Rab1b-GBF1-ARF1 Secretory Pathway Axis Is Required for Birnavirus Replication.

Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). Here, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. By analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP ribosylation factor 1 (ARF1), is required for IBDV replication, since inhibiting its activity by treatment with brefeldin A (BFA) or golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative mutant T31N overexpression hampered IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnavirus-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, with the lack of a transcriptionally active core being the main differential feature. This structural trait, among others that resemble those of the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses has been argued. Here, we present original data showing that IBDV-induced GC reorganization and the cross talk between IBDV and the Rab1b-GBF1-ARF1 mediate the intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnavirus-host cell interactions and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.

14-3-3ß isoform is specifically acetylated at Lys51 during differentiation to the osteogenic lineage.

The 14-3-3 protein family binds and regulates hundreds of serine/threonine phosphorylated proteins as an essential component of many signaling networks. Specific biological functions are currently been discovered for each of its seven isoforms in mammals. These proteins have been traditionally considered unregulated; however, its acetylation in an essential lysine residue, causing its inactivation, was recently published. Here, we studied the acetylation state of this lysine 49/51 during the osteogenic differentiation of human adipose-derived stem cells. We found that during this process, the levels of 14-3-3ß (but not its isoform 14-3-3γ) acK49/51 increase, representing the first report linking this PTM to a specific isoform and a cellular process. Our results suggested that this posttranslational modification could be catalyzed by the HBO1 acetyltransferase, as overexpression of HBO1 increased specifically 14-3-3 acK49/51 acetylation. Acetylated 14-3-3 proteins are located primarily in the nucleus, where their active state has been described to bind H3 histones and many transcription factors. The inhibition of the expression of different isoforms showed that the specific silencing of the 14-3-3ß gene, but not γ, increased significantly the osteogenic potential of the cells. This result correlated to the increase in acetylation of 14-3- 3ß Lys 49/51 during osteogenesis. The possible role of this PTM in osteogenesis is discussed.

Human adipose mesenchymal stromal cells growing into PCL-nHA electrospun scaffolds undergo hypoxia adaptive ultrastructural changes.

Human Adipose-Derived Mesenchymal Stem/Stromal Cells (hAD-MSCs) have great potential for tissue regeneration. Since transplanted hAD-MSCs are likely to be placed in a hypoxic environment, culturing the cells under hypoxic conditions might improve their post-transplantation survival and regenerative performance. The combination of hAD-MSCs and PCL-nHA nanofibers synergically improves the contribution of both components for osteoblast differentiation. In this work, we hypothesized that this biomaterial constitutes a hypoxic environment for hAD-MSCs. We studied the cellular re-arrangement and the subcellular ultrastructure by Transmission Electron Microscopy (TEM) of hAD-MSCs grown into PCL-nHA nanofibers, and we compared them with the same cells grown in two-dimensional cultures, over tissue culture-treated plastic, or glass coverslips. Among the most evident changes, PCL-nHA grown cells showed enlarged mitochondria, and accumulation of glycogen granules, consistent with a hypoxic environment. We observed a 3.5 upregulation (p = 0.0379) of Hypoxia Inducible Factor (HIF)-1A gene expression in PCL-nHA grown cells. This work evidences for the first time intra-cellular changes in three-dimensional compared to two-dimensional cultures, which are adaptive responses of the cells to an environment more closely resembling that of the in vivo niche after transplantation, thus PCL-nHA nanofibers are adequate for hAD-MSCs pre-conditioning.

Infectious Bursal Disease Virus Assembly Causes Endoplasmic Reticulum Stress and Lipid Droplet Accumulation.

Gumboro illness is caused by the highly contagious immunosuppressive infectious bursal disease virus (IBDV), which affects the poultry industry globally. We have previously shown that IBDV hijacks the endocytic pathway to construct viral replication complexes on endosomes linked to the Golgi complex (GC). Then, analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b, the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), and its substrate, the small GTPase ADP-ribosylation factor 1 (ARF1), for IBDV replication. In the current work, we focused on elucidating the IBDV assembly sites. We show that viral assembly occurs within single-membrane compartments closely associated with endoplasmic reticulum (ER) membranes, though we failed to elucidate the exact nature of the virus-wrapping membranes. Additionally, we show that IBDV infection promotes the stress of the ER, characterized by an accumulation of the chaperone binding protein (BiP) and lipid droplets (LDs) in the host cells. Overall, our results represent further original data showing the interplay between IBDV and the secretory pathway, making a substantial contribution to the field of birnaviruses-host cell interactions.