title: Biochemical characterization of a recombinant TRIM5alpha protein that restricts human immunodeficiency virus type 1 replication.
abstract: The rhesus monkey intrinsic immunity factor TRIM5alpha(rh) recognizes incoming capsids from a variety of retroviruses, including human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV), and inhibits the accumulation of viral reverse transcripts. However, direct interactions between restricting TRIM5alpha proteins and retroviral capsids have not previously been demonstrated using pure recombinant proteins. To facilitate structural and mechanistic studies of retroviral restriction, we have developed methods for expressing and purifying an active chimeric TRIM5alpha(rh) protein containing the RING domain from the related human TRIM21 protein. This recombinant TRIM5-21R protein was expressed in SF-21 insect cells and purified through three chromatographic steps. Two distinct TRIM5-21R species were purified and shown to correspond to monomers and dimers, as analyzed by analytical ultracentrifugation. Chemically cross-linked recombinant TRIM5-21R dimers and mammalian-expressed TRIM5-21R and TRIM5alpha proteins exhibited similar sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities, indicating that mammalian TRIM5alpha proteins are predominantly dimeric. Purified TRIM5-21R had ubiquitin ligase activity and could autoubquitylate with different E2 ubiquitin conjugating enzymes in vitro. TRIM5-21R bound directly to synthetic capsids composed of recombinant HIV-1 CA-NC proteins and to authentic EIAV core particles. HIV-1 CA-NC assemblies bound dimeric TRIM5-21R better than either monomeric TRIM5-21R or TRIM5-21R constructs that lacked the SPRY domain or its V1 loop. Thus, our studies indicate that TRIM5alpha proteins are dimeric ubiquitin E3 ligases that recognize retroviral capsids through direct interactions mediated by the SPRY domain and demonstrate that these activities can be recapitulated in vitro using pure recombinant proteins.
authors.affiliation: Department of Biochemistry, University of Utah, Salt Lake City, UT 84112-5650, USA.
authors.forename: Charles R
authors.initials: CR
authors.lastname: Langelier
authors.forename: Virginie
authors.initials: V
authors.lastname: Sandrin
authors.forename: Debra M
authors.initials: DM
authors.lastname: Eckert
authors.forename: Devin E
authors.initials: DE
authors.lastname: Christensen
authors.forename: Viswanathan
authors.initials: V
authors.lastname: Chandrasekaran
authors.forename: Steven L
authors.initials: SL
authors.lastname: Alam
authors.forename: Christopher
authors.initials: C
authors.lastname: Aiken
authors.forename: John C
authors.initials: JC
authors.lastname: Olsen
authors.forename: Alak Kanti
authors.initials: AK
authors.lastname: Kar
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Sodroski
authors.forename: Wesley I
authors.initials: WI
authors.lastname: Sundquist
mesh_terms: D002213:Capsid / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000502:physiology; D019281:Dimerization; D015497:HIV-1 / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry* / Q000502:physiology; D019251:Nucleocapsid / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012261:Ribonucleoproteins; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D014779:Virus Replication; D000099207:SS-A Antigen
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D000099207:SS-A Antigen; C482587:TRIM5(alpha) protein, rhesus monkey
references.citation: J Mol Biol. 2008 Mar 21;377(2):431-49
references.pmid: 18272178
references.citation: J Virol. 2002 Jun;76(11):5667-77
references.pmid: 11991995
references.citation: Mol Cell. 2006 Mar 17;21(6):873-80
references.pmid: 16543155
references.citation: J Biol Chem. 1990 Sep 15;265(26):15813-7
references.pmid: 2203788
references.citation: Retrovirology. 2004 May 14;1:9
references.pmid: 15169567
references.citation: EMBO J. 1986 Dec 1;5(12):3133-42
references.pmid: 3102226
references.citation: Nat Immunol. 2004 Nov;5(11):1109-15
references.pmid: 15496950
references.citation: Cell. 1996 Dec 27;87(7):1285-94
references.pmid: 8980234
references.citation: Virology. 2006 Aug 1;351(2):404-19
references.pmid: 16650449
references.citation: Proc Natl Acad Sci U S A. 2006 May 9;103(19):7465-70
references.pmid: 16648264
references.citation: Virology. 2007 Apr 10;360(2):419-33
references.pmid: 17156811
references.citation: EMBO J. 2003 Apr 1;22(7):1707-15
references.pmid: 12660176
references.citation: Nat Struct Mol Biol. 2007 Oct;14(10):941-8
references.pmid: 17873885
references.citation: J Virol. 2005 Nov;79(22):14446-50
references.pmid: 16254380
references.citation: Virology. 2006 Sep 30;353(2):396-409
references.pmid: 16828831
references.citation: J Virol. 2000 Feb;74(3):1168-77
references.pmid: 10627527
references.citation: J Virol. 2000 Jul;74(13):6198-202
references.pmid: 10846106
references.citation: Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9564-8
references.pmid: 7937806
references.citation: Gene Ther. 2005 Jan;12(1):22-9
references.pmid: 15550928
references.citation: J Virol. 2005 Aug;79(16):10571-7
references.pmid: 16051849
references.citation: Nature. 2000 Sep 21;407(6802):409-13
references.pmid: 11014200
references.citation: J Virol. 2006 Sep;80(17):8554-65
references.pmid: 16912305
references.citation: Methods Enzymol. 2005;399:21-36
references.pmid: 16338346
references.citation: J Virol. 2006 Oct;80(19):9754-60
references.pmid: 16973579
references.citation: Virology. 2007 Dec 20;369(2):400-10
references.pmid: 17920096
references.citation: Mol Cell. 2005 Nov 23;20(4):525-38
references.pmid: 16307917
references.citation: Gene. 2005 Dec 5;362:109-16
references.pmid: 16226405
references.citation: Virology. 2006 Jun 5;349(2):300-15
references.pmid: 16472833
references.citation: Proc Natl Acad Sci U S A. 2006 Apr 4;103(14):5514-9
references.pmid: 16540544
references.citation: Exp Cell Res. 2003 Aug 1;288(1):84-93
references.pmid: 12878161
references.citation: Proc Natl Acad Sci U S A. 2007 Apr 10;104(15):6200-5
references.pmid: 17400754
references.citation: J Mol Biol. 1999 Feb 12;286(1):83-93
references.pmid: 9931251
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10780-5
references.pmid: 15249687
references.citation: J Virol. 2005 Jan;79(2):869-75
references.pmid: 15613315
references.citation: J Biol Chem. 2005 Mar 18;280(11):10548-55
references.pmid: 15644320
references.citation: Proc Natl Acad Sci U S A. 2005 Feb 22;102(8):2832-7
references.pmid: 15689398
references.citation: FEBS J. 2008 Apr;275(7):1540-1555
references.pmid: 18312418
references.citation: J Virol. 1995 Oct;69(10):6487-97
references.pmid: 7666550
references.citation: Virology. 2007 Sep 1;365(2):302-14
references.pmid: 17493656
references.citation: J Virol. 2007 Oct;81(19):10362-78
references.pmid: 17626085
references.citation: Science. 1999 Jan 1;283(5398):80-3
references.pmid: 9872746
references.citation: PLoS Pathog. 2008 May 23;4(5):e1000074
references.pmid: 18497858
references.citation: Proc Natl Acad Sci U S A. 2004 Jul 20;101(29):10786-91
references.pmid: 15249690
references.citation: J Virol. 2007 Mar;81(5):2138-48
references.pmid: 17135314
references.citation: Curr Opin Struct Biol. 2008 Apr;18(2):203-17
references.pmid: 18406133
references.citation: J Virol. 1998 Mar;72(3):1902-9
references.pmid: 9499042
references.citation: Mol Immunol. 2006 Feb;43(6):588-98
references.pmid: 15916807
references.citation: Virology. 2006 Jul 5;350(2):493-500
references.pmid: 16624363
references.citation: J Cell Biol. 2008 Feb 11;180(3):549-61
references.pmid: 18250195
references.citation: Cell. 2007 Oct 5;131(1):70-9
references.pmid: 17923088
references.citation: J Virol. 2006 Jul;80(14):6738-44
references.pmid: 16809279
references.citation: Methods Enzymol. 2004;384:212-32
references.pmid: 15081689
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 10;101(32):11827-32
references.pmid: 15280539
references.citation: Retrovirology. 2007 Jun 12;4:40
references.pmid: 17565686
references.citation: Virology. 2006 Sep 15;353(1):234-46
references.pmid: 16808955
references.citation: J Mol Biol. 1997 Jun 27;269(5):780-95
references.pmid: 9223641
references.citation: Mol Immunol. 2007 Mar;44(9):2406-14
references.pmid: 17118455
references.citation: J Virol. 1999 Oct;73(10):8824-30
references.pmid: 10482638
references.citation: J Virol. 2005 Mar;79(5):3139-45
references.pmid: 15709033
references.citation: Curr Biol. 2005 Jan 11;15(1):73-8
references.pmid: 15649369
references.citation: J Biol Chem. 2005 Jul 22;280(29):26933-40
references.pmid: 15897199
references.citation: J Virol. 2006 Apr;80(8):4061-7
references.pmid: 16571822
references.citation: PLoS Pathog. 2007 Dec;3(12):e197
references.pmid: 18159944
references.citation: J Virol. 2005 Jul;79(14):8969-78
references.pmid: 15994791
references.citation: Nature. 2004 Feb 26;427(6977):848-53
references.pmid: 14985764
references.citation: Biochim Biophys Acta. 2001 Dec 5;1568(2):155-61
references.pmid: 11750763
references.citation: Biochem Biophys Res Commun. 1989 Apr 28;160(2):486-94
references.pmid: 2541703
references.citation: Virology. 2005 Dec 20;343(2):201-11
references.pmid: 16183097
references.citation: J Virol. 2005 May;79(10):6111-21
references.pmid: 15857996
references.citation: Retrovirology. 2005 Jun 20;2:40
references.pmid: 15967037
references.citation: Mol Cell. 2006 Dec 28;24(6):967-76
references.pmid: 17189197
references.citation: J Biol Chem. 2006 Jan 6;281(1):528-42
references.pmid: 16195228
references.citation: EMBO J. 2003 Feb 3;22(3):385-94
references.pmid: 12554640