ORF018R, a highly abundant virion protein from Singapore grouper iridovirus, is involved in serine/threonine phosphorylation and virion assembly

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.