The molecular mechanisms behind infection and propagation of human restricted pathogens such as human norovirus (HuNoV) have defied interrogation because they were previously unculturable. However, human intestinal enteroids (HIEs) have emerged to offer unique ex vivo models for targeted studies of intestinal biology, including inflammatory and infectious diseases. Carbohydrate-dependent histo-blood group antigens (HBGAs) are known to be critical for clinical infection. To explore whether HBGAs of glycosphingolipids contribute to HuNoV infection, we obtained HIE cultures established from stem cells isolated from jejunal biopsies of six individuals with different ABO, Lewis and secretor genotypes. We analyzed their glycerolipid and sphingolipid compositions and quantified interaction kinetics and the affinity of HuNoV virus-like particles (VLPs) to lipid vesicles produced from the individual HIE-lipid extracts. All HIEs had a similar lipid and glycerolipid composition. Sphingolipids included HBGA-related type 1 chain glycosphingolipids (GSLs), with HBGA epitopes corresponding to the geno- and phenotypes of the different HIEs. As revealed by single particle interaction studies of Sydney GII.4 VLPs with glycosphingolipid-containing HIE membranes, both binding kinetics and affinities explain the patterns of susceptibility towards GII.4 infection for individual HIEs. This is the first time norovirus VLPs have been shown to interact specifically with secretor gene dependent GSLs embedded in lipid membranes of HIEs that propagate GII.4 HuNoV ex vivo, highlighting the potential of HIEs for advanced future studies of intestinal glycobiology and host-pathogen interactions.
Research in the field of extracellular vesicles is rapidly expanding and finding footholds in many areas of medical science. However, the availability of methodologies to quantify the concentration of membrane material present in a sample remains limited. Herein, we present a novel approach for the quantification of vesicle material, specifically the quantification of the total lipid membrane surface area, found in a sample using Forster resonance energy transfer (FRET). In this assay, sonication is used to drive the fusion between vesicles in the sample to be quantified and liposomes containing a pair of FRET fluorophores. The change in emission spectrum upon vesicle fusion is directly related to the total membrane surface area of the sample added, and a calibration curve allows for the quantification of a variety of vesicle species, including enveloped viruses, bacterial outer membrane vesicles, and mammalian extracellular vesicles. Without extensive optimization of experimental parameters, we were able to quantify down to ∼10 9 vesicles/mL, using as little as 60 μL of the sample. The assay precision was comparable to that of a commercial nanoparticle tracking analysis system. While its limit of detection was slightly higher, the FRET assay is superior for the detection of small vesicles, as its performance is vesicle-size-independent. Taken together, the FRET assay is a simple, robust, and versatile method for the quantification of a variety of purified vesicle samples.
Over two decades, epidemiological studies have revealed that interactions between human polymorphic apolipoprotein 4 (ApoE, isoform 4) and herpes simplex virus type 1 (HSV1) associate with higher risk of Alzheimer's disease, a serious and increasing issue among elder populations worldwide. Nevertheless, little is known about the mechanisms behind ApoE-HSV1 interactions at molecular levels. Here, we investigate the effects of ApoE on the HSV1 infectious life cycle in in vitro cell experiments. Analysis of HSV1 growth curves shows that HSV1 production is promoted in presence of any of the three ApoE isoforms, with ApoE3 or 4 demonstrating more efficient pro-viral effects than ApoE2. When added prior to infection, ApoE inhibits viral attachment but does not affect viral entry. qPCR-based quantification reveals that harbouring ApoE2, 3, or 4 leads to an increase of HSV1 extracellular release but unchanged levels of viral genome copies within cells or on the cell surface, indicating that virus replication, assembly, or transport to cell membrane are not affected. Further test of virus release directly demonstrates that HSV1 detachment from the cell surface is promoted by ApoE. Subsequent results reveal that ApoE is not only present in purified HSV1 particles produced in ApoE-expressing cells after ultra-centrifugation but also able to incorporate into HSV1 particles after purification, suggesting that harbouring ApoE may promote HSV1 detachment. With the help of total internal reflection microscopy (TIRFM), this hypothesis was tested by quantifying interaction kinetics and apparent affinity between of HSV1 and native supported lipid bilayer. HSV1 particle decorated with ApoE demonstrates higher dissociation rate constants (koff) and less irreversible binding to the membrane, which is in line with the promoted virus release. Similar interaction kinetics have also been tested between non-decorated HSV1 particles and membranes harbouring ApoE but revealed no difference in the kinetics of virus particles on membranes with ApoE present or absent. Overall, our results provide new insights into the roles of ApoE during HSV1 infections, which is worth to be considered when studying their involvement during AD development.
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