The autophagy machinery interacts with EBV capsids during viral envelope release.

Autophagy serves as a defense mechanism against intracellular pathogens, but several microorganisms exploit it for their own benefit. Accordingly, certain herpesviruses include autophagic membranes into their infectious virus particles. In this study, we analyzed the composition of purified virions of the Epstein-Barr virus (EBV), a common oncogenic γ-herpesvirus. In these, we found several components of the autophagy machinery, including membrane-associated LC3B-II, and numerous viral proteins, such as the capsid assembly proteins BVRF2 and BdRF1. Additionally, we showed that BVRF2 and BdRF1 interact with LC3B-II via their common protein domain. Using an EBV mutant, we identified BVRF2 as essential to assemble mature capsids and produce infectious EBV. However, BdRF1 was sufficient for the release of noninfectious viral envelopes as long as autophagy was not compromised. These data suggest that BVRF2 and BdRF1 are not only important for capsid assembly but together with the LC3B conjugation complex of ATG5-ATG12-ATG15L1 are also critical for EBV envelope release.

Measuring oxidation within LC3-associated phagosomes that optimizes MHC class II restricted antigen presentation.

LC3-associated phagocytosis (LAP) uses components of the molecular machinery of macroautophagy and is involved in the presentation of extracellular antigens by Major Histocompatibility Complex (MHC) class II molecules. It is initiated by receptor-mediated phagocytosis and results in the formation of LAPosomes: single-membrane vesicles that are decorated with the macroautophagy protein LC3B. LAPosomes have been described to prolong antigen presentation in macrophages but the molecular mechanism of this process is just beginning to be understood. Known key regulators of LAPosome formation are Reactive Oxygen Species (ROS), which can modulate the pH and the oxidative state within LAPosomes. Here, we present two complementary methods to monitor oxidation in LAPosomes and to study its function in MHC class II restricted antigen presentation, both in primary human macrophages: (I) Coating the LAP-trigger zymosan with OxyBURST allows semi-quantitative assessment of oxidation levels within LAPosomes by confocal microscopy. (II) The co-culture of macrophages with CD4+T cells to assess the effects of LAP on Candida albicans antigen presentation by measuring IL-17A and IFN-γ secretion.

Oxidation inhibits autophagy protein deconjugation from phagosomes to sustain MHC class II restricted antigen presentation.

LC3-associated phagocytosis (LAP) contributes to a wide range of cellular processes and notably to immunity. The stabilization of phagosomes by the macroautophagy machinery in human macrophages can maintain antigen presentation on MHC class II molecules. However, the molecular mechanisms involved in the formation and maturation of the resulting LAPosomes are not completely understood. Here, we show that reactive oxygen species (ROS) produced by NADPH oxidase 2 (NOX2) stabilize LAPosomes by inhibiting LC3 deconjugation from the LAPosome cytosolic surface. NOX2 residing in the LAPosome membrane generates ROS to cause oxidative inactivation of the protease ATG4B, which otherwise releases LC3B from LAPosomes. An oxidation-insensitive ATG4B mutant compromises LAP and thereby impedes sustained MHC class II presentation of exogenous Candida albicans antigens. Redox regulation of ATG4B is thereby an important mechanism for maintaining LC3 decoration of LAPosomes to support antigen processing for MHC class II presentation.

Seamless Combination of Fluorescence-Activated Cell Sorting and Hanging-Drop Networks for Individual Handling and Culturing of Stem Cells and Microtissue Spheroids.

Open microfluidic cell culturing devices offer new possibilities to simplify loading, culturing, and harvesting of individual cells or microtissues due to the fact that liquids and cells/microtissues are directly accessible. We present a complete workflow for microfluidic handling and culturing of individual cells and microtissue spheroids, which is based on the hanging-drop network concept: The open microfluidic devices are seamlessly combined with fluorescence-activated cell sorting (FACS), so that individual cells, including stem cells, can be directly sorted into specified culturing compartments in a fully automated way and at high accuracy. Moreover, already assembled microtissue spheroids can be loaded into the microfluidic structures by using a conventional pipet. Cell and microtissue culturing is then performed in hanging drops under controlled perfusion. On-chip drop size control measures were applied to stabilize the system. Cells and microtissue spheroids can be retrieved from the chip by using a parallelized transfer method. The presented methodology holds great promise for combinatorial screening of stem-cell and multicellular-spheroid cultures.