Host factors associated with the Sindbis virus RNA-dependent RNA polymerase: role for G3BP1 and G3BP2 in virus replication.
Sindbis virus (SINV) is the prototype member of the Alphavirus genus, whose members cause severe human diseases for which there is no specific treatment. To ascertain host factors important in the replication of the SINV RNA genome, we generated a SINV expressing nsP4, the viral RNA-dependent RNA polymerase, with an in-frame 3xFlag epitope tag. Proteomic analysis of nsP4-containing complexes isolated from cells infected with the tagged virus revealed 29 associated host proteins. Of these, 10 proteins were associated only at a later time of infection (12 h), 14 were associated both early and late, and five were isolated only at the earlier time (6 h postinfection). These results demonstrate the dynamic nature of the virus-host interaction that occurs over the course of infection and suggest that different host proteins may be required for the multiple functions carried out by nsP4. Two related proteins found in association with nsP4 at both times of infection, GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) and G3BP2 were also previously identified as associated with SINV nsP2 and nsP3. We demonstrate a likely overlapping role for these host factors in limiting SINV replication events. The present study also identifies 10 host factors associated with nsP4 6 h after infection that were not found to be associated with nsP2 or nsP3. These factors are candidates for playing important roles in the RNA replication process. Identifying host factors essential for replication should lead to new strategies to interrupt alphavirus replication.
Cristea
Ileana M
IM
Department of Molecular Biology, Princeton University, Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey 08544, USA.
Rozjabek
Heather
H
Molloy
Kelly R
KR
Karki
Sophiya
S
White
Laura L
LL
Rice
Charles M
CM
Rout
Michael P
MP
Chait
Brian T
BT
MacDonald
Margaret R
MR
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D006224:Cricetinae; D054884:Host-Pathogen Interactions; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D051381:Rats; D012845:Sindbis Virus / Q000502:physiology*; D013997:Time Factors; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
D002352:Carrier Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase
J Virol. 2003 Nov;77(21):11555-62
14557641
J Virol. 1998 Aug;72(8):6546-53
9658098
J Virol. 2005 Aug;79(15):9397-409
16014903
J Cell Biochem. 2001;84(1):173-87
11746526
Anal Chem. 2000 Jun 1;72(11):2482-9
10857624
Methods. 2007 Jul;42(3):298-305
17532517
Nat Rev Mol Cell Biol. 2005 Jun;6(6):449-61
15928709
J Cell Biol. 1988 Dec;107(6 Pt 1):2075-86
2904446
J Virol. 1987 Dec;61(12):3809-19
3479621
Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2679-83
1372987
Mol Cell Proteomics. 2005 Dec;4(12):1933-41
16155292
Mol Biotechnol. 2001 Nov;19(3):313-21
11721627
J Cell Biol. 2003 Mar 17;160(6):823-31
12642610
J Virol. 2008 Oct;82(20):10088-101
18684830
Microbiol Rev. 1994 Sep;58(3):491-562
7968923
Virology. 2007 Feb 5;358(1):221-32
16979682
J Biol Chem. 2006 Oct 6;281(40):30269-78
16895903
Prog Nucleic Acid Res Mol Biol. 2002;71:187-222
12102555
J Virol. 2006 Jun;80(12):5686-96
16731907
Nature. 1993 Feb 25;361(6414):739-42
8441470
J Virol. 2000 Oct;74(20):9802-7
11000258
J Am Soc Mass Spectrom. 2010 Jan;21(1):34-46
19822444
Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8967-71
1924357
J Virol. 2002 Nov;76(22):11254-64
12388685
J Virol. 2001 Apr;75(8):3509-19
11264340
J Biol Chem. 2000 Nov 17;275(46):36441-9
10969074
PLoS One. 2007 Apr 04;2(4):e358
17406682
Proc Natl Acad Sci U S A. 2009 Jun 23;106(25):10207-12
19509342
Biochem Soc Trans. 2002 Nov;30(Pt 6):963-9
12440955
Nucleic Acids Res. 1999 Feb 1;27(3):817-21
9889278
Cell Host Microbe. 2007 Nov 15;2(5):295-305
18005751
J Virol. 2007 May;81(10):5046-57
17329335
Proc Natl Acad Sci U S A. 1983 Sep;80(17):5271-5
6577423
Virology. 2009 Feb 5;384(1):201-8
19036396
Virology. 1976 Oct 1;74(1):154-68
62444
J Virol. 1999 May;73(5):3854-65
10196280
J Virol. 1999 May;73(5):3682-91
10196260
Biochem Biophys Res Commun. 2006 Nov 10;350(1):174-8
16996479
J Virol. 2006 Oct;80(20):9962-9
17005674
J Virol. 2006 Apr;80(8):4122-34
16571828
J Cell Biol. 2005 Jun 20;169(6):871-84
15967811
J Biol Chem. 2002 Mar 8;277(10):8243-7
11756452
Circ Res. 2008 Aug 29;103(5):485-92
18669920