Enhanced Uptake and Endosomal Release of LbL Microcarriers Functionalized with Reversible Fusion Proteins.

The efficient application of smart drug-delivery systems requires further improvement of their cellular uptake and in particular their release from endolysosomal compartments into the cytoplasm of target cells. The usage of virus proteins allows for such developments, as viruses have evolved efficient entry mechanisms into the cell, mediated by their fusion proteins. In our investigations, the transferability of the glycoprotein G which is a fusion protein of the vesicular stomatitis virus (VSV-G) onto the surface of a layer-by-layer (LbL) designed microcarrier was investigated. The assembly of VSV-G as a reversible viral fusion protein onto LbL microcarriers indeed induced an enhanced uptake rate on Vero cells as well as a fast and efficient release of the intact carriers from endolysosomes into the cytoplasm. Additionally, neither virus-associated effects on cellular viability nor activation of an interferon response were detected. Our study emphasizes the suitability of VSV-G as an efficient surface functionalization of drug-delivery systems.

Teratogenic Rubella Virus Alters the Endodermal Differentiation Capacity of Human Induced Pluripotent Stem Cells.

The study of congenital virus infections in humans requires suitable ex vivo platforms for the species-specific events during embryonal development. A prominent example for these infections is rubella virus (RV) which most commonly leads to defects in ear, heart, and eye development. We applied teratogenic RV to human induced pluripotent stem cells (iPSCs) followed by differentiation into cells of the three embryonic lineages (ecto-, meso-, and endoderm) as a cell culture model for blastocyst- and gastrulation-like stages. In the presence of RV, lineage-specific differentiation markers were expressed, indicating that lineage identity was maintained. However, portrait analysis of the transcriptomic expression signatures of all samples revealed that mock- and RV-infected endodermal cells were less related to each other than their ecto- and mesodermal counterparts. Markers for definitive endoderm were increased during RV infection. Profound alterations of the epigenetic landscape including the expression level of components of the chromatin remodeling complexes and an induction of type III interferons were found, especially after endodermal differentiation of RV-infected iPSCs. Moreover, the eye field transcription factors RAX and SIX3 and components of the gene set vasculogenesis were identified as dysregulated transcripts. Although iPSC morphology was maintained, the formation of embryoid bodies as three-dimensional cell aggregates and as such cellular adhesion capacity was impaired during RV infection. The correlation of the molecular alterations induced by RV during differentiation of iPSCs with the clinical signs of congenital rubella syndrome suggests mechanisms of viral impairment of human development.

Rubella Virus Strain-Associated Differences in the Induction of Oxidative Stress Are Independent of Their Interferon Activation.

Rubella virus (RV) infection impacts cellular metabolic activity in a complex manner with strain-specific nutritional requirements. Here we addressed whether this differential metabolic influence was associated with differences in oxidative stress induction and subsequently with innate immune response activation. The low passaged clinical isolates of RV examined in this study induced oxidative stress as validated through generation of the reactive oxygen species (ROS) cytoplasmic hydrogen peroxide and mitochondrial superoxide. The addition of the cytoplasmic and mitochondrial ROS scavengers N-acetyl-l-cysteine and MitoTEMPO, respectively, reduced RV-associated cytopathogenicity and caspase activation. While the degree of oxidative stress induction varied among RV clinical isolates, the level of innate immune response and interferon-stimulated gene activation was comparable. The type III IFNs were highly upregulated in all cell culture systems tested. However, only pre-stimulation with IFN ß slightly reduced RV replication indicating that RV appears to have evolved the ability to counteract innate immune response mechanisms. Through the data presented, we showed that the ability of RV to induce oxidative stress was independent of its capacity to stimulate and counteract the intrinsic innate immune response.

Rubella Viruses Shift Cellular Bioenergetics to a More Oxidative and Glycolytic Phenotype with a Strain-Specific Requirement for Glutamine.

The flexible regulation of cellular metabolic pathways enables cellular adaptation to changes in energy demand under conditions of stress such as posed by a virus infection. To analyze such an impact on cellular metabolism, rubella virus (RV) was used in this study. RV replication under selected substrate supplementation with glucose, pyruvate, and glutamine as essential nutrients for mammalian cells revealed its requirement for glutamine. The assessment of the mitochondrial respiratory (based on the oxygen consumption rate) and glycolytic (based on the extracellular acidification rate) rate and capacity by respective stress tests through Seahorse technology enabled determination of the bioenergetic phenotype of RV-infected cells. Irrespective of the cellular metabolic background, RV infection induced a shift of the bioenergetic state of epithelial cells (Vero and A549) and human umbilical vein endothelial cells to a higher oxidative and glycolytic level. Interestingly there was a RV strain-specific, but genotype-independent demand for glutamine to induce a significant increase in metabolic activity. While glutaminolysis appeared to be rather negligible for RV replication, glutamine could serve as donor of its amide nitrogen in biosynthesis pathways for important metabolites. This study suggests that the capacity of RVs to induce metabolic alterations could evolve differently during natural infection. Thus, changes in cellular bioenergetics represent an important component of virus-host interactions and could complement our understanding of the viral preference for a distinct host cell population.IMPORTANCE RV pathologies, especially during embryonal development, could be connected with its impact on mitochondrial metabolism. With bioenergetic phenotyping we pursued a rather novel approach in virology. For the first time it was shown that a virus infection could shift the bioenergetics of its infected host cell to a higher energetic state. Notably, the capacity to induce such alterations varied among different RV isolates. Thus, our data add viral adaptation of cellular metabolic activity to its specific needs as a novel aspect to virus-host evolution. In addition, this study emphasizes the implementation of different viral strains in the study of virus-host interactions and the use of bioenergetic phenotyping of infected cells as a biomarker for virus-induced pathological alterations.