Nucleocytoplasmic shuttling of BEFV M protein-modulated by lamin A/C and chromosome maintenance region 1 through a transcription-, carrier- and energy-dependent pathway.

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.

Bovine ephemeral fever rhabdovirus α1 protein has viroporin-like properties and binds importin ß1 and importin 7.

Bovine ephemeral fever virus (BEFV) is an arthropod-borne rhabdovirus that is classified as the type species of the genus Ephemerovirus. 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 (α1, α2/α3, ß, and γ) encoding proteins of unknown function. We show that the 10.5-kDa BEFV α1 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 α1-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 α1-deficient BEFV strain) and in cells expressing a BEFV α1-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 α1, 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 α1 was observed to interact specifically with importin ß1 and importin 7 but not with importin α3. These data suggest that, in addition to its function as a viroporin, BEFV α1 may modulate components of nuclear trafficking pathways, but the specific role thereof remains unclear. Although rhabdovirus accessory genes occur commonly among arthropod-borne rhabdoviruses, little is known of their functions. Here, we demonstrate that the BEFV α1 ORF encodes a protein which has the structural and functional characteristics of a viroporin. We show that α1 localizes in the Golgi complex and increases cellular permeability. We also show that BEFV α1 binds importin ß1 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.