Frog Virus 3 Open Reading Frame 97R Localizes to the Endoplasmic Reticulum and Induces Nuclear Invaginations

Abstract

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 (ORFs). The FV3 ORF 97R contains a B-cell lymphoma 2 (Bcl-2) homology 1 (BH1) domain and has sequence similarity to the myeloid cell leukemia-1 (Mcl-1) protein, 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 localized to the endoplasmic reticulum (ER) at 24 h posttransfection. However, at 35 h posttransfection, 97R localized to the ER but also began to form concentrated pockets continuous with the nuclear membrane. After 48 h posttransfection, 97R was still localized to the ER, but we began 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 abolished 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.

Tet2 restrains inflammatory gene expression in macrophages

“…Western blotting was performed against Tet2, Arginase 1, IκBα, Erk1/ 2, phospho-Erk1/2, Jun, phospho-cJun, and β-actin and immunofluorescence against Tet2 using standard techniques [26,27]. Antibodies and conditions are described in the Supplementary Methods.…”

“…Immunofluorescence. Cells were plated on coverslips and fixed in 3.7% paraformaldehyde (Bioshop) as previously described [27]. A Tet2 antibody (S-13, Santa Cruz) was used at 1:50 and cells were mounted with Vectashield Mounting Medium with DAPI (Vector Laboratories, Burlington, ON, Canada).…”

Localization of Frog Virus 3 Conserved Viral Proteins 88R, 91R, and 94L

“…For localization studies, it is much easier to use mammalian cell lines, where a variety of intracellular organelle markers exist. In addition, we have used BGMK cells in the past to localize a variety of FV3 genes, and have found that the localization in BGMK cells mimics the localization in amphibian cell lines [30,31]. It is noteworthy that BGMK cells are infectible by FV3 when the cells are incubated at 30 °C [32].…”

The Bcl-2 Family in Host-Virus Interactions

“…Many vBcl-2 orthologues, including the first viral Bcl-2 orthologue found, adenovirus E1B 19K, though without an obvious hydrophobic C-terminal region, are closely associated with intracellular membranes, the ER and nuclear envelope [ 86 ] and the association with membranes is required for its function [ 87 ]. Frog virus 3 Bcl-2 orthologue 97R localises to the ER and deletion of the C-terminal 29 residues inactivates the protein [ 88 ]. The African swine fever virus Bcl-2 orthologue A179L, is closely associated with viral factories, and though it lacks an obvious TM region, it is associated with the ER and mitochondrial membranes.…”