A fungal biofilm reactor based on metal structured packing improves the quality of a Gla::GFP fusion protein produced by Aspergillus oryzae.

Fungal biofilm is known to promote the excretion of secondary metabolites in accordance with solid-state-related physiological mechanisms. This work is based on the comparative analysis of classical submerged fermentation with a fungal biofilm reactor for the production of a Gla::green fluorescent protein (GFP) fusion protein by Aspergillus oryzae. The biofilm reactor comprises a metal structured packing allowing the attachment of the fungal biomass. Since the production of the target protein is under the control of the promoter glaB, specifically induced in solid-state fermentation, the biofilm mode of culture is expected to enhance the global productivity. Although production of the target protein was enhanced by using the biofilm mode of culture, we also found that fusion protein production is also significant when the submerged mode of culture is used. This result is related to high shear stress leading to biomass autolysis and leakage of intracellular fusion protein into the extracellular medium. Moreover, 2-D gel electrophoresis highlights the preservation of fusion protein integrity produced in biofilm conditions. Two fungal biofilm reactor designs were then investigated further, i.e. with full immersion of the packing or with medium recirculation on the packing, and the scale-up potentialities were evaluated. In this context, it has been shown that full immersion of the metal packing in the liquid medium during cultivation allows for a uniform colonization of the packing by the fungal biomass and leads to a better quality of the fusion protein.

The HTLV-1 Tax protein inhibits formation of stress granules by interacting with histone deacetylase 6.

Human T cell leukemia virus type-1 (HTLV-1) is the causative agent of a fatal adult T-cell leukemia. Through deregulation of multiple cellular signaling pathways the viral Tax protein has a pivotal role in T-cell transformation. In response to stressful stimuli, cells mount a cellular stress response to limit the damage that environmental forces inflict on DNA or proteins. During stress response, cells postpone the translation of most cellular mRNAs, which are gathered into cytoplasmic mRNA-silencing foci called stress granules (SGs) and allocate their available resources towards the production of dedicated stress-management proteins. Here we demonstrate that Tax controls the formation of SGs and interferes with the cellular stress response pathway. In agreement with previous reports, we observed that Tax relocates from the nucleus to the cytoplasm in response to environmental stress. We found that the presence of Tax in the cytoplasm of stressed cells prevents the formation of SGs and counteracts the shutoff of specific host proteins. Unexpectedly, nuclear localization of Tax promotes spontaneous aggregation of SGs, even in the absence of stress. Mutant analysis revealed that the SG inhibitory capacity of Tax is independent of its transcriptional abilities but relies on its interaction with histone deacetylase 6, a critical component of SGs. Importantly, the stress-protective effect of Tax was also observed in the context of HTLV-1 infected cells, which were shown to be less prone to form SGs and undergo apoptosis under arsenite exposure. These observations identify Tax as the first virally encoded inhibitory component of SGs and unravel a new strategy developed by HTLV-1 to deregulate normal cell processes. We postulate that inhibition of the stress response pathway by Tax would favor cell survival under stressful conditions and may have an important role in HTLV-1-induced cellular transformation.

Genetic determinants of bovine leukemia virus pathogenesis.

The understanding of HTLV-induced disease is hampered by the lack of a suitable animal model allowing the study of both viral replication and leukemogenesis in vivo. Although valuable information has been obtained in different species, such as rabbits, mice, rats, and monkeys, none of these systems was able to conciliate topics as different as viral infectivity, propagation within the host, and generation of leukemic cells. An alternate strategy is based on the understanding of diseases induced by viruses closely related to HTLV-1, like bovine leukemia virus (BLV). Both viruses indeed belong to the same subfamily of retroviruses, harbor a similar genomic organization, and infect and transform cells of the hematopoietic system. The main advantage of the BLV system is that it allows direct experimentation in two different species, cattle and sheep.

Discordance between bovine leukemia virus tax immortalization in vitro and oncogenicity in vivo.

Bovine leukemia virus (BLV) Tax protein, a transcriptional activator of viral expression, is essential for viral replication in vivo. Tax is believed to be involved in leukemogenesis because of its second function, immortalization of primary cells in vitro. These activities of Tax can be dissociated on the basis of point mutations within specific regions of the protein. For example, mutation of the phosphorylation sites at serines 106 and 293 abrogates immortalization potential in vitro but maintains transcriptional activity. This type of mutant is thus particularly useful for unraveling the role of Tax immortalization activity during leukemogenesis independently of viral replication. In this report, we describe the biological properties of BLV recombinant proviruses mutated in the Tax phosphorylation sites (BLVTax106+293). Titration of the proviral loads by semiquantitative PCR revealed that the BLV mutants propagated at wild-type levels in vivo. Furthermore, two animals (sheep 480 and 296) infected with BLVTax106+293 developed leukemia or lymphosarcoma after 16 and 36 months, respectively. These periods of time are within the normal range of latencies preceding the onset of pathogenesis induced by wild-type viruses. The phenotype of the mutant-infected cells was characteristic of a B lymphocyte (immunoglobulin M positive) expressing CD11b and CD5 (except at the final stage for the latter marker), a pattern that is typical of wild-type virus-infected target cells. Interestingly, the transformed B lymphocytes from sheep 480 also coexpressed the CD8 marker, a phenotype rarely observed in tumor biopsies from chronic lymphocytic leukemia patients. Finally, direct sequencing of the tax gene demonstrated that the leukemic cells did not harbor revertant proviruses. We conclude that viruses expressing a Tax mutant unable to transform primary cells in culture are still pathogenic in the sheep animal model. Our data thus provide a clear example of the discordant conclusions that can be drawn from in vitro immortalization assays and in vivo experiments. These observations could be of interest for other systems, such as the related human T-cell leukemia virus type 1, which currently lack animal models allowing the study of the leukemogenic process.

Phosphorylation of bovine leukemia virus Tax protein is required for in vitro transformation but not for transactivation.

The Tax proteins of the oncovirinae viruses are phosphorylated transcriptional activators that exhibit oncogenic potential. The role of phosphorylation in their functional activities remains unknown. As a model for the Human T-cell leukemia virus type I (HTLV-I), Bovine Leukemia Virus (BLV) permits the characterization of viral replication and leukemogenesis in vivo. Here, we show that the BLV Tax protein is phosphorylated on serine residues 106 and 293 both in insect and in mammalian cells. These sites can also be efficiently phosphorylated by the cdc2 and MAP kinases in vitro. Mutation of these residues does not affect the capacity of the Tax protein to function as a transactivator. Indeed, the Tax proteins mutated at one or both serines increase LTR-directed viral transcription at levels similar to those obtained with wild-type Tax in cell culture. Moreover, inhibition of Tax phosphorylation by W7, a calmodulin antagonist, does not alter its transactivation activity. Thus, phosphorylation on serines 106 and 293 is not required for transactivation by Tax. However, simultaneous substitution of both serines into alanine residues destroys the capacity of Tax to cooperate with the Ha-ras oncogene to transform primary rat embryo fibroblasts and induce tumors in nude mice. When the serines were replaced with aspartic acid residues, the oncogenic potential of Tax was maintained indicating that the negative charge rather than the phosphate group itself was required for Tax oncogenicity. Finally, to assess the role of the serine residues in vivo, recombinant viruses which express the Tax mutants were constructed and injected into sheep. It appeared that the mutated proviruses replicate at levels similar to the wild-type virus in vivo. We conclude that Tax phosphorylation is dispensable for transactivation and viral replication in vivo but is required for its oncogenic potential in vitro.