Identification of viral genes essential for replication of murine gamma-herpesvirus 68 using signature-tagged mutagenesis.
Gamma-herpesviruses, Epstein-Barr virus, and Kaposi's sarcoma-associated herpesvirus are important human pathogens, because they are involved in tumor development. Murine gamma-herpesvirus-68 (MHV-68 or gammaHV-68) has emerged as a small animal model system for the study of gamma-herpesvirus pathogenesis and host-virus interactions. To identify the genes required for viral replication in vitro and in vivo, we generated 1,152 mutants using signature-tagged transposon mutagenesis on an infectious bacterial artificial chromosome of MHV-68. Almost every ORF was mutated by random insertion. For each ORF, a mutant with an insertion proximal to the N terminus of each ORF was examined for the ability to grow in fibroblasts. Our results indicate that 41 genes are essential for in vitro growth, whereas 26 are nonessential and 6 attenuated. Replication-competent mutants were pooled to infect mice, which led to the discovery of ORF 54 being important for MHV-68 to replicate in the lung. This genetic analysis of a tumor-associated herpesvirus at the whole genome level validates signature-tagged transposon mutagenesis screening as an effective genetic system to identify important virulent genes in vivo and define interactions with the host immune system.
Song
Moon Jung
MJ
Department of Microbiology, College of Medicine, Hallym University, Chuncheon 200-702, Republic of Korea.
Hwang
Seungmin
S
Wong
Wendy H
WH
Wu
Ting-Ting
TT
Lee
Sangmi
S
Liao
Hsiang-I
HI
Sun
Ren
R
D000818:Animals; D005814:Genes, Viral / Q000502:physiology*; D051379:Mice; D016296:Mutagenesis; D009154:Mutation; D041681:NIH 3T3 Cells; D016366:Open Reading Frames; D011755:Pyrophosphatases / Q000502:physiology; D018144:Rhadinovirus / Q000235:genetics* / Q000502:physiology; D014779:Virus Replication
D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase
J Virol. 1997 Aug;71(8):5894-904
9223479
J Virol. 2000 Feb;74(4):1973-84
10644370
J Gen Virol. 1998 Jan;79 ( Pt 1):149-53
9460936
J Exp Med. 1998 Jun 15;187(12):1941-51
9625754
Trends Microbiol. 1998 Jul;6(7):276-82
9717216
J Virol. 1999 Apr;73(4):3273-83
10074181
Genome Res. 1999 Mar;9(3):308-15
10077537
Biotechniques. 1999 Mar;26(3):473-8, 480
10090989
Nucleic Acids Res. 1999 Jul 1;27(13):2777-84
10373596
J Virol. 1999 Sep;73(9):7710-21
10438861
Curr Opin Immunol. 1999 Aug;11(4):371-9
10448140
J Virol. 2000 Aug;74(16):7451-61
10906198
Genome Res. 2001 Jan;11(1):43-54
11156614
Philos Trans R Soc Lond B Biol Sci. 2001 Apr 29;356(1408):569-79
11313012
Philos Trans R Soc Lond B Biol Sci. 2001 Apr 29;356(1408):581-93
11313013
J Exp Med. 2001 Aug 6;194(3):301-12
11489949
J Virol. 2002 Feb;76(4):1790-801
11799175
J Exp Med. 2002 Apr 1;195(7):931-40
11927636
Nat Immunol. 2002 Aug;3(8):733-40
12101398
J Gen Virol. 2002 Oct;83(Pt 10):2339-45
12237414
Curr Protein Pept Sci. 2001 Dec;2(4):381-8
12374097
J Virol. 2003 Feb;77(4):2410-7
12551978
Virology. 2003 Mar 15;307(2):179-90
12667789
J Virol. 2003 May;77(10):5731-9
12719566
J Virol. 2003 Sep;77(17):9700-9
12915582
J Virol. 2003 Oct;77(19):10295-303
12970414
Proc Natl Acad Sci U S A. 2003 Oct 14;100(21):12396-401
14519856
Annu Rev Genet. 2003;37:3-29
14616054
J Gen Virol. 2003 Dec;84(Pt 12):3405-16
14645921
J Virol. 2003 Dec;77(24):13425-32
14645600
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14223-8
14623981
J Virol. 2004 May;78(10):5103-12
15113892
J Virol. 2004 Jun;78(12):6610-20
15163752
J Virol. 2004 Oct;78(19):10282-90
15367594
J Cell Biochem. 1990 Aug;43(4):339-53
2168898
J Virol. 1992 Nov;66(11):6706-13
1328686
J Gen Virol. 1992 Dec;73 ( Pt 12):3275-9
1469366
J Virol. 1994 Jan;68(1):289-97
8254740
Science. 1995 Jul 21;269(5222):400-3
7618105
J Virol. 1996 Oct;70(10):6775-80
8794315
Mol Microbiol. 1997 Oct;26(2):399-407
9383163
Curr Opin Microbiol. 1999 Aug;2(4):403-9
10458986
Annu Rev Microbiol. 1999;53:129-54
10547688
J Gen Virol. 1997 Jun;78 ( Pt 6):1425-33
9191940