title: A Tyr residue in the reverse transcriptase domain can mimic the protein-priming Tyr residue in the terminal protein domain of a hepadnavirus P protein.
abstract: Hepadnaviruses are the only known viruses that replicate by protein-primed reverse transcription. Beyond the conserved reverse transcriptase (RT) and RNase H domains, their polymerases (P proteins) carry a unique terminal protein (TP) domain that provides a specific Tyr residue, Tyr96 in duck hepatitis B virus (DHBV), to which the first nucleotide of minus-strand DNA is autocatalytically attached and extended by three more nucleotides. In vitro reconstitution of this priming reaction with DHBV P protein and cellular chaperones had revealed strict requirements for the Dε RNA stem-loop as a template and for catalytic activity of the RT domain plus RNA-binding competence of the TP domain. Chaperone dependence can be obviated by using a truncated P protein (miniP). Here, we found that miniP with a tobacco etch virus (TEV) protease cleavage site between TP and RT (miniP(TEV)) displayed authentic priming activity when supplied with α-(32)P-labeled deoxynucleoside triphosphates; however, protease cleavage revealed, surprisingly, that the RT domain was also labeled. RT labeling had identical requirements as priming at Tyr96 and originated from dNMP transfer to a unique Tyr residue identified as Tyr561 in the presumed RT primer grip motif. Mutating Tyr561 did not affect Tyr96 priming in vitro and only modestly reduced replication competence of an intact DHBV genome; hence, deoxynucleotidylated Tyr561 is not an obligate intermediate in TP priming. However, as a first alternative substrate for the exquisitely complex protein-priming reaction, dNMP transfer to Tyr561 is a novel tool to further clarify the mechanism of hepadnaviral replication initiation and suggests that specific priming inhibitors can be found.
authors: University Hospital Freiburg, Internal Medicine II/Molecular Biology, Hugstetter Str. 55, D-79016 Freiburg, Germany.
authors: Jürgen
authors: J
authors: Beck
authors: Michael
authors: M
authors: Nassal
references: J Virol. 2007 Dec;81(24):13354-64
references: 17913810
references: J Virol. 1996 Sep;70(9):5741-50
references: 8709189
references: Nucleic Acids Res. 2007;35(18):6124-36
references: 17804463
references: Mob DNA. 2011 Jan 21;2(1):1
references: 21255388
references: J Virol. 2005 Aug;79(16):10164-70
references: 16051809
references: EMBO J. 1991 Nov;10(11):3533-40
references: 1915307
references: Mol Cell Biol. 1998 Nov;18(11):6265-72
references: 9774643
references: EMBO J. 2006 Aug 9;25(15):3458-68
references: 16900098
references: Curr Opin Struct Biol. 2009 Dec;19(6):752-8
references: 19914060
references: Annu Rev Biochem. 1991;60:39-71
references: 1883199
references: J Biol Chem. 2003 Jan 10;278(2):1201-5
references: 12444091
references: Biochemistry. 1998 Jul 7;37(27):9630-40
references: 9657675
references: Virology. 2009 Jul 20;390(1):130-8
references: 19467554
references: Nucleic Acids Res. 1995 Dec 25;23(24):4954-62
references: 8559651
references: J Biol Chem. 2006 Nov 24;281(47):35794-801
references: 17005569
references: Gastroenterology. 2009 Nov;137(5):1593-608.e1-2
references: 19737565
references: Antivir Ther. 2004 Apr;9(2):149-60
references: 15134177
references: J Virol. 1993 Nov;67(11):6507-12
references: 7692081
references: J Virol. 2007 Dec;81(23):13230-4
references: 17881436
references: J Virol. 1997 Apr;71(4):2996-3004
references: 9060659
references: Nucleic Acids Res. 2002 Apr 1;30(7):1679-87
references: 11917030
references: J Virol. 2008 Jun;82(12):5703-14
references: 18400846
references: World J Gastroenterol. 2007 Jan 7;13(1):48-64
references: 17206754
references: J Gen Virol. 2004 May;85(Pt 5):1077-1093
references: 15105525
references: Adv Virus Res. 2004;63:1-70
references: 15530560
references: J Virol. 1997 Jul;71(7):5345-54
references: 9188604
references: J Virol. 1994 Jun;68(6):3536-43
references: 8189492
references: EMBO J. 1992 Sep;11(9):3413-20
references: 1380455
references: J Virol. 2006 Mar;80(5):2141-50
references: 16474122
references: Nucleic Acids Res. 1996 Nov 1;24(21):4364-6
references: 8932398
references: J Biol Chem. 1999 Jun 18;274(25):17395-8
references: 10364165
references: J Virol. 1996 May;70(5):2764-73
references: 8627750
references: Methods Enzymol. 2009;463:259-82
references: 19892177
references: J Virol. 1998 Jul;72(7):5789-96
references: 9621038
references: J Virol. 1994 May;68(5):2994-9
references: 7512155
references: J Virol. 2003 Jan;77(2):891-904
references: 12502805
references: Virus Res. 2008 Jun;134(1-2):235-49
references: 18339439
references: J Virol. 2011 Aug;85(15):7754-65
references: 21593164
references: J Virol. 1994 Dec;68(12):8437-42
references: 7525990
references: J Mol Med (Berl). 2011 Jan;89(1):5-12
references: 20820752
references: J Biol Chem. 2003 Sep 19;278(38):36128-38
references: 12851401
references: Immunol Rev. 2009 Jan;227(1):75-86
references: 19120477
references: J Virol. 1994 Jan;68(1):6-13
references: 7504742
references: Mol Cell Biol. 1981 Feb;1(2):165-78
references: 6100962
references: J Virol. 2001 Aug;75(16):7410-9
references: 11462013
references: J Virol. 2003 Apr;77(8):4471-80
references: 12663754
references: Curr Opin Struct Biol. 2009 Feb;19(1):31-8
references: 19157855
references: Proteins. 2006 Jan 1;62(1):24-45
references: 16287116
references: Nucleic Acids Res. 2008 Nov;36(20):6633-44
references: 18940872
references: J Mol Biol. 2009 Jan 23;385(3):693-713
references: 19022262
references: J Virol. 2011 Mar;85(5):2449-57
references: 21177813
references: J Biol Chem. 2008 Nov 28;283(48):33508-15
references: 18826949
references: J Virol. 2001 May;75(10):4771-9
references: 11312349
references: J Virol. 2006 Aug;80(15):7405-15
references: 16840321
references: Proc Natl Acad Sci U S A. 1999 Sep 14;96(19):10818-23
references: 10485909
references: Nucleic Acids Res. 2007;35(10):3375-82
references: 17478507
references: J Mol Biol. 2007 Jul 13;370(3):471-80
references: 17531265
references: Cell. 1992 Nov 13;71(4):663-70
references: 1384989
references: Curr Protoc Protein Sci. 2005 Jun;Chapter 11:Unit 11.4
references: 18429274
references: EMBO J. 2006 Mar 22;25(6):1335-43
references: 16511564
references: J Virol. 2002 Jan;76(1):269-79
references: 11739692