Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus.
Infectious salmon anaemia virus (ISAV) is a piscine orthomyxovirus causing a serious disease in farmed Atlantic salmon (Salmo salar L.). The virus surface glycoprotein hemagglutinin-esterase (HE) is responsible for both viral attachment and release. Similarity to bovine and porcine torovirus hemagglutinin-esterase (BToV HE, PToV HE), bovine coronavirus HE (BCoV HE) and influenza C hemagglutinin-esterase-fusion (InfC HEF) proteins were exploited in a computational homology-based structure analysis of ISAV HE. The analysis resolved structural aspects of the protein and identified important features of relevance to ISAV HE activity. By recombinant expression and purification of secretory HE (recHE) proteins, receptor-binding and quantitative analyses of enzymatic activities displayed by ISAV HE molecules are presented for the first time. Three different recHE molecules were constructed: one representing a high virulent isolate, one a low virulent, while in the third a Ser(32) to Ala(32) amino acid substitution was introduced in the enzymatic catalytic site as inferred from the model. The three amino acid differences between the high and low virulent variants, of which two localized to the putative receptor-binding domain and one in the esterase domain, had no impact on receptor-binding or -release activities. In contrast, the Ser(32) amino acid substitution totally abolished enzymatic activity while receptor binding increased, as observed by agglutination of Atlantic salmon red blood cells. This demonstrates the essential role of a serine in the enzyme's catalytic site. In conclusion, structural analysis of ISAV HE in combination with selected recHE proteins gave insights into structure-function relationships and opens up for further studies aiming at dissecting molecular determinants of ISAV virulence.
Müller
Anita
A
Department of Marine Biotechnology, Norwegian College of Fishery Science, Breivika, N-9037 Tromsø, Norway. anita.mueller@nfh.uit.no
Markussen
Turhan
T
Drabløs
Finn
F
Gjøen
Tor
T
Jørgensen
Trond Ø
TØ
Solem
Stein Tore
ST
Mjaaland
Siri
S
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D019295:Computational Biology; D017938:Coronavirus, Bovine / Q000235:genetics; D006389:Hemagglutinins, Viral / Q000235:genetics* / Q000378:metabolism*; D009982:Gammainfluenzavirus / Q000235:genetics; D046829:Isavirus / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011991:Receptors, Virus / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera; D017003:Torovirus / Q000235:genetics; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D053585:Virus Attachment; D057074:Virus Release
D006389:Hemagglutinins, Viral; D011991:Receptors, Virus; D014760:Viral Fusion Proteins; C060814:hemagglutinin esterase
BMC Vet Res. 2008 Aug 04;4:28
18680586
Bioinformatics. 2003 May 22;19(8):1015-8
12761065
Nature. 1993 Jun 3;363(6428):418-23
8502295
Dis Aquat Organ. 2009 Apr 6;84(2):105-14
19476280
J Virol. 2005 Jun;79(11):6763-71
15890915
Nucleic Acids Res. 2010 Jan;38(Database issue):D5-16
19910364
Virology. 1996 Apr 1;218(1):127-33
8615014
J Virol. 2001 Aug;75(15):6825-34
11435561
Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13961-6
9391135
J Mol Biol. 1977 May 15;112(2):343-7
875021
Glycoconj J. 2009 Jan;26(1):99-109
18661232
J Virol. 2001 Jun;75(11):5352-6
11333916
J Gen Virol. 1985 Feb;66 ( Pt 2):295-301
3968541
J Virol. 1995 Feb;69(2):1099-106
7815489
Science. 1987 Jun 5;236(4806):1293-5
3589663
Virus Res. 2007 Sep;128(1-2):159-63
17521765
Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15897-902
19721004
Virus Res. 2001 Nov 5;79(1-2):177-85
11551658
Virology. 2002 Dec 20;304(2):379-91
12504577
N Engl J Med. 2005 Sep 29;353(13):1363-73
16192481
Electrophoresis. 1997 Dec;18(15):2714-23
9504803
Protein Eng. 1993 Jan;6(1):37-40
8433969
Virology. 2005 Aug 15;339(1):12-20
15950996
J Virol. 2004 Mar;78(6):3063-71
14990725
Biomed Pharmacother. 2000 May;54(4):196-209
10872718
J Gen Virol. 2002 Feb;83(Pt 2):421-428
11807235
Nature. 1983 May 5-11;303(5912):41-4
6188957
Nucleic Acids Res. 2000 Jan 1;28(1):235-42
10592235
J Biol Chem. 1986 May 5;261(13):5947-51
3700379
Arch Virol. 2007 Jan;152(1):151-79
16941061
Annu Rev Biochem. 1987;56:365-94
3304138
BMC Struct Biol. 2008 Dec 26;8:55
19111067
J Virol. 1997 Dec;71(12):9016-23
9371558
Bioinformatics. 2003 Oct;19 Suppl 2:ii246-55
14534198
Virology. 1974 Oct;61(2):397-410
4472498
Nature. 1998 Nov 5;396(6706):92-6
9817207
Microbiol Rev. 1992 Mar;56(1):61-79
1579113
J Virol. 2005 Oct;79(19):12544-53
16160182
Protein Expr Purif. 2008 Dec;62(2):206-15
18799134
J Virol. 2004 Mar;78(6):3055-62
14990724
J Gen Virol. 2001 Jul;82(Pt 7):1757-1765
11413388
Bioinformatics. 2001 Aug;17(8):750-1
11524381
J Biol Chem. 2004 Dec 31;279(53):55840-9
15492014
Proc Natl Acad Sci U S A. 2008 Jul 1;105(26):9065-9
18550812
EMBO J. 1985 Jun;4(6):1503-6
2411539
J Virol. 1997 Oct;71(10):7681-6
9311851
Structure. 2000 Apr 15;8(4):373-83
10801485
Virology. 1995 Sep 10;212(1):77-83
7676651
J Gen Virol. 2002 Nov;83(Pt 11):2693-2697
12388804
Biopolymers. 1983 Dec;22(12):2577-637
6667333
FEBS Lett. 1993 May 24;323(1-2):96-8
8495755
Biochemistry (Mosc). 2003 Nov;68(11):1225-30
14640965
Virology. 1998 May 10;244(2):315-21
9601502
Rev Med Virol. 2002 May-Jun;12(3):159-66
11987141
J Gen Virol. 1988 Apr;69 ( Pt 4):839-46
3356980
J Virol. 2000 Jul;74(13):6015-20
10846083
J Gen Virol. 2001 Mar;82(Pt 3):631-640
11172105
Virology. 1993 Feb;192(2):679-82
8421907
J Virol. 2004 Nov;78(22):12665-7
15507653
Nucleic Acids Res. 2003 Jul 1;31(13):3305-7
12824313
Dis Aquat Organ. 1998 Oct 8;34(2):77-85
9828403
Protein Sci. 2000 Feb;9(2):232-41
10716175
Arch Virol. 2005 Nov;150(11):2195-216
16012784
J Gen Virol. 1995 Oct;76 ( Pt 10):2529-37
7595356
Virology. 2008 May 10;374(2):515-27
18280528