Role for amino acids 212KLR214 of Ebola virus VP40 in assembly and budding.
Ebola virus VP40 is able to produce virus-like particles (VLPs) in the absence of other viral proteins. At least three domains within VP40 are thought to be required for efficient VLP release: the late domain (L-domain), membrane association domain (M-domain), and self-interaction domain (I-domain). While the L-domain of Ebola VP40 has been well characterized, the exact mechanism by which VP40 mediates budding through the M- and I-domains remains unclear. To identify additional domains important for VP40 assembly/budding, amino acids (212)KLR(214) were targeted for mutagenesis based on the published crystal structure of VP40. These residues are part of a loop connecting two beta sheets in the C-terminal region and thus are potentially important for overall structure and/or oligomerization of VP40. A series of alanine substitutions were generated in the KLR region of VP40, and these mutants were examined for VLP budding, intracellular localization, and oligomerization. Our results indicated that (i) (212)KLR(214) residues of VP40 are important for efficient release of VP40 VLPs, with Leu213 being the most critical; (ii) VP40 KLR mutants displayed altered patterns of cellular localization compared to that of wild-type VP40 (VP40-WT); and (iii) self-assembly of VP40 KLR mutants into oligomers was altered compared to that of VP40-WT. These results suggest that (12)KLR(214) residues of VP40 are important for proper assembly/oligomerization of VP40 which subsequently leads to efficient budding of VLPs.
McCarthy
Sarah E
SE
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA.
Johnson
Reed F
RF
Zhang
Yong-An
YA
Sunyer
J Oriol
JO
Harty
Ronald N
RN
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019281:Dimerization; D029043:Ebolavirus / Q000737:chemistry / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009698:Nucleoproteins / Q000502:physiology*; D017434:Protein Structure, Tertiary; D014758:Viral Core Proteins / Q000502:physiology*; D014771:Virion; D019065:Virus Assembly
D009698:Nucleoproteins; D014758:Viral Core Proteins; C084819:nucleoprotein VP40, Ebola virus
Virology. 2001 Sep 30;288(2):308-14
11601902
J Virol. 2001 Jun;75(11):5205-14
11333902
J Virol. 2002 May;76(10):4855-65
11967302
J Virol. 2003 Feb;77(3):1812-9
12525615
Virology. 2003 Aug 1;312(2):359-68
12919741
Proc Natl Acad Sci U S A. 2003 Dec 23;100(26):15936-41
14673115
J Virol. 2004 Jul;78(14):7344-51
15220407
Lancet Infect Dis. 2004 Aug;4(8):487-98
15288821
J Gen Virol. 1977 Jan;34(1):127-36
188976
Virology. 1980 Oct 15;106(1):41-9
6251620
Virology. 1982 Jul 30;120(2):349-59
6285608
Proc Natl Acad Sci U S A. 1982 Oct;79(20):6232-6
6292897
Virology. 1985 Nov;147(1):169-76
4060597
Virology. 1989 Nov;173(1):311-6
2815585
Virology. 1995 Jan 10;206(1):28-37
7831783
Arch Virol Suppl. 1996;11:77-100
8800808
J Mol Biol. 1997 Dec 19;274(5):816-25
9405160
Virus Res. 2004 Dec;106(2):181-8
15567496
J Struct Biol. 2005 Jul;151(1):30-40
15908231
Virology. 2006 Jan 5;344(1):64-70
16364737
J Virol. 2006 Jun;80(11):5135-44
16698994
Virol J. 2006;3:31
16719918
J Virol. 2007 Feb;81(3):1472-8
17108052
Curr Top Microbiol Immunol. 1999;235:1-21
9893375
J Mol Biol. 2000 Jun 30;300(1):103-12
10864502
EMBO J. 2000 Aug 15;19(16):4228-36
10944105
Proc Natl Acad Sci U S A. 2000 Dec 5;97(25):13871-6
11095724
EMBO J. 2000 Dec 15;19(24):6732-41
11118208
Virology. 2001 Apr 25;283(1):1-6
11312656
J Exp Med. 2002 Mar 4;195(5):593-602
11877482