IFITM3 directly engages and shuttles incoming virus particles to lysosomes.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) have emerged as important innate immune effectors that prevent diverse virus infections in vertebrates. However, the cellular mechanisms and live-cell imaging of these small membrane proteins have been challenging to evaluate during viral entry of mammalian cells. Using CRISPR-Cas9-mediated IFITM-mutant cell lines, we demonstrate that human IFITM1, IFITM2 and IFITM3 act cooperatively and function in a dose-dependent fashion in interferon-stimulated cells. Through site-specific fluorophore tagging and live-cell imaging studies, we show that IFITM3 is on endocytic vesicles that fuse with incoming virus particles and enhances the trafficking of this pathogenic cargo to lysosomes. IFITM3 trafficking is specific to restricted viruses, requires S-palmitoylation and is abrogated with loss-of-function mutants. The site-specific protein labeling and live-cell imaging approaches described here should facilitate the functional analysis of host factors involved in pathogen restriction as well as their mechanisms of regulation.

The crystal structure of the PB2 cap-binding domain of influenza B virus reveals a novel cap recognition mechanism.

The influenza RNA-dependent RNA polymerase is a core enzyme required for both transcription and replication of the virus RNA genome, making it a potential drug target for the influenza virus. To detect the feature of cap-dependent transcription of influenza B virus (FluB) polymerase, we determined the crystal structures of the wild-type FluB polymerase PB2 subunit cap-binding domain (PB2cap) with bound GDP and the mutant FluB Q325F PB2cap with bound m(7)GDP or GDP. These structures revealed that, distinct from influenza A virus (FluA) PB2cap, the guanine and ribose moieties of substrates invert in FluB PB2caps. Moreover, we characterized the substrate specificity and affinity of the PB2caps using isothermal titration calorimetry. FluB PB2cap has a weaker affinity for m(7)GDP than FluA PB2cap. Unlike FluA PB2cap that has a preference for m(7)GDP in comparison with GDP, FluB PB2cap shows an analogous affinity for both substrates. Replacement of FluB PB2 Glu(325) by Phe, the corresponding residue of FluA PB2, increased the binding affinity of FluB PB2cap for m(7)GDP to a level approximate to that of FluA PB2cap and caused a significant higher affinity to GDP. This study indicated that FluB PB2cap has a unique cap recognition mechanism compared with FluA PB2cap, providing molecular insight into inhibitor design targeting FluB PB2cap.

Nmnat exerts neuroprotective effects in dendrites and axons.

Dendrites can be maintained for extended periods of time after they initially establish coverage of their receptive field. The long-term maintenance of dendrites underlies synaptic connectivity, but how neurons establish and then maintain their dendritic arborization patterns throughout development is not well understood. Here, we show that the NAD synthase Nicotinamide mononucleotide adenylyltransferase (Nmnat) is cell-autonomously required for maintaining type-specific dendritic coverage of Drosophila dendritic arborization (da) sensory neurons. In nmnat heterozygous mutants, dendritic arborization patterns of class IV da neurons are properly established before increased retraction and decreased growth of terminal branches lead to progressive defects in dendritic coverage during later stages of development. Although sensory axons are largely intact in nmnat heterozygotes, complete loss of nmnat function causes severe axonal degeneration, demonstrating differential requirements for nmnat dosage in the maintenance of dendritic arborization patterns and axonal integrity. Overexpression of Nmnat suppresses dendrite maintenance defects associated with loss of the tumor suppressor kinase Warts (Wts), providing evidence that Nmnat, in addition to its neuroprotective role in axons, can function as a protective factor against progressive dendritic loss. Moreover, motor neurons deficient for nmnat show progressive defects in both dendrites and axons. Our studies reveal an essential role for endogenous Nmnat function in the maintenance of both axonal and dendritic integrity and present evidence of a broad neuroprotective role for Nmnat in the central nervous system.

Glucosyltransferase Activity of Clostridium difficile Toxin B Triggers Autophagy-mediated Cell Growth Arrest.

Autophagy is a bulk cell-degradation process that occurs through the lysosomal machinery, and many reports have shown that it participates in microbial pathogenicity. However, the role of autophagy in Clostridium difficile infection (CDI), the leading cause of antibiotics-associated diarrhea, pseudomembranous colitis and even death in severe cases, is not clear. Here we report that the major virulent factor toxin B (TcdB) of Clostridium difficile elicits a strong autophagy response in host cells through its glucosyltransferase activity. Using a variety of autophagy-deficient cell lines, i.e. HeLa/ATG7 -/-, MEF/atg7 -/-, MEF/tsc2 -/-, we demonstrate that toxin-triggered autophagy inhibits host cell proliferation, which contributes to TcdB-caused cytopathic biological effects. We further show that both the PI3K complex and mTOR pathway play important roles in this autophagy induction process and consequent cytopathic event. Although the glucosyltransferase activity of TcdB is responsible for inducing both cell rounding and autophagy, there is no evidence suggesting the causal relationship between these two events. Taken together, our data demonstrate for the first time that the glucosyltransferase enzymatic activity of a pathogenic bacteria is responsible for host autophagy induction and the following cell growth arrest, providing a new paradigm for the role of autophagy in host defense mechanisms upon pathogenic infection.

Divergent roles of BECN1 in LC3 lipidation and autophagosomal function.

BECN1/Beclin 1 is regarded as a critical component in the class III phosphatidylinositol 3-kinase (PtdIns3K) complex to trigger autophagy in mammalian cells. Despite its significant role in a number of cellular and physiological processes, the exact function of BECN1 in autophagy remains controversial. Here we created a BECN1 knockout human cell line using the TALEN technique. Surprisingly, the complete loss of BECN1 had little effect on LC3 (MAP1LC3B/LC3B) lipidation, and LC3B puncta resembling autophagosomes by fluorescence microscopy were still evident albeit significantly smaller than those in the wild-type cells. Electron microscopy (EM) analysis revealed that BECN1 deficiency led to malformed autophagosome-like structures containing multiple layers of membranes under amino acid starvation. We further confirmed that the PtdIns3K complex activity and autophagy flux were disrupted in BECN1(-/-) cells. Our results demonstrate the essential role of BECN1 in the functional formation of autophagosomes, but not in LC3B lipidation.

Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B.

As a gram-positive, spore-forming anaerobic bacillus, Clostridium difficile (C. difficile) is responsible for severe and fatal pseudomembranous colitis, and poses the most urgent antibiotic resistance threat worldwide. Epidemic C. difficile is the leading cause of antibiotic-associated diarrhoea globally, especially diarrhoea due to the emergence of hypervirulent strains associated with high mortality and morbidity. TcdB, one of the key virulence factors secreted by this bacterium, enters host cells through a poorly understood mechanism to elicit its pathogenic effect. Here we report the first identification of the TcdB cellular receptor, chondroitin sulfate proteoglycan 4 (CSPG4). CSPG4 was initially isolated from a whole-genome human shRNAmir library screening, and its role was confirmed by both TALEN- and CRISPR/Cas9-mediated gene knockout in human cells. CSPG4 is critical for TcdB binding to the cell surface, inducing cytoskeleton disruption and cell death. A direct interaction between the N-terminus of CSPG4 and the C-terminus of TcdB was confirmed, and the soluble peptide of the toxin-binding domain of CSPG4 could protect cells from the action of TcdB. Notably, the complete loss of CSPG4/NG2 decreased TcdB-triggered interleukin-8 induction in mice without significantly affecting animal mortality. Based on both the in vitro and in vivo studies, we propose a dual-receptor model for TcdB endocytosis. The discovery of the first TcdB receptor reveals a previously unsuspected role for CSPG4 and provides a new therapeutic target for the treatment of C. difficile infection.