Pseudorevertants of a Semliki forest virus fusion-blocking mutation reveal a critical interchain interaction in the core trimer.
Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells by a low-pH-triggered membrane fusion reaction mediated by the viral E1 protein. E1 inserts into target membranes and refolds to a hairpin-like homotrimer containing a central core trimer and an outer layer composed of domain III and the juxtamembrane stem region. The key residues involved in mediating E1 trimerization are not well understood. We recently showed that aspartate 188 in the interface of the core trimer plays a critical role. Substitution with lysine (D188K) blocks formation of the core trimer and E1 trimerization and strongly inhibits virus fusion and infection. Here, we have isolated and characterized revertants that rescued the fusion and growth defects of D188K. These revertants included pseudorevertants containing acidic or polar neutral residues at E1 position 188 and a second-site revertant containing an E1 K176T mutation. Computational analysis using multiconformation continuum electrostatics revealed an important interaction bridging D188 of one chain with K176 of the adjacent chain in the core trimer. E1 K176 is completely conserved among the alphaviruses, and mutations of K176 to threonine (K176T) or isoleucine (K176I) produced similar fusion phenotypes as D188 mutants. Together, our data support a model in which a ring of three salt bridges formed by D188 and K176 stabilize the core trimer, a key intermediate of the alphavirus fusion protein.
Liu
Catherine Y
CY
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Besanceney
Christen
C
Song
Yifan
Y
Kielian
Margaret
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D006224:Cricetinae; D008561:Membrane Fusion; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D055503:Protein Multimerization; D012672:Semliki forest virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; 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
Nat Rev Microbiol. 2006 Jan;4(1):67-76
16357862
J Virol. 2006 Jun;80(12):6115-22
16731950
Proteins. 1991;11(4):281-96
1758883
J Virol. 2002 Dec;76(24):12691-702
12438595
J Virol. 2002 Apr;76(7):3267-75
11884551
J Virol. 2006 Oct;80(19):9599-607
16973563
J Virol. 1990 Oct;64(10):4614-24
2118964
Nature. 2004 Jan 22;427(6972):320-5
14737160
Physiology (Bethesda). 2007 Feb;22:30-9
17289928
Virology. 1997 Nov 10;238(1):14-21
9375004
J Comput Chem. 2002 Jan 15;23(1):128-37
11913378
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
J Virol. 2004 Apr;78(7):3312-8
15016852
Trends Microbiol. 2009 Nov;17(11):514-21
19796949
J Virol. 1991 Aug;65(8):4292-300
2072453
J Virol. 2009 Nov;83(21):11298-306
19692469
J Comput Chem. 2009 Nov 15;30(14):2231-47
19274707
Cell. 2003 Sep 5;114(5):573-83
13678581
J Virol. 2002 Nov;76(22):11645-58
12388725
Nat Struct Mol Biol. 2008 Jul;15(7):690-8
18596815
Adv Virus Res. 2005;64:231-61
16139596
Nature. 2004 Jan 22;427(6972):313-9
14737159
Biophys J. 1997 May;72(5):2075-93
9129810
J Cell Biol. 2005 Oct 10;171(1):111-20
16216925
Cell Host Microbe. 2008 Dec 11;4(6):600-8
19064260
Mol Cell. 2000 Feb;5(2):255-66
10882067
J Virol. 2009 May;83(9):4670-7
19244325
Cell. 2001 Apr 6;105(1):137-48
11301009
Viruses. 2010 Mar 26;2(4):796-825
21546978
Biophys J. 2002 Oct;83(4):1731-48
12324397
Structure. 2006 Jan;14(1):75-86
16407067
Adv Virus Res. 2005;64:263-84
16139597
J Virol. 2004 Dec;78(24):13543-52
15564465
Proteins. 2005 Dec 1;61(4):704-21
16231289
J Virol. 2006 Nov;80(22):11362-9
16971447