Frog virus 3- 97R localizes to the endoplasmic reticulum and induces nuclear invaginations.

Frog virus 3 (FV3) is the type species of the genus Ranavirus, family Iridoviridae. The genome of FV3 is 105,903 bases in length and encodes 97 open reading frames (ORF). The FV3 ORF 97R contains a Bcl-2 homology 1 (BH1) domain and has sequence similarity to Mcl-1, suggesting a potential role in apoptosis. To begin to understand the role of 97R, we characterized 97R through immunofluorescence and mutagenesis. Here we demonstrated that 97R localizes to the endoplasmic reticulum (ER) at 24 hours post-transfection. However, at 35 hours post-transfection 97R localizes to the ER but also begins to form concentrated pockets, continuous with the nuclear membrane. After 48 hours post-transfection 97R still localizes to the ER, but we begin to observe the ER and the outer nuclear membrane invaginating into the nucleus. To further explore 97R targeting to the ER we created a series of C-terminal transmembrane domain deletion mutants. We found that deletion of 29 amino acids from the C-terminus of 97R abolishes localization to the ER. In contrast, deletion of 12 amino acids from the C-terminus of 97R did not affect 97R localization to the ER. In addition a hybrid protein containing the 97R C-terminal 33 amino acids was similarly targeted to the ER. These data indicate that the C-terminal 33 amino acids of 97R are necessary and sufficient for ER targeting.