title: Artificial Recruitment of UAF1-USP Complexes by a PHLPP1-E1 Chimeric Helicase Enhances Human Papillomavirus DNA Replication.
abstract: The E1 helicase from anogenital human papillomavirus (HPV) types interacts with the cellular WD repeat-containing protein UAF1 in complex with the deubiquitinating enzyme USP1, USP12, or USP46. This interaction stimulates viral DNA replication and is required for maintenance of the viral episome in keratinocytes. E1 associates with UAF1 through a short UAF1-binding site (UBS) located within the N-terminal 40 residues of the protein. Here, we investigated if the E1 UBS could be replaced by the analogous domain from an unrelated protein, the pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1). We found that PHLPP1 and E1 interact with UAF1 in a mutually exclusive manner and mapped the minimal PHLPP1 UBS (PUBS) to a 100-amino-acid region sufficient for assembly into UAF1-USP complexes. Similarly to the E1 UBS, overexpression of PUBS in trans inhibited HPV DNA replication, albeit less efficiently. Characterization of a PHLPP1-E1 chimeric helicase revealed that PUBS could partially substitute for the E1 UBS in enhancing viral DNA replication and that the stimulatory effect of PUBS likely involves recruitment of UAF1-USP complexes, as it was abolished by mutations that weaken UAF1-binding and by overexpression of catalytically inactive USPs. Although functionally similar to the E1 UBS, PUBS is larger in size and requires both the WD repeat region and C-terminal ubiquitin-like domain of UAF1 for interaction, in contrast to E1, which does not contact the latter. Overall, this comparison of two heterologous UBSs indicates that these domains function as transferable protein interaction modules and provide further evidence that the association of E1 with UAF1-containing deubiquitinating complexes stimulates HPV DNA replication.
  
  
  OBJECTIVE
  The E1 protein from anogenital HPV types interacts with the UAF1-associated deubiquitinating enzymes USP1, USP12, and USP46 to stimulate replication of the viral genome. Little is known about the molecular nature of the E1-UAF1 interaction and, more generally, how UAF1-USP complexes recognize their substrate proteins. To address this question, we characterized the UAF1-binding site (UBS) of PHLPP1, a protein unrelated to E1. Using a PHLPP1-E1 chimeric helicase, we show that the PHLPP1 UBS (PUBS) can partially substitute for the E1 UBS in stimulating HPV DNA replication. This stimulation required conserved sequences in PUBS that meditate its interaction with UAF1, including a motif common to the E1 UBS. These results indicate that UAF1-binding sequences function as transferable protein interaction modules and provide further evidence that UAF1-USP complexes stimulate HPV DNA replication.
authors.affiliation: Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: David
authors.initials: D
authors.lastname: Gagnon
authors.affiliation: Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Michaël
authors.initials: M
authors.lastname: Lehoux
authors.affiliation: Institut de Recherches Cliniques de Montréal (IRCM), Montreal, Quebec, Canada Department of Biochemistry and Molecular Medicine, Université de Montréal, Montreal, Quebec, Canada jacques.archambault@ircm.qc.ca.
authors.forename: Jacques
authors.initials: J
authors.lastname: Archambault
mesh_terms: D002460:Cell Line; D004261:DNA Replication; D054884:Host-Pathogen Interactions; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D027383:Papillomaviridae / Q000502:physiology*; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D009687:Nuclear Proteins; D010749:Phosphoprotein Phosphatases; D011994:Recombinant Proteins; D014764:Viral Proteins; C118863:E1 protein, Human papillomavirus type 31; C526165:WDR48 protein, human; C400601:PHLPP1 protein, human
references.citation: Virology. 2010 Mar 30;399(1):65-76
references.pmid: 20079917
references.citation: Virology. 2009 Feb 20;384(2):360-8
references.pmid: 19141359
references.citation: J Biol Chem. 2010 Apr 9;285(15):11252-7
references.pmid: 20147737
references.citation: Structure. 2010 Aug 11;18(8):966-75
references.pmid: 20696397
references.citation: J Virol. 2011 Jun;85(11):5287-300
references.pmid: 21450828
references.citation: J Virol. 2011 Sep;85(17):8996-9012
references.pmid: 21734051
references.citation: Mol Syst Biol. 2011;7:539
references.pmid: 21988835
references.citation: Biophys J. 2011 Nov 16;101(10):2525-34
references.pmid: 22098752
references.citation: J Virol. 2012 Apr;86(7):3486-500
references.pmid: 22278251
references.citation: Biochemistry. 2012 Apr 3;51(13):2829-39
references.pmid: 22439892
references.citation: J Cancer Res Clin Oncol. 2012 Jul;138(7):1231-8
references.pmid: 22426999
references.citation: Nat Protoc. 2012 Aug;7(8):1511-22
references.pmid: 22814390
references.citation: Vaccine. 2012 Nov 20;30 Suppl 5:F12-23
references.pmid: 23199955
references.citation: Oncogene. 2013 Jan 24;32(4):471-8
references.pmid: 22391563
references.citation: Structure. 2013 Apr 2;21(4):665-71
references.pmid: 23523425
references.citation: Virology. 2013 Oct;445(1-2):35-56
references.pmid: 24029589
references.citation: J Biol Chem. 2013 Nov 29;288(48):34545-54
references.pmid: 24145035
references.citation: Annu Rev Pharmacol Toxicol. 2014;54:537-58
references.pmid: 24392697
references.citation: Nucleic Acids Res. 2014 Jul;42(Web Server issue):W325-30
references.pmid: 24880689
references.citation: J Virol. 2014 Aug;88(15):8545-55
references.pmid: 24850727
references.citation: Oncotarget. 2014 Aug 30;5(16):7081-92
references.pmid: 25216524
references.citation: Methods Mol Biol. 2015;1249:305-16
references.pmid: 25348316
references.citation: Nucleic Acids Res. 2015 Jan;43(Database issue):D339-44
references.pmid: 25348404
references.citation: Mol Cell. 2007 Dec 14;28(5):786-97
references.pmid: 18082604
references.citation: Virology. 2000 Jun 20;272(1):137-50
references.pmid: 10873756
references.citation: Immunity. 2002 Aug;17(2):221-33
references.pmid: 12196293
references.citation: Genome Res. 2004 Jun;14(6):1188-90
references.pmid: 15173120
references.citation: J Gen Virol. 1998 Jul;79 ( Pt 7):1651-8
references.pmid: 9680127
references.citation: J Virol. 1999 Feb;73(2):1001-9
references.pmid: 9882301
references.citation: Proc Natl Acad Sci U S A. 2007 Dec 4;104(49):19541-6
references.pmid: 18048335
references.citation: J Virol. 2008 Feb;82(3):1271-83
references.pmid: 18032488
references.citation: BMC Genomics. 2008;9:308
references.pmid: 18588675
references.citation: Trends Endocrinol Metab. 2008 Aug;19(6):223-30
references.pmid: 18511290
references.citation: J Biol Chem. 2009 Feb 20;284(8):5343-51
references.pmid: 19075014
references.citation: Nat Protoc. 2009;4(3):363-71
references.pmid: 19247286
references.citation: Cell. 2009 Jul 23;138(2):389-403
references.pmid: 19615732
references.citation: J Virol. 2011 Sep;85(17):8981-95
references.pmid: 21734054
references.citation: Genes Dev. 2011 Sep 1;25(17):1847-58
references.pmid: 21896657
references.citation: J Biol Chem. 2010 Apr 2;285(14):10362-9
references.pmid: 20147293