Iridovirus is an important pathogen causing serious diseases among wild, cultured and ornamental fish. Previous studies have shown that Singapore grouper iridovirus (SGIV) contains 162 open reading frames (ORFs) from which 51 viral proteins have been confirmed by proteomics studies. ORF018R, which is conserved among vertebrate iridoviruses, is an abundant virion protein identified from SGIV. Here, immunofluorescence staining showed that ORF018R occurred at high abundance throughout SGIV-infected cells. The function of ORF018R was explored using antisense morpholino oligonucleotides (asMOs). Knockdown of ORF018R expression resulted in a reduction in the expression of viral late genes, distortion of viral particle assembly and inhibition of SGIV infection in grouper embryonic cells. Western blotting with phosphoserine-specific antibody indicated that serine phosphorylation was significantly enhanced for proteins of molecular masss 17–32 kDa by SDS-PAGE when ORF018R expression was eliminated. These proteins were analysed further by two-dimensional gel electrophoresis, and numerous protein spots were found to shift to a lower pI and higher molecular mass as a result of the loss of ORF018R function. Five proteins with enhanced phosphorylation were identified by matrix-assisted laser desorption/ionization time-of-flight (TOF)-TOF mass spectrometry, including three viral proteins: ORF049L (dUTPase), ORF075R and ORF086R, and two host proteins: subunit 12 of eukaryotic translation factor 3 and natural killer enhancing factor. These findings suggest that ORF018R is involved in serine/threonine phosphorylation in SGIV-infected late-stage cells and plays an important role in expression of viral late genes and virion assembly.
BiX.,
LinQ.,
FooT. W.,
JoshiS.,
YouT.,
ShenH. M.,
OngC. N.,
CheahP. Y.,
EuK. W.,
HewC. L.2006; Proteomics analysis of colorectal cancer reveals alterations in metabolic pathways: mechanism of tumorigenesis. Mol Cell Proteomics 5:1119–1130[CrossRef]
ChenL. M.,
WangF.,
SongW. J.,
HewC. L.2006; Temporal and differential gene expression of Singapore grouper iridovirus. J Gen Virol 87:2907–2915[CrossRef]
DaszakP.,
BergerL.,
CunninghamA. A.,
HyattA. D.,
GreenD. E.,
SpeareR.1999; Emerging infectious diseases and amphibian population declines. Emerg Infect Dis 5:735–748[CrossRef]
EatonH. E.,
MetcalfJ.,
PennyE.,
TcherepanovV.,
UptonC.,
BrunettiC. R.2007; Comparative genomic analysis of the family Iridoviridae : re-annotating and defining the core set of iridovirus genes. Virol J 4:11[CrossRef]
GoorhaR.,
MurtiK. G.1982; The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant. Proc Natl Acad Sci U S A 79:248–252[CrossRef]
HamiltonM. A.,
RussoR. C.,
ThurstonR. V.1977; Trimmed Spearman–Karber method for estimating median lethal concentrations in toxicity bioassays. Environ Sci Technol 11:714–719[CrossRef]
HuangC.,
ZhangX.,
LinQ.,
XuX.,
HuZ.,
HewC. L.2002; Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466. Mol Cell Proteomics 1:223–231[CrossRef]
HuangX. H.,
HuangY. H.,
YuanX. P.,
HewC. L.2006; Electron microscopic examination of the viromatrix of Rana grylio virus in a fish cell line. J Virol Methods 133:117–123[CrossRef]
KomarovaA. V.,
RealE.,
BormanA. M.,
BrocardM.,
EnglandP.,
TordoN.,
HersheyJ. W.,
KeanK. M.,
JacobY.2007; Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis. Nucleic Acids Res 35:1522–1532[CrossRef]
LeopardiR.,
SantC. V.,
RoizmanB.1997; The herpes simplex virus protein kinase US3 is required for protection from apoptosis induced by the virus. Proc Natl Acad Sci U S A 94:7891–7896[CrossRef]
QinQ. W.,
LamT. J.,
SinY. M.,
ShenH.,
ChangS. F.,
NgohG. H.,
ChenC. L.2001; Electron microscopic observations of a marine fish iridovirus isolated from brown-spotted grouper, Epinephelus tauvina . J Virol Methods 98:17–24[CrossRef]
SampleR.,
BryanL.,
LongS.,
MajjiS.,
HoskinsG.,
SinningA.,
OlivierJ.,
ChincharV. G.2007; Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II. Virology 358:311–320[CrossRef]
SarreT. F.1989; The phosphorylation of eukaryotic initiation factor 2: a principle of translational control in mammalian cells. Biosystems 22:311–325[CrossRef]
SummertonJ. E.2007; Morpholino, siRNA, and S-DNA compared: impact of structure and mechanism of action on off-target effects and sequence specificity. Curr Top Med Chem 7:651–660[CrossRef]
YanX. D.,
OlsonN. H.,
Van EttenJ. L.,
BergoinM.,
RossmannM. G.,
BakerT. S.2000; Structure and assembly of large lipid-containing dsDNA viruses. Nat Struct Biol 7:101–103[CrossRef]