Cholesterol is a critical component of cellular membranes, regulating assembly and function of membrane-based protein/lipid complexes. Many RNA viruses, including enteroviruses, remodel host membranes to generate organelles with unique lipid blueprints on which they assemble replication complexes and synthesize viral RNA. Here we find that clathrin-mediated endocytosis (CME) is harnessed by enteroviruses to traffic cholesterol from the plasma membrane (PM) and extracellular medium to replication organelles where cholesterol then regulates viral polyprotein processing and facilitates genome synthesis. When CME is disrupted, cellular cholesterol pools are instead stored in lipid droplets; cholesterol cannot be trafficked to replication organelles; and replication is inhibited. In contrast, replication is stimulated in cholesterol-elevated cells like those lacking caveolins or those from Niemann-Pick disease patients. Our findings indicate cholesterol as a critical determinant for enteroviral replication and outline roles for the endocytic machinery in both the enteroviral lifecycle and host cell cholesterol homeostasis.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell–mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

32The immune systems of free-living mammals such as humans and wild mice display a 33 heightened degree of activation compared with laboratory mice maintained under artificial 34 conditions. Here, we demonstrate that releasing inbred laboratory mice into an outdoor enclosure 35to mimic life in a natural environment alters the state of immunity. In addition to enhancing the 36 differentiation of T cell populations previously associated with pathogen exposure, we found that 37 outdoor release of mice led to an increase in circulating granulocytes. However, rewilded mice 38 were not infected by pathogens previously implicated in immune activation. Rather, changes to 39 the immune system were associated with an altered composition of the microbiota, and fungi 40 isolated from rewilded mice were sufficient to increase circulating granulocytes. These findings 41 establish an experimental procedure to investigate the impact of the natural environment on 42 immune development and identify a role for sustained fungal exposure in determining 43 granulocyte numbers. 44 45 46 48 research and has enabled fundamental advances in basic immunology. Yet, this ubiquitous model 49 fails to recreate certain aspects of human immunity. Inbred laboratory mice and adult humans 50 differ in the proportion of leukocyte subsets, transcriptional responses to microbial challenges, 51 and other immune parameters (Masopust et al., 2017; Tao and Reese, 2017). Such differences 52 may limit the predictive value of experiments with mice when studying complex inflammatory 53 and infectious diseases, resulting in significant shortcomings in translating laboratory 54 observations to humans. 55Recent findings suggest that this shortcoming of the rodent model may be due to the 56 specific pathogen free (SPF) environment in which they are maintained. Wild mice and pet store 57 mice, both of which are exposed to a litany of pathogens that are typically excluded from SPF 58 facilities, display an abundance of differentiated memory T cells that more closely resembles the 59 state of immunity in adult humans (Abolins et al., 2017; Beura et al., 2016; Choi et al., 2019). 60Similarly, transferring embryos from lab mice into wild mice generates commensal-and 61 pathogen-exposed offspring (wildlings) that more faithfully recreate human immunity than 62 standard SPF mice, including the unresponsiveness to immunotherapies that failed in clinical 63 trials (Rosshart et al., 2019). Sequentially infecting SPF mice with 3 viruses and a helminth shifts 64 the gene expression profile of peripheral blood mononuclear cells (PBMCs) towards that of pet 65 store mice and adult humans (Reese et al., 2016), further highlighting the role for pathogen 66 experience in normalizing the immune system. SPF mice are also distinguished from free-living 67 mammals by the lack of exposure to potentially immuno-stimulatory members of the microbiota 68 that are absent in a laboratory animal facility. For example, the offspring of germ-free mice 69 inoculated with ileocecal contents from ...

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.

A common paradigm holds that during cell-to-cell transmission, viruses behave as lone soldiers. Recently, we discovered not only that enteroviruses are transmitted via vesicles as populations of viral particles but also that this type of transmission enhances their infection efficiency (Y. H. Chen et al., Cell 160:619 -630, 2015). This mechanism could be advantageous for the overall fitness of the viral population, promoting genetic interplay by enabling viral quasispecies to collectively infect a susceptible host cell. Here, we discuss these findings in the context of viral pathogenesis and also propose that this novel type of vesicular transmission is widespread among different virus families and includes populations of both viral particles and naked viral genomes.