title: ORF73-null murine gammaherpesvirus 68 reveals roles for mLANA and p53 in virus replication.
abstract: Gammaherpesviruses establish lifelong, latent infections in host lymphocytes, during which a limited subset of viral gene products facilitates maintenance of the viral episome. Among the gamma-2-herpesvirus (rhadinovirus) subfamily, this includes expression of the conserved ORF73-encoded LANA proteins. We previously demonstrated by loss-of-function mutagenesis that the murine gammaherpesvirus 68 (MHV68) ORF73 gene product, mLANA, is required for the establishment of latency following intranasal inoculation of mice (N. J. Moorman, D. O. Willer, and S. H. Speck, J. Virol. 77:10295-10303, 2003). mLANA-deficient viruses also exhibited a defect in acute virus replication in the lungs of infected mice. The latter observation led us to examine the role of mLANA in productive viral replication. We assessed the capacity of mLANA-deficient virus (73.Stop) to replicate in cell culture at low multiplicities of infection (MOIs) and found that 73.Stop growth was impaired in murine fibroblasts but not in Vero cells. A recombinant virus expressing an mLANA-green fluorescent protein (GFP) fusion revealed that mLANA is expressed throughout the virus replication cycle. In addition, 73.Stop infection of murine fibroblasts at high MOIs was substantially more cytotoxic than infection with a genetically repaired marker rescue virus (73.MR), a phenotype that correlated with enhanced kinetics of viral gene expression and increased activation of p53. Notably, augmented cell death, viral gene expression, and p53 induction were independent of viral DNA replication. Expression of a mLANA-GFP fusion protein in fibroblasts correlated with both reduced p53 stabilization and reduced cell death following treatment with p53-inducing agonists. In agreement, accentuated cell death associated with 73.Stop infection was reduced in p53-deficient murine embryonic fibroblasts. Additionally, replication of 73.Stop in p53-deficient cells was restored to levels comparable to those of 73.MR. More remarkably, the absence of p53 led to an overall delay in replication for both 73.Stop and 73.MR viruses, which correlated with delayed viral gene expression, indicating a role for p53 in MHV68 replication. Consistent with these findings, the expression of replication-promoting viral genes was positively influenced by p53 overexpression or treatment with the p53 agonist etoposide. Overall, these data demonstrate the importance of mLANA in MHV68 replication and suggest that LANA proteins limit the induction of cellular stress responses to regulate the viral gene expression cascade and limit host cell injury.
authors: Department of Microbiology and immunology, Emory Vaccine Center, Emory University School of Medicine, 1462 Clifton Rd., Atlanta, GA 30322, USA.
authors: J Craig
authors: JC
authors: Forrest
authors: Clinton R
authors: CR
authors: Paden
authors: Robert D
authors: RD
authors: Allen
authors: Julie
authors: J
authors: Collins
authors: Samuel H
authors: SH
authors: Speck
mesh_terms: D000818:Animals; D000951:Antigens, Neoplasm / Q000502:physiology*; D002460:Cell Line; D002522:Chlorocebus aethiops; D005347:Fibroblasts / Q000378:metabolism; D018142:Gammaherpesvirinae; D015967:Gene Expression Regulation, Viral; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D058965:MART-1 Antigen; D051379:Mice; D041681:NIH 3T3 Cells; D009363:Neoplasm Proteins / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
chemical_list: D000951:Antigens, Neoplasm; D058965:MART-1 Antigen; C548667:MLANA protein, human; C548668:Mlana protein, mouse; D009363:Neoplasm Proteins; D016159:Tumor Suppressor Protein p53; D014764:Viral Proteins; D049452:Green Fluorescent Proteins
references: J Virol. 2004 Aug;78(15):8068-77
references: 15254178
references: Virology. 2005 Oct 25;341(2):271-83
references: 16102793
references: Apoptosis. 2002 Aug;7(4):321-8
references: 12101391
references: J Virol. 1995 Nov;69(11):6697-704
references: 7474079
references: Gene. 2000 Apr 18;247(1-2):1-15
references: 10773440
references: J Exp Med. 1998 Jun 15;187(12):1941-51
references: 9625754
references: Nature. 1970 Aug 15;227(5259):680-5
references: 5432063
references: Proc Natl Acad Sci U S A. 1996 Oct 15;93(21):11321-6
references: 8876134
references: Cell. 1997 Mar 7;88(5):593-602
references: 9054499
references: Genes Dev. 2000 Aug 15;14(16):2015-27
references: 10950866
references: Oncogene. 2002 Dec 5;21(55):8379-87
references: 12466958
references: J Virol. 2003 Jul;77(13):7308-18
references: 12805429
references: Nature. 1999 Dec 23-30;402(6764):889-94
references: 10622254
references: J Virol. 2006 Jan;80(2):697-709
references: 16378973
references: Cancer Cell. 2002 Jul;2(1):55-65
references: 12150825
references: J Clin Invest. 2006 Mar;116(3):735-42
references: 16498502
references: Virology. 2007 Sep 1;365(2):250-9
references: 17477952
references: PLoS Pathog. 2005 Sep;1(1):e10
references: 16201011
references: Biochem Cell Biol. 1997;75(4):337-49
references: 9493956
references: J Immunol. 2000 Jul 15;165(2):1074-81
references: 10878386
references: Methods. 2001 Dec;25(4):402-8
references: 11846609
references: J Virol. 2003 Aug;77(15):8310-21
references: 12857900
references: Cancer Surv. 1986;5(2):389-404
references: 2946407
references: J Virol. 2001 Feb;75(3):1487-506
references: 11152521
references: Oncogene. 2005 Apr 18;24(17):2899-908
references: 15838523
references: J Gen Virol. 2003 Dec;84(Pt 12):3405-16
references: 14645921
references: J Clin Invest. 2005 Mar;115(3):642-52
references: 15765147
references: Proc Natl Acad Sci U S A. 2005 Mar 8;102(10):3805-10
references: 15738413
references: J Virol. 2004 Jun;78(12):6585-94
references: 15163750
references: J Virol. 2000 Aug;74(16):7451-61
references: 10906198
references: J Virol. 1997 Aug;71(8):5894-904
references: 9223479
references: Science. 2004 Feb 13;303(5660):1010-4
references: 14963330
references: J Biol Chem. 1996 Feb 16;271(7):3534-40
references: 8631958
references: J Virol. 2003 May;77(10):5731-9
references: 12719566
references: J Virol. 2004 Oct;78(19):10336-47
references: 15367600
references: Proc Natl Acad Sci U S A. 2006 Feb 21;103(8):2821-6
references: 16477038
references: Virology. 2006 Apr 25;348(1):19-34
references: 16455125
references: J Virol. 2006 Aug;80(16):7965-75
references: 16873253
references: J Virol. 2006 Nov;80(21):10802-12
references: 16956952
references: Oncogene. 1992 May;7(5):935-42
references: 1349164
references: Nature. 1991 Jan 31;349(6308):429-31
references: 1671528
references: J Virol. 2000 Aug;74(15):6964-74
references: 10888635
references: Cell Death Differ. 2006 Jun;13(6):941-50
references: 16601750
references: Oncogene. 2005 Apr 18;24(17):2796-809
references: 15838516
references: J Clin Invest. 1997 Nov 15;100(10):2606-10
references: 9366576
references: J Virol. 1997 Apr;71(4):3146-60
references: 9060678
references: J Gen Virol. 1992 Dec;73 ( Pt 12):3275-9
references: 1469366
references: J Virol. 2001 Feb;75(4):1870-8
references: 11160686
references: J Virol. 2007 Feb;81(4):1912-22
references: 17121789
references: J Virol. 2003 May;77(10):5911-25
references: 12719584
references: Cell. 2004 Feb 20;116(4):487-9
references: 14980216
references: Antimicrob Agents Chemother. 1998 Jan;42(1):170-2
references: 9449280
references: J Virol. 2006 Sep;80(17):8390-401
references: 16912290
references: J Virol. 2003 Oct;77(19):10295-303
references: 12970414
references: J Virol. 2005 Mar;79(5):3127-38
references: 15709032
references: Cancer Cell. 2007 Feb;11(2):175-89
references: 17292828
references: J Virol. 2003 Oct;77(19):10488-503
references: 12970434
references: EMBO J. 2006 Sep 20;25(18):4284-92
references: 16957780
references: J Virol. 1999 Apr;73(4):3273-83
references: 10074181
references: J Virol. 2006 Dec;80(24):11946-59
references: 17005668
references: Nature. 2006 Nov 30;444(7119):638-42
references: 17136094
references: J Virol. 1998 Mar;72(3):2033-9
references: 9499057
references: J Virol. 2004 Mar;78(5):2169-78
references: 14963113
references: J Gen Virol. 1999 Oct;80 ( Pt 10):2737-40
references: 10573168
references: J Virol. 2004 Aug;78(15):8359-71
references: 15254208
references: Microbiol Mol Biol Rev. 2002 Jun;66(2):179-202
references: 12040123
references: Genes Dev. 1998 Oct 1;12(19):3008-19
references: 9765203
references: J Virol. 2002 Jun;76(12):6244-56
references: 12021358
references: J Virol. 1996 Oct;70(10):6775-80
references: 8794315
references: Nature. 2000 Nov 16;408(6810):307-10
references: 11099028
references: J Gen Virol. 2005 Mar;86(Pt 3):561-74
references: 15722515
references: J Virol. 1985 Nov;56(2):558-70
references: 2997476
references: J Virol. 2000 Feb;74(4):2029-37
references: 10644377
references: J Biol Chem. 2005 Feb 4;280(5):3862-74
references: 15525642
references: J Biol Chem. 2005 Aug 26;280(34):30336-41
references: 15964848
references: J Virol. 1999 Jun;73(6):5110-22
references: 10233974
references: Nat Med. 2000 Oct;6(10):1121-7
references: 11017143
references: Nature. 2006 Nov 30;444(7119):633-7
references: 17136093
references: Gene. 2002 May 15;290(1-2):19-34
references: 12062798
references: Genes Dev. 1993 Apr;7(4):535-45
references: 8384579