Restricted growth of U-type infectious haematopoietic necrosis virus (IHNV) in rainbow trout cells may be linked to casein kinase II activity.
Previously, we demonstrated that a representative M genogroup type strain of infectious haematopoietic necrosis virus (IHNV) from rainbow trout grows well in rainbow trout-derived RTG-2 cells, but a U genogroup type strain from sockeye salmon has restricted growth, associated with reduced genome replication and mRNA transcription. Here, we analysed further the mechanisms for this growth restriction of U-type IHNV in RTG-2 cells, using strategies that assessed differences in viral genes, host immune regulation and phosphorylation. To determine whether the viral glycoprotein (G) or non-virion (NV) protein was responsible for the growth restriction, four recombinant IHNV viruses were generated in which the G gene of an infectious IHNV clone was replaced by the G gene of U- or M-type IHNV and the NV gene was replaced by NV of U- or M-type IHNV. There was no significant difference in the growth of these recombinants in RTG-2 cells, indicating that G and NV proteins are not major factors responsible for the differential growth of the U- and M-type strains. Poly I:C pretreatment of RTG-2 cells suppressed the growth of both U- and M-type IHNV, although the M virus continued to replicate at a reduced level. Both viruses induced type 1 interferon (IFN1) and the IFN1 stimulated gene Mx1, but the expression levels in M-infected cells were significantly higher than in U-infected cells and an inhibitor of the IFN1-inducible protein kinase PKR, 2-aminopurine (2-AP), did not affect the growth of U- or M-type IHNV in RTG-2 cells. These data did not indicate a role for the IFN1 system in the restricted growth of U-type IHNV in RTG-2 cells. Prediction of kinase-specific phosphorylation sites in the viral phosphoprotein (P) using the NetPhosK program revealed differences between U- and M-type P genes at five phosphorylation sites. Pretreatment of RTG-2 cells with a PKC inhibitor or a p38MAPK inhibitor did not affect the growth of the U- and M-type viruses. However, 100 μm of the casein kinase II (CKII) inhibitor, 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB), reduced the titre of the U type 8.3-fold at 24 h post-infection. In contrast, 100 μm of the CKII inhibitor reduced the titre of the M type only 1.3-fold at 48 h post-infection. Our data suggest that the different growth of U- and M-type IHNV in RTG-2 cells may be linked to a differential requirement for cellular protein kinases such as CKII for their growth.
US Geological Survey, Western Fisheries Research Center, Seattle, WA, USA.
J W
JW
Park
C H
CH
Moon
A
A
Harmache
A R
AR
Wargo
M K
MK
Purcell
M
M
Bremont
G
G
Kurath
D015075:2-Aminopurine / Q000378:metabolism; D000818:Animals; D047390:Casein Kinase II / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D004004:Dichlororibofuranosylbenzimidazole / Q000378:metabolism; D005393:Fish Diseases / Q000276:immunology / Q000378:metabolism / Q000821:virology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D006023:Glycoproteins / Q000378:metabolism; D054884:Host-Pathogen Interactions; D029142:Infectious hematopoietic necrosis virus / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D007370:Interferon Type I / Q000378:metabolism; D008969:Molecular Sequence Data; D064547:Myxovirus Resistance Proteins; D017686:Oncorhynchus mykiss / Q000821:virology*; D011070:Poly I-C / Q000378:metabolism; D018353:Rhabdoviridae Infections / Q000276:immunology / Q000662:veterinary* / Q000821:virology; D014764:Viral Proteins / Q000378:metabolism; D014779:Virus Replication
D006023:Glycoproteins; D007370:Interferon Type I; D064547:Myxovirus Resistance Proteins; D014764:Viral Proteins; D015075:2-Aminopurine; D004004:Dichlororibofuranosylbenzimidazole; D047390:Casein Kinase II; D019204:GTP-Binding Proteins; D011070:Poly I-C
J Immunol. 2007 Sep 15;179(6):3859-71
17785823
Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):5952-7
15069200
J Mol Biol. 1982 Apr 15;156(3):609-31
6288961
Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6570-4
1321444
Virology. 2006 Jan 5;344(1):119-30
16364743
J Virol. 2000 Dec;74(23):11247-53
11070023
J Virol. 2004 Jun;78(12):6420-30
15163735
J Gen Virol. 2003 Apr;84(Pt 4):803-814
12655081
J Am Vet Med Assoc. 1997 Sep 1;211(5):556-60
9290819
J Virol. 2003 Jan;77(1):732-8
12477878
Fish Shellfish Immunol. 2004 Nov;17(5):447-62
15313511
Nat Med. 2004 Dec;10(12 Suppl):S77-81
15577935
J Virol. 2007 Jan;81(2):492-502
17065214
J Virol. 1997 Jul;71(7):5304-11
9188599
Nat Rev Immunol. 2008 Jul;8(7):559-68
18575461
J Virol. 1985 Feb;53(2):469-76
3838192
J Virol. 1975 Jun;15(6):1348-56
167189
J Virol. 2003 Apr;77(8):4646-57
12663771
Virology. 2005 Mar 15;333(2):215-25
15721356
J Fish Dis. 2010 Jul;33(7):583-91
20367739
J Endocrinol. 2001 Apr;169(1):71-8
11250648
Proc Natl Acad Sci U S A. 1983 Jan;80(1):70-4
6185960
Mol Immunol. 2007 May;44(12):3137-46
17391766
Adv Exp Med Biol. 2001;494:173-81
11774465
J Fish Dis. 2009 Jul;32(7):619-31
19486239
Virus Res. 1995 Oct;38(2-3):175-92
8578857
Proc Natl Acad Sci U S A. 1986 Nov;83(21):8122-6
3095828
Fish Shellfish Immunol. 2006 Sep;21(3):272-8
16580844
Immunity. 2000 Jul;13(1):129-41
10933401
J Virol. 2001 Oct;75(19):9121-8
11533176
Proteomics. 2004 Jun;4(6):1633-49
15174133
Science. 2006 May 12;312(5775):879-82
16690858
Mol Immunol. 2008 Mar;45(5):1439-48
17928055
J Gen Virol. 1972 Sep;16(3):391-8
4342823
J Gen Virol. 2009 Sep;90(Pt 9):2172-82
19474249
Curr Top Microbiol Immunol. 2002;262:139-57
11987804
J Virol. 2008 Jan;82(1):105-14
17959676
Dev Comp Immunol. 2004 Jun;28(7-8):793-801
15043947
J Interferon Cytokine Res. 1995 Aug;15(8):691-702
8528941
J Aquat Anim Health. 2006 Dec;18(4):232-43
26599159
Fish Shellfish Immunol. 2002 May;12(5):389-97
12194451
J Gen Virol. 1999 May;80 ( Pt 5):1221-1229
10355769
J Virol. 2003 Jul;77(14):8039-47
12829843
Biochim Biophys Acta. 2006 Jun;1760(6):941-50
16650600
Fish Shellfish Immunol. 2006 Feb;20(2):172-91
15939626
Virology. 2007 Jun 5;362(2):304-13
17292937
J Virol. 2004 Apr;78(8):4098-107
15047826
Cytokine Growth Factor Rev. 2007 Oct-Dec;18(5-6):425-33
17683972