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Noroviruses (NoVs) are a leading cause of gastroenteritis globally, yet the host factors required for NoV infection are poorly understood. We identified host molecules that are essential for murine NoV (MNoV)-induced cell death, including CD300lf as a proteinaceous receptor. We found that CD300lf is essential for MNoV binding and replication in cell lines and primary cells. Additionally, Cd300lf-/- mice are resistant to MNoV infection. Expression of CD300lf in human cells breaks the species barrier that would otherwise restrict MNoV replication. The crystal structure of the CD300lf ectodomain reveals a potential ligand-binding cleft composed of residues that are critical for MNoV infection. Therefore, the presence of a proteinaceous receptor is the primary determinant of MNoV species tropism, whereas other components of cellular machinery required for NoV replication are conserved between humans and mice.

Caliciviruses are significant agricultural and human pathogens that are poorly understood due to the dearth of molecular tools, including reporter systems. We report the development of a robust luciferase-based reporter system for a model calicivirus, murine norovirus (MNoV). Genetic insertion of a HiBiT tag, an 11 amino acid fragment of nanolucifersase, at the junction of the nonstructural proteins NS4 and NS5 yields infectious virus. The resultant MNoV-HiBiT produces a robust signal that is detected early in infection and occurs only in cells susceptible to MNoV infection. The MNoV-HiBiT reporter is effective at monitoring acute infection in STAT1 deficient mice. Furthermore, we used this tool to characterize two unappreciated host directed anti-MNoV compounds. The use of the MNoV-HiBiT virus enables new mechanistic studies by a rapid and quantitative means of measuring MNoV replication. The HiBiT insertion strategy we describe may be useful for the generation of other calicivirus reporters.

Murine norovirus (MNoV) is closely related to human norovirus (HNoV), an infectious agent responsible for acute gastroenteritis worldwide. Here we report the X-ray crystal structure of the dimeric MNoV VP1 protruding (P) domain in complex with its cellular receptor CD300lf. CD300lf binds the P domain with a 2:2 stoichiometry, engaging a cleft between the AB and DE loops of the P2 subdomain at a site that overlaps the epitopes of neutralizing antibodies. We also identify that bile acids are cofactors enhancing MNoV cell-binding and infectivity. Structures of CD300lf–P domain in complex with glycochenodeoxycholic acid (GCDCA) and lithocholic acid (LCA) reveal two bile acid binding sites at the P domain dimer interface distant from receptor binding sites. The structural determinants for receptor and bile acid binding are supported by numerous biophysical assays utilizing interface residue mutations. We find that the monomeric affinity of CD300lf for the P domain is low and is divalent cation dependent. We have also determined the crystal structure of CD300lf in complex with phosphocholine, revealing that MNoV engages its receptor in a manner mimicking host ligands including similar metal coordination. Docking of the cocomplex structures onto a cryo-EM–derived model of MNoV suggests that each virion can make multiple CD300lf engagements, and thus, infection may be driven by the avidity of cell surface clustered CD300lf. These studies identify multiple potential modulators of norovirus infection that may act to regulate the interaction between the viral capsid P domain and its cognate cellular receptor.

Interferon (IFN) induced activities are critical, early determinants of immune responses and infection outcomes. A key facet of IFN responses is the upregulation of hundreds of mRNAs termed interferon-stimulated genes (ISGs) that activate intrinsic and cell-mediated defenses. While primary interferon signaling is well-delineated, other layers of regulation are less explored but implied by aberrant ISG expression signatures in many diseases in the absence of infection. Consistently, our examination of tonic ISG levels across uninfected human tissues and individuals revealed three ISG subclasses. As tissue identity and many comorbidities with increased virus susceptibility are characterized by differences in metabolism, we characterized ISG responses in cells grown in media known to favor either aerobic glycolysis (glucose) or oxidative phosphorylation (galactose supplementation). While these conditions over time had a varying impact on the expression of ISG RNAs, the differences were typically greater between treatments than between glucose/galactose. Interestingly, extended interferon-priming led to divergent expression of two ISG proteins: upregulation of IRF1 in IFN-γ/glucose and increased IFITM3 in galactose by IFN-α and IFN-γ. In agreement with a hardwired response, glucose/galactose regulation of interferon-γ induced IRF1 is conserved in unrelated mouse and cat cell types. In galactose conditions, proteasome inhibition restored interferon-γ induced IRF1 levels to that of glucose/interferon-γ. Glucose/interferon-γ decreased replication of the model poxvirus vaccinia at low MOI and high MOIs. Vaccinia replication was restored by IRF1 KO. In contrast, but consistent with differential regulation of IRF1 protein by glucose/galactose, WT and IRF1 KO cells in galactose media supported similar levels of vaccinia replication regardless of IFN-γ priming. Also associated with glucose/galactose is a seemingly second block at a very late stage in viral replication which results in reductions in herpes- and poxvirus titers but not viral protein expression. Collectively, these data illustrate a novel layer of regulation for the key ISG protein, IRF1, mediated by glucose/galactose and imply unappreciated subprograms embedded in the interferon response. In principle, such cellular circuitry could rapidly adapt immune responses by sensing changing metabolite levels consumed during viral replication and cell proliferation.

Membrane-bound organelles serve as platforms for the assembly of multi-protein complexes that function as hubs of signal transduction in eukaryotic cells. Microbial pathogens have evolved virulence factors that reprogram these host signaling responses, but the underlying molecular mechanisms are poorly understood. Here we test the ability of ~200 type III and type IV effector proteins from six Gram-negative bacterial species to interact with the eukaryotic plasma membrane and intracellular organelles. We show that over 30% of the effectors localize to yeast and mammalian cell membranes, including a subset of previously uncharacterized Legionella effectors that appear to be able to regulate yeast vacuolar fusion. A combined genetic, cellular, and biochemical approach supports that some of the tested bacterial effectors can bind to membrane phospholipids and may regulate membrane trafficking. Finally, we show that the type III effector IpgB1 from Shigella flexneri may bind to acidic phospholipids and regulate actin filament dynamics. Microbial pathogens secrete effector proteins into host cells to affect cellular functions. Here, the authors use a yeast-based screen to study around 200 effectors from six bacterial species, showing that over 30% of them interact with the eukaryotic plasma membrane or intracellular organelles.

Murine norovirus (MNoV) is an important model of human norovirus (HNoV) and mucosal virus infection more broadly. Viral receptor utilization is a major determinant of cell tropism, host range, and pathogenesis. The bona fide receptor for HNoV is unknown. Recently, we identified CD300lf as a proteinaceous receptor for MNoV. Interestingly, its paralogue CD300ld was also sufficient for MNoV infection in vitro. Here we explored whether CD300lf is the sole physiologic receptor in vivo and whether HNoV can use a CD300 ortholog as an entry receptor. We report that both CD300ld and CD300lf are sufficient for infection by diverse MNoV strains in vitro. We further demonstrate that CD300lf is essential for both oral and parenteral MNoV infection and to elicit anti-MNoV humoral responses in vivo. In mice deficient in STAT1 signaling, CD300lf is required for MNoV-induced lethality. Finally, we demonstrate that human CD300lf (huCD300lf) is not essential for HNoV infection, nor does huCD300lf inhibit binding of HNoV virus-like particles to glycans. Thus, we report huCD300lf is not a receptor for HNoV.

A core set of autophagy proteins is required for gamma interferon (IFN-γ)-mediated clearance of Toxoplasma gondii in the mouse because of their control of several downstream effectors, including immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs). However, these effectors are absent (i.e., IRGs) from or nonessential (i.e., GBPs) in IFN-γ-activated human cells, raising the question of how these cells control parasite replication. Here, we define a novel role for ubiquitination and recruitment of autophagy adaptors in the strain-specific control of T. gondii replication in IFN-γ-activated human cells. Vacuoles containing susceptible strains of T. gondii became ubiquitinated, recruited the adaptors p62 and NDP52, and were decorated with LC3. Parasites within LC3-positive vacuoles became enclosed in multiple layers of host membranes, resulting in stunting of parasite replication. However, LC3-positive T. gondii -containing vacuoles did not fuse with endosomes and lysosomes, indicating that this process is fundamentally different from xenophagy, a form of autophagy involved in the control of intracellular bacterial pathogens. Genetic knockout of ATG16L or ATG7 reverted the membrane encapsulation and restored parasite replication, indicating that core autophagy proteins involved in LC3 conjugation are important in the control of parasite growth. Despite a role for the core autophagy machinery in this process, upstream activation through Beclin 1 was not sufficient to enhance the ubiquitination of T. gondii -containing vacuoles, suggesting a lack of reliance on canonical autophagy. These findings demonstrate a new mechanism for IFN-γ-dependent control of T. gondii in human cells that depends on ubiquitination and core autophagy proteins that mediate membrane engulfment and restricted growth. IMPORTANCE Autophagy is a process of cellular remodeling that allows the cell to recycle senescent organelles and recapture nutrients. During innate immune responses in the mouse, autophagy is recruited to help target intracellular pathogens and thus eliminate them. However, the antimicrobial mediators that depend on autophagy in the mouse are not conserved in humans, raising the issue of how human cells control intracellular pathogens. Our study defines a new pathway for the control of the ubiquitous intracellular parasite T. gondii in human cells activated by IFN-γ. Recruitment of autophagy adaptors resulted in engulfment of the parasite in multiple membranes and growth impairment. Although susceptible type 2 and 3 stains of T. gondii were captured by this autophagy-dependent pathway, type 1 strains were able to avoid entrapment. Autophagy is a process of cellular remodeling that allows the cell to recycle senescent organelles and recapture nutrients. During innate immune responses in the mouse, autophagy is recruited to help target intracellular pathogens and thus eliminate them. However, the antimicrobial mediators that depend on autophagy in the mouse are not conserved in humans, raising the issue of how human cells control intracellular pathogens. Our study defines a new pathway for the control of the ubiquitous intracellular parasite T. gondii in human cells activated by IFN-γ. Recruitment of autophagy adaptors resulted in engulfment of the parasite in multiple membranes and growth impairment. Although susceptible type 2 and 3 stains of T. gondii were captured by this autophagy-dependent pathway, type 1 strains were able to avoid entrapment.

Cellular susceptibility to viral infections is in part determined by the presence of a host cellular receptor. Here we use murine norovirus as a model to uncover an unappreciated connection between an intracellular lipid biosynthetic enzyme and a receptor conformation that is permissive for viral infection. The serine palmitoyltransferase complex is required for de novo sphingolipid biosynthesis and we find that its absence impairs the ability of murine norovirus to bind and enter cells. Although the serine palmitoyltransferase complex is dispensable for the surface expression of the norovirus receptor, CD300lf, serine palmitoyltransferase activity is required for CD300lf to adopt a conformation permissive for viral binding. Addition of extracellular ceramide to serine palmitoyltransferase-deficient cells chemically complements both the conformational changes of CD300lf and the cellular susceptibility to murine norovirus infection. Taken together, these data indicate that intracellular sphingolipid biosynthesis regulates the conformation of the murine norovirus receptor and therefore the tropism of murine norovirus. This indicates that intracellular biosynthetic pathways can regulate viral tropism even when the receptor for a virus is expressed on the target cell surface. The serine palmitoyltransferase (SPT) complex, which is required for de novo sphingolipid biosynthesis, is required for the murine norovirus receptor, CD300lf, to adopt a conformation permissive for viral binding.

Non-enveloped viruses require cell lysis to release new virions from infected cells, suggesting that these viruses require mechanisms to induce cell death. Noroviruses are one such group of viruses, but there is no known mechanism that causes norovirus infection-triggered cell death and lysis 1 – 3 . Here we identify a molecular mechanism of norovirus-induced cell death. We found that the norovirus-encoded NTPase NS3 contains an N-terminal four-helix bundle domain homologous to the membrane-disruption domain of the pseudokinase mixed lineage kinase domain-like (MLKL). NS3 has a mitochondrial localization signal and thus induces cell death by targeting mitochondria. Full-length NS3 and an N-terminal fragment of the protein bound the mitochondrial membrane lipid cardiolipin, permeabilized the mitochondrial membrane and induced mitochondrial dysfunction. Both the N-terminal region and the mitochondrial localization motif of NS3 were essential for cell death, viral egress from cells and viral replication in mice. These findings suggest that noroviruses have acquired a host MLKL-like pore-forming domain to facilitate viral egress by inducing mitochondrial dysfunction. The murine norovirus NTPase NS3 induces mitochondrial disruption, resulting in cell death, which is required for viral egress.

CLEC16A is in a locus genetically linked to autoimmune diseases including multiple sclerosis, but the function of this gene in the nervous system is unknown. Here we show that two mouse strains carrying independent Clec16a mutations developed neurodegenerative disease characterized by motor impairments and loss of Purkinje cells. Neurons from Clec16a -mutant mice exhibited increased expression of the autophagy substrate p62, accumulation of abnormal intra-axonal membranous structures bearing the autophagy protein LC3 and abnormal Golgi morphology. Multiple aspects of endocytosis, lysosome and Golgi function were normal in Clec16a-deficient murine embryonic fibroblasts and HeLa cells. However, these cells displayed abnormal bulk autophagy despite unimpaired autophagosome formation. Cultured Clec16a-deficient cells exhibited a striking accumulation of LC3 and LAMP-1 positive autolysosomes containing undigested cytoplasmic contents. Therefore Clec16a, an autophagy protein that is critical for autolysosome function and clearance, is required for Purkinje cell survival.