Picornavirus genome replication: roles of precursor proteins and rate-limiting steps in oriI-dependent VPg uridylylation.
The 5' ends of all picornaviral RNAs are linked covalently to the genome-encoded peptide, VPg (or 3B). VPg linkage is thought to occur in two steps. First, VPg serves as a primer for production of diuridylylated VPg (VPg-pUpU) in a reaction catalyzed by the viral polymerase that is templated by an RNA element (oriI). It is currently thought that the viral 3AB protein is the source of VPg in vivo. Second, VPg-pUpU is transferred to the 3' end of plus- and/or minus-strand RNA and serves as primer for production of full-length RNA. Nothing is known about the mechanism of transfer. We present biochemical and biological evidence refuting the use of 3AB as the donor for VPg uridylylation. Our data are consistent with precursors 3BC and/or 3BCD being employed for uridylylation. This conclusion is supported by in vitro uridylylation of these proteins, the ability of a mutant replicon incapable of producing processed VPg to replicate in HeLa cells and cell-free extracts and corresponding precursor processing profiles, and the demonstration of 3BC-linked RNA in mutant replicon-transfected cells. These data permit elaboration of our model for VPg uridylylation to include the use of precursor proteins and invoke a possible mechanism for location of the diuridylylated, VPg-containing precursor at the 3' end of plus- or minus-strand RNA for production of full-length RNA. Finally, determinants of VPg uridylylation efficiency suggest formation and/or collapse or release of the uridylylated product as the rate-limiting step in vitro depending upon the VPg donor employed.
Pathak
Harsh B
HB
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Oh
Hyung Suk
HS
Goodfellow
Ian G
IG
Arnold
Jamie J
JJ
Cameron
Craig E
CE
D002474:Cell-Free System / Q000378:metabolism / Q000821:virology; D016679:Genome, Viral / Q000502:physiology*; D006023:Glycoproteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008954:Models, Biological; D017955:Poliovirus / Q000502:physiology*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012367:RNA, Viral / Q000378:metabolism; D014529:Uridine / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
D006023:Glycoproteins; D012367:RNA, Viral; D014764:Viral Proteins; D014529:Uridine
J Biol Chem. 2004 Mar 26;279(13):12659-67
14711816
J Biol Chem. 1995 Jun 16;270(24):14430-8
7782305
EMBO J. 2006 Feb 22;25(4):880-8
16456546
Biochemistry. 2004 May 11;43(18):5138-48
15122879
Virology. 1992 Nov;191(1):309-20
1329322
J Virol. 2007 Jun;81(11):5669-84
17360746
EMBO J. 1996 Jan 2;15(1):23-33
8598203
J Virol. 2002 Aug;76(15):7485-94
12097561
J Virol. 2006 Oct;80(19):9865-75
16973591
J Virol. 2006 Aug;80(15):7405-15
16840321
EMBO J. 2004 Sep 1;23(17):3462-71
15306852
J Virol. 2002 Oct;76(19):9686-94
12208947
Arch Virol. 2002 Apr;147(4):731-44
12038684
J Virol. 2007 Sep;81(17):8919-32
17581994
J Biol Chem. 2007 Jun 1;282(22):16202-13
17392285
Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5428-32
8516284
Cell Cycle. 2007 Jan 1;6(1):36-8
17245115
J Virol. 2005 Jun;79(12):7803-11
15919933
EMBO J. 1995 Mar 1;14(5):894-907
7889939
J Virol. 2003 Apr;77(8):4739-50
12663781
J Virol. 2008 May;82(9):4363-70
18305026
J Biol Chem. 2007 Jun 1;282(22):16135-45
17400557
J Virol. 1995 Sep;69(9):5516-27
7636997
Virus Res. 1997 Oct;51(2):151-7
9498613
J Biol Chem. 2008 Jan 11;283(2):875-88
17993457
J Virol. 1996 Sep;70(9):5954-61
8709217
Nucleic Acids Res. 2006 May 31;34(10):2953-65
16738134
Genes Dev. 1998 Aug 1;12(15):2293-304
9694795
J Virol. 2003 Jan;77(2):891-904
12502805
J Biol Chem. 2000 Feb 25;275(8):5329-36
10681506
J Biol Chem. 1999 Apr 30;274(18):12867-76
10212275
J Virol. 2007 Apr;81(7):3583-96
17251299
J Virol. 2003 May;77(9):5136-44
12692216
Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12688-93
15314212
Dev Cell. 2006 Aug;11(2):191-201
16890159
J Virol. 2003 May;77(9):5152-66
12692218
J Virol. 2007 Nov;81(22):12485-95
17855535
J Biol Chem. 2002 Aug 30;277(35):31551-62
12077141
J Virol. 2000 Nov;74(22):10359-70
11044080
J Biol Chem. 1999 Dec 24;274(52):37060-9
10601264
J Biol Chem. 2003 Nov 7;278(45):43951-60
12937178
J Virol. 2005 Jun;79(12):7698-706
15919922
J Virol. 2000 Nov;74(22):10371-80
11044081
Virology. 1966 Jun;29(2):179-89
4287327
J Virol. 2001 Nov;75(22):10979-90
11602738
Nature. 1998 May 21;393(6682):280-4
9607767
J Gen Virol. 2006 Jan;87(Pt 1):103-113
16361422