VAPYRIN-like is required for development of the moss Physcomitrella patens.

The VAPYRIN (VPY) gene in Medicago truncatula and Petunia hybrida is required for arbuscular mycorrhizal (AM) symbiosis. The moss Physcomitrella patens has a close homolog (VPY-like, VPYL), although it does not form AM. Here, we explore the phylogeny of VPY and VPYL in land plants, and study the expression and developmental function of VPYL in Ppatens We show that VPYL is expressed primarily in the protonema, the early filamentous stage of moss development, and later in rhizoids arising from the leafy gametophores and in adult phyllids. Knockout mutants have specific phenotypes in branching of the protonema and in cell division of the leaves (phyllids) in gametophores. The mutants are responsive to auxin and strigolactone, which are involved in regulation of protonemal branching, indicating that hormonal signaling in the mutants is not affected in hormonal signaling. Taken together, these results suggest that VPYL exerts negative regulation of protonemal branching and cell division in phyllids. We discuss VPY and VPYL phylogeny and function in land plants in the context of AM symbiosis in angiosperms and development in the moss.

Transient vitamin B5 starving improves mammalian cell homeostasis and protein production.

Maintaining a metabolic steady state is essential for an organism's fitness and survival when confronted with environmental stress, and metabolic imbalance can be reversed by exposing the organism to fasting. Here, we attempted to apply this physiological principle to mammalian cell cultures to improve cellular fitness and consequently their ability to express recombinant proteins. We showed that transient vitamin B5 deprivation, an essential cofactor of central cellular metabolism, can quickly and irreversibly affect mammalian cell growth and division. A selection method was designed that relies on mammalian cell dependence on vitamin B5 for energy production, using the co-expression of the B5 transporter SLC5A6 and a gene of interest. We demonstrated that vitamin B5 selection persistently activates peroxisome proliferator-activated receptors (PPAR), a family of transcription factors involved in energy homeostasis, thereby altering lipid metabolism, improving cell fitness and therapeutic protein production. Thus, stable PPAR activation may constitute a cellular memory of past deprivation state, providing increased resistance to further potential fasting events. In other words, our results imply that cultured cells, once exposed to metabolic starvation, may display an improved metabolic fitness as compared to non-exposed cells, allowing increased resistance to cellular stress.

Influence of cytoskeleton organization on recombinant protein expression by CHO cells.

In this study, we assessed the importance of cytoskeleton organization in the mammalian cells used to produce therapeutic proteins. Two cytoskeletal genes, Actin alpha cardiac muscle 1 (ACTC1) and a guanosine triphosphate GTPase-activating protein (TAGAP), were found to be upregulated in highly productive therapeutic protein-expressing Chinese hamster ovary (CHO) cells selected by the deprivation of vitamin B5. We report here that the overexpression of the ACTC1 protein was able to improve significantly recombinant therapeutic production, as well as to decrease the levels of toxic lactate metabolic by-products. ACTC1 overexpression was accompanied by altered as well as decreased polymerized actin, which was associated with high protein production by CHO cell cultured in suspension. We suggest that the depolymerization of actin and the possible modulation of integrin signaling, as well as changes in basal metabolism, may be driving the increase of protein secretion by CHO cells.

Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release.

The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical proteins are known to contain type-C endogenous retrovirus (ERV) sequences in their genome and to release retroviral-like particles. Although evidence for their infectivity is missing, this has raised safety concerns. As the genomic origin of these particles remained unclear, we characterized type-C ERV elements at the genome, transcriptome, and viral particle RNA levels. We identified 173 type-C ERV sequences clustering into three functionally conserved groups. Transcripts from one type-C ERV group were full-length, with intact open reading frames, and cognate viral genome RNA was loaded into retroviral-like particles, suggesting that this ERV group may produce functional viruses. CRISPR-Cas9 genome editing was used to disrupt the gag gene of the expressed type-C ERV group. Comparison of CRISPR-derived mutations at the DNA and RNA level led to the identification of a single ERV as the main source of the release of RNA-loaded viral particles. Clones bearing a Gag loss-of-function mutation in this ERV showed a reduction of RNA-containing viral particle release down to detection limits, without compromising cell growth or therapeutic protein production. Overall, our study provides a strategy to mitigate potential viral particle contaminations resulting from ERVs during biopharmaceutical manufacturing.