 1993 Jan;64(1):16-25
references.pmid: 7679295
references.citation: J Virol. 1999 Apr;73(4):3438-42
references.pmid: 10074199
references.citation: J Biol Chem. 2012 Jul 13;287(29):24671-89
references.pmid: 22621926
references.citation: Virology. 2006 Sep 30;353(2):294-306
references.pmid: 16815524
references.citation: J Biol Chem. 2011 Nov 25;286(47):40659-70
references.pmid: 21917931
references.citation: J Biol Chem. 2009 Feb 6;284(6):3873-84
references.pmid: 19073598
references.citation: Biophys J. 2006 Aug 1;91(3):938-47
references.pmid: 16698774
references.citation: Virology. 2011 Jul 5;415(2):69-82
references.pmid: 21524776
references.citation: Nature. 2005 Dec 15;438(7070):975-80
references.pmid: 16355215
references.citation: J Virol. 2007 Aug;81(15):8361-6
references.pmid: 17494063
references.citation: J Magn Reson. 2012 Jan;214(1):111-8
references.pmid: 22079194
references.citation: J Virol. 1997 Dec;71(12):8973-82
references.pmid: 9371553
references.citation: J Virol. 2009 Sep;83(17):8744-58
references.pmid: 19553314
references.citation: J Virol. 2011 Jan;85(1):32-42
references.pmid: 20980504
references.citation: J Virol. 1989 May;63(5):2019-29
references.pmid: 2649692
references.citation: J Mol Biol. 2001 Jan 19;305(3):567-80
references.pmid: 11152613
references.citation: J Virol. 2003 Mar;77(6):3371-83
references.pmid: 12610112
references.citation: Thorax. 2013 Jan;68(1):66-75
references.pmid: 23229815
references.citation: Virology. 2000 Jul 20;273(1):210-8
references.pmid: 10891423
references.citation: J Gen Virol. 2005 Feb;86(Pt 2):375-384
references.pmid: 15659757
references.citation: PLoS Pathog. 2009 Jul;5(7):e1000511
references.pmid: 19593379
references.citation: Proteins. 2005 Aug 1;60(2):224-31
references.pmid: 15981269
references.citation: FEBS Lett. 2004 Jan 16;557(1-3):99-103
references.pmid: 14741348
references.citation: Virology. 2012 Oct 25;432(2):485-94
references.pmid: 22832120
references.citation: J Gen Virol. 1990 Jul;71 ( Pt 7):1571-6
references.pmid: 2374008
references.citation: J Infect Dis. 1996 Sep;174(3):456-62
references.pmid: 8769600
references.citation: J Gen Virol. 1993 Jul;74 ( Pt 7):1445-50
references.pmid: 8336126
references.citation: PLoS One. 2013 Apr 30;8(4):e62954
references.pmid: 23646163
references.citation: J Mol Biol. 2002 May 24;319(1):209-27
references.pmid: 12051947
references.citation: J Biomol NMR. 2001 Dec;21(4):321-33
references.pmid: 11824752
references.citation: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W363-7
references.pmid: 15980490
references.citation: Protein Sci. 2008 May;17(5):813-20
references.pmid: 18369195