title: Influenza hemagglutinin (HA) stem region mutations that stabilize or destabilize the structure of multiple HA subtypes.
abstract: Influenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles.
  
  
  OBJECTIVE
  The influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. We show here that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, since the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza virus subtypes to improve the stability of vaccine strains or components.
authors: Department of Microbiology and Immunology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia, USA.
authors: Lauren
authors: L
authors: Byrd-Leotis
authors: Department of Microbiology and Immunology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia, USA.
authors: Summer E
authors: SE
authors: Galloway
authors: Department of Microbiology and Immunology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia, USA.
authors: Evangeline
authors: E
authors: Agbogu
authors: Department of Microbiology and Immunology, Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia, USA dsteinh@emory.edu.
authors: David A
authors: DA
authors: Steinhauer
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D004252:DNA Mutational Analysis; D014644:Genetic Variation; D006388:Hemagglutinins / Q000737:chemistry / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D009980:Influenza A virus / Q000235:genetics*; D008156:Luciferases; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011487:Protein Conformation; D055550:Protein Stability; D013045:Species Specificity; D014709:Vero Cells
chemical_list: D006388:Hemagglutinins; D008156:Luciferases
references: J Virol. 2013 May;87(9):4826-34
references: 23449784
references: N Engl J Med. 2013 May 16;368(20):1888-97
references: 23577628
references: J Gen Virol. 2013 Jun;94(Pt 6):1220-9
references: 23486663
references: Nature. 2013 Sep 26;501(7468):556-9
references: 23842497
references: PLoS Pathog. 2014 Jan;10(1):e1003831
references: 24391498
references: J Pharm Sci. 2014 Mar;103(3):821-7
references: 24425059
references: Biochim Biophys Acta. 2014 Apr;1838(4):1153-68
references: 24161712
references: N Engl J Med. 2005 Sep 29;353(13):1374-85
references: 16192482
references: Vaccine. 2006 Jan 23;24(4):525-31
references: 16150515
references: J Membr Biol. 2006;211(3):139-50
references: 17091214
references: Virology. 2008 Jan 20;370(2):403-14
references: 17936324
references: Proc Natl Acad Sci U S A. 2008 Nov 18;105(46):17736-41
references: 19004788
references: Vaccine. 2008 Sep 12;26 Suppl 4:D54-8
references: 19230161
references: J Virol. 2009 Apr;83(8):3568-80
references: 19193808
references: J Virol. 2010 Feb;84(3):1527-35
references: 19923184
references: J Virol. 2010 Jul;84(13):6570-7
references: 20427525
references: PLoS One. 2011;6(4):e18577
references: 21490925
references: PLoS Pathog. 2011 Dec;7(12):e1002398
references: 22144894
references: J Virol. 2000 Sep;74(18):8502-12
references: 10954551
references: Annu Rev Biochem. 2000;69:531-69
references: 10966468
references: Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6883-8
references: 11371620
references: EMBO J. 2001 Aug 15;20(16):4432-42
references: 11500371
references: EMBO J. 2002 Mar 1;21(5):865-75
references: 11867515
references: Am J Physiol Cell Physiol. 2002 Apr;282(4):C736-43
references: 11880261
references: Annu Rev Genet. 2002;36:305-32
references: 12429695
references: Virology. 2004 Aug 1;325(2):287-96
references: 15246268
references: J Gen Virol. 1974 Dec;25(3):351-7
references: 4475092
references: Virology. 1975 Dec;68(2):426-39
references: 173078
references: Virology. 1975 Dec;68(2):440-54
references: 128196
references: Nature. 1977 Dec 15;270(5638):617-8
references: 593388
references: Nature. 1978 Jun 29;273(5665):778-9
references: 661986
references: Virology. 1978 Nov;91(1):79-85
references: 569394
references: Arch Virol. 1978;58(4):323-33
references: 736796
references: Nature. 1981 Jan 29;289(5796):366-73
references: 7464906
references: Cell. 1985 Feb;40(2):431-9
references: 3967299
references: Arch Virol. 1985;85(1-2):1-11
references: 4015405
references: EMBO J. 2014 Apr 16;33(8):823-41
references: 24668228
references: Proc Natl Acad Sci U S A. 2014 Jul 29;111(30):11175-80
references: 25024224
references: Biologicals. 2014 Sep;42(5):237-59
references: 24996452
references: J Virol. 2014 Nov;88(21):12374-84
references: 25122789
references: Curr Top Microbiol Immunol. 2014;385:3-34
references: 25031010
references: Curr Top Microbiol Immunol. 2014;385:137-55
references: 25048542
references: Curr Top Microbiol Immunol. 2014;385:63-91
references: 25078920
references: Virology. 1994 Nov 15;205(1):17-23
references: 7975212
references: Proc Natl Acad Sci U S A. 1996 Nov 12;93(23):12873-8
references: 8917512
references: Nature. 1997 Oct 9;389(6651):554
references: 9335492
references: Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14306-13
references: 9405608
references: Lancet. 1998 Feb 14;351(9101):472-7
references: 9482438
references: Cell. 1998 Oct 30;95(3):409-17
references: 9814710
references: N Engl J Med. 2004 Dec 2;351(23):2363-5
references: 15575048
references: J Clin Microbiol. 1985 Nov;22(5):719-25
references: 4056002
references: Vaccine. 1985 Sep;3(3 Suppl):215-8
references: 4060851
references: Virology. 1985 Dec;147(2):287-94
references: 2416114
references: Virology. 1986 Dec;155(2):484-97
references: 3788061
references: J Gen Virol. 1986 Dec;67 ( Pt 12):2813-7
references: 3794667
references: J Virol. 1987 Sep;61(9):2857-63
references: 2441080
references: Annu Rev Biochem. 1987;56:365-94
references: 3304138
references: J Virol. 1988 Feb;62(2):488-95
references: 3336068
references: Vaccine. 1988 Apr;6(2):129-33
references: 2838984
references: EMBO J. 1990 Jan;9(1):17-24
references: 2295311
references: Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11525-9
references: 1763066
references: Microbiol Rev. 1992 Mar;56(1):152-79
references: 1579108
references: Biochemistry. 1993 Mar 30;32(12):2967-78
references: 8457561
references: Nature. 1994 Sep 1;371(6492):37-43
references: 8072525
references: Vaccine. 2012 Jan 20;30(5):872-8
references: 22172510
references: J Gen Virol. 2012 May;93(Pt 5):970-9
references: 22258863
references: Vaccine. 2012 Jun 19;30(29):4369-76
references: 22682293
references: Nature. 2012 Jun 21;486(7403):420-8
references: 22722205
references: Science. 2012 Jun 22;336(6088):1534-41
references: 22723413
references: Science. 2012 Jun 22;336(6088):1541-7
references: 22723414
references: PLoS Pathog. 2013 Feb;9(2):e1003151
references: 23459660