E1 mutants identify a critical region in the trimer interface of the Semliki forest virus fusion protein.
The alphavirus Semliki Forest virus (SFV) uses a membrane fusion reaction to infect host cells. Fusion of the virus and cell membranes is triggered by low pH in the endosome and is mediated by the viral membrane protein E1. During fusion, E1 inserts into the target membrane, trimerizes, and refolds into a hairpin conformation. Formation of the E1 homotrimer is critical to membrane fusion, but the mechanism of trimerization is not understood. The crystal structure of the postfusion E1 trimer shows that an aspartate residue, D188, is positioned in the central core trimer interface. D188 is conserved in all reported alphavirus E1 sequences. We tested the contribution of this amino acid to trimerization and fusion by replacing D188 with alanine (D188A) or lysine (D188K) in an SFV infectious clone. These mutations were predicted to disrupt specific interactions at this position and/or change their pH dependence. Our results indicated that the D188K mutation blocked SFV fusion and infection. At low pH, D188K E1 inserted into target membranes but was trapped as a target membrane-inserted monomer that did not efficiently form the stable core trimer. In contrast, the D188A mutant was infectious, although trimerization and fusion required a lower pH. While there are extensive contacts between E1 subunits in the homotrimer, the D188K mutant identifies an important "hot spot" for protein-protein interactions within the core trimer.
Liu
Catherine Y
CY
Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.
Kielian
Margaret
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006863:Hydrogen-Ion Concentration; D008561:Membrane Fusion / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D055503:Protein Multimerization; D012672:Semliki forest virus / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014760:Viral Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D053586:Virus Internalization
C494127:E1 glycoprotein, Semliki forest virus; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins
EMBO J. 2004 Feb 25;23(4):728-38
14963486
J Cell Biol. 1996 Aug;134(4):863-72
8769412
Curr Opin Struct Biol. 2009 Apr;19(2):189-96
19356922
J Virol. 1995 Apr;69(4):2471-9
7884895
J Biol Chem. 2003 Feb 28;278(9):7189-98
12493775
J Virol. 2006 Jun;80(12):6115-22
16731950
J Virol. 1999 Dec;73(12):10029-39
10559317
Nature. 2004 Jan 22;427(6972):320-5
14737160
Nat Rev Microbiol. 2005 Jan;3(1):13-22
15608696
Virology. 2006 Jan 5;344(1):38-47
16364734
Science. 2007 Feb 9;315(5813):843-8
17289996
Annu Rev Biochem. 2001;70:777-810
11395423
J Virol. 1990 Oct;64(10):4614-24
2118964
J Virol. 2006 Nov;80(22):11362-9
16971447
Nat Rev Microbiol. 2006 Jan;4(1):67-76
16357862
Bioinformatics. 1999 Apr;15(4):327-32
10320401
Virology. 1997 Nov 10;238(1):14-21
9375004
J Virol. 1991 Aug;65(8):4107-13
2072446
Crit Rev Biochem Mol Biol. 2008 May-Jun;43(3):189-219
18568847
EMBO J. 1993 Feb;12(2):693-701
8440260
Nat Struct Mol Biol. 2008 Jul;15(7):675-83
18596814
J Virol. 2004 Apr;78(7):3312-8
15016852
J Cell Biol. 1998 Jan 12;140(1):91-9
9425157
J Virol. 1991 Aug;65(8):4292-300
2072453
Nat Struct Mol Biol. 2008 Jul;15(7):690-8
18596815
J Gen Virol. 2006 Oct;87(Pt 10):2755-2766
16963734
Adv Virus Res. 2005;64:231-61
16139596
J Cell Biol. 2008 Oct 20;183(2):353-61
18936253
Nature. 2004 Jan 22;427(6972):313-9
14737159
J Virol. 1989 Dec;63(12):4991-7
2479769
J Cell Biol. 2005 Oct 10;171(1):111-20
16216925
J Cell Biol. 1992 Jan;116(2):339-48
1370493
Cell Host Microbe. 2008 Dec 11;4(6):600-8
19064260
Science. 2006 Jul 14;313(5784):187-91
16840692
J Virol. 2002 Dec;76(24):12712-22
12438597
J Virol. 2000 Sep;74(17):7772-80
10933683
Cell. 2001 Apr 6;105(1):137-48
11301009
Structure. 2006 Jan;14(1):75-86
16407067
J Virol. 2004 Dec;78(24):13543-52
15564465
Subcell Biochem. 2000;34:379-408
10808339