Bovine Ephemeral Fever Rhabdovirus α1 Protein Has Viroporin-Like Properties and Binds Importin β1 and Importin 7

Abstract

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genusEphemerovirus. In addition to the five canonical rhabdovirus structural proteins (N, P, M, G, and L), the large and complex BEFV genome contains several open reading frames (ORFs) between the G and L genes (␣1, ␣2/␣3, ␤, and ␥) encoding proteins of unknown function. We show that the 10.5-kDa BEFV ␣1 protein is expressed in infected cells and, consistent with previous predictions based on its structure, has the properties of a viroporin. Expression of a BEFV ␣1-maltose binding protein (MBP) fusion protein in Escherichia coli was observed to inhibit cell growth and increase membrane permeability to hygromycin B. Increased membrane permeability was also observed in BEFV-infected mammalian cells (but not cells infected with an ␣1-deficient BEFV strain) and in cells expressing a BEFV ␣1-green fluorescent protein (GFP) fusion protein, which was shown by confocal microscopy to localize to the Golgi complex. Furthermore, the predicted C-terminal cytoplasmic domain of ␣1, which contains a strong nuclear localization signal (NLS), was translocated to the nucleus when expressed independently, and in an affinity chromatography assay employing a GFP trap, the full-length ␣1 was observed to interact specifically with importin ␤1 and importin 7 but not with importin ␣3. These data suggest that, in addition to its function as a viroporin, BEFV ␣1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. IMPORTANCEAlthough rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV ␣1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that ␣1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV ␣1 binds importin ␤1 and importin 7, suggesting that it may have a yet unknown role in modulating nuclear trafficking. This is the first functional analysis of an ephemerovirus accessory protein and of a rhabdovirus viroporin.

Oxygen-dependent hydroxylation by Factor Inhibiting HIF (FIH) regulates the TRPV3 ion channel

“…Purified protein was then separated by SDS-PAGE and subjected to in-gel tryptic digestion as described previously (Hastie et al, 2012;Peet et al, 2004). In-gel digests were subjected to capillary high performance liquid chromatography (HPLC) and either arrayed onto matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF)/TOF targets for MALDI-TOF/ TOF-MS and -MS/MS (Dave et al, 2011) or sprayed directly into the ion-source of a High-Performance LTQ-Orbitrap Elite mass spectrometer essentially as described previously (Joubert et al, 2014). Quantification of hydroxylated versus nonhydroxylated peptides was calculated from the area under peaks in MS/MS.…”

Ankyrin Repeat Proteins of Orf Virus Influence the Cellular Hypoxia Response Pathway

“…For example, while ov126 bound FIH with a relatively high affinity and was efficiently hydroxylated in vitro, ov129 bound FIH with a low affinity but displayed efficient hydroxylation. This is reminiscent of other FIH substrates whose binding does not correlate with hydroxylation, including the ANK protein Notch4, which is able to bind FIH but is not hydroxylated (37). It is also important that the hydroxylation data reflect hydroxylation of the isolated ARDs by purified FIH and may not reflect the efficiency of hydroxylation of full-length ORFV ANK proteins in a cellular context.…”

“…After the final gradient, the column was held at 95% solvent B for 5 min. The LTQ-Orbitrap-Velos Pro mass spectrometer acquisition and database search parameters were essentially as described previously (37). Asparagine hydroxylation was included as a variable modification, and the Sequest HT database search engine in Proteome Discoverer…”

Proteomic changes occurring in the malaria mosquitoes Anopheles gambiae and Anopheles stephensi during aging

“…In-gel tryptic digests were analysed by either MALDI-TOF/TOF mass spectrometry [18] or separated by CapHPLC and sprayed directly into the ion source of an LTQ-Orbitrap XL hybrid MS [19].…”