Characterization of a membrane protein (VP001L) from infectious spleen and kidney necrosis virus (ISKNV).

Infectious spleen and kidney necrosis virus (ISKNV) is the type species of megalocytivirus, Iridoviridae. A novel membrane protein corresponding to the first open reading frame (ORF001L) of ISKNV genome was identified. This 378-residue protein, termed the VP001L protein, has a high content of hydrophobic sequences and contains 10-11 putative transmembrane domains, indicating it may be a membrane protein. The VP001L mRNA start site was extended 433 bp upstream of the start codon and the temporal analysis showed that the VP001L gene was first transcribed at 8 h post-infection (h.p.i.). VP001L protein was detected on the plasma membrane of ISKNV infected cells by immunofluresence. In order to further investigate different transmembrane domains' influence on subcellular localization of VP001L, series of truncated or deleted mutants were constructed with GFP at the C terminus. The transfection results indicated that the second putative transmembrane domain played a determinative role in VP001L's membrane localization and the translocation of the first and third transmembrane domains depended on their interactions with the second one. Therefore, this novel VP001L protein is considered to serve as a model for analyzing the topology and roles of different hydrophobic regions in multi-transmembrane proteins.