Identification of two novel membrane proteins from the Tiger frog virus (TFV)

The Tiger frog virus (TFV) belongs to the genus Ranavirus in the family Iridoviridae, and its genome was completely sequenced in 2002. In order to better understand the viral structure and functional genes involved in infection and virus–host interactions, two candidate genes, ORF001L and ORF020R, were selected for our study. ORF001L and ORF020R were analyzed by genomic comparison and by using the TMHMM software. Both genes were conserved in the genus Ranavirus, may encode putative membrane proteins, and were determined as late genes by temporal analysis. In order to identify whether these two proteins were structural proteins or not, ORF001L and ORF020R were cloned and expressed in the pET32a (+) vector. Antisera against the two proteins were prepared by immunization of mice with purified proteins. Western blot analysis suggested that both ORF001L and ORF020R were structural proteins. Indirect immunofluorescence assay (IFA) revealed that the subcellular location of the two proteins was confined to the cytoplasm, especially at the viral assembly site (AS). Immunogold electron microscopy (IEM) further localized these two proteins, showing that they were envelope proteins.