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Rana grylio virus (RGV) is a pathogenic iridovirus that has resulted in high mortality in cultured frog. Here, an envelope protein gene, 2L, was identified from RGV and its possible role in virus infection was investigated. Database searches found that RGV 2L had homologues in all sequenced iridoviruses and is a core gene of iridoviruses. Western blotting detection of purified RGV virions confirmed that 2L protein was associated with virion membrane. Fluorescence localization revealed that 2L protein co-localized with viral factories in RGV infected cells. In co-transfected cells, 2L protein co-localized with two other viral envelope proteins, 22R and 53R. However, 2L protein did not co-localize with the major capsid protein of RGV in co-transfected cells. Meanwhile, fluorescence observation showed that 2L protein co-localized with endoplasmic reticulum, but did not co-localize with mitochondria and Golgi apparatus. Moreover, a conditional lethal mutant virus containing the lac repressor/operator system was constructed to investigate the role of RGV 2L in virus infection. The ability to form plaques and the virus titres were strongly reduced when expression of 2L was repressed. Therefore, the current data showed that 2L protein is essential for virus infection. Our study is the first report, to our knowledge, of co-localization between envelope proteins in iridovirus and provides new insights into the understanding of envelope proteins in iridovirus.

Rana grylio virus (RGV) envelope protein 2L: subcellular localization and essential roles in virus infectivity revealed by conditional lethal mutant