Publications

Associate Professor of Microbiology and Immunology

Publications

  • MLKL Requires the Inositol Phosphate Code to Execute Necroptosis. Molecular cell Dovey, C. M., Diep, J., Clarke, B. P., Hale, A. T., McNamara, D. E., Guo, H., Brown, N. W., Cao, J. Y., Grace, C. R., Gough, P. J., Bertin, J., Dixon, S. J., Fiedler, D., Mocarski, E. S., Kaiser, W. J., Moldoveanu, T., York, J. D., Carette, J. E. 2018; 70 (5): 936–48.e7

    Abstract

    Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.

    View details for DOI 10.1016/j.molcel.2018.05.010

    View details for PubMedID 29883610

    View details for PubMedCentralID PMC5994928

  • A CRISPR toolbox to study virus-host interactions NATURE REVIEWS MICROBIOLOGY Puschnik, A. S., Majzoub, K., Ooi, Y. S., Carette, J. E. 2017; 15 (6): 351-364

    Abstract

    Viruses depend on their hosts to complete their replication cycles; they exploit cellular receptors for entry and hijack cellular functions to replicate their genome, assemble progeny virions and spread. Recently, genome-scale CRISPR-Cas screens have been used to identify host factors that are required for virus replication, including the replication of clinically relevant viruses such as Zika virus, West Nile virus, dengue virus and hepatitis C virus. In this Review, we discuss the technical aspects of genome-scale knockout screens using CRISPR-Cas technology, and we compare these screens with alternative genetic screening technologies. The relative ease of use and reproducibility of CRISPR-Cas make it a powerful tool for probing virus-host interactions and for identifying new antiviral targets.

    View details for DOI 10.1038/nrmicro.2017.29

    View details for Web of Science ID 000401062000010

    View details for PubMedID 28420884

  • A Small-Molecule Oligosaccharyltransferase Inhibitor with Pan-flaviviral Activity. Cell reports Puschnik, A. S., Marceau, C. D., Ooi, Y. S., Majzoub, K., Rinis, N., Contessa, J. N., Carette, J. E. 2017; 21 (11): 3032–39

    Abstract

    The mosquito-borne flaviviruses include important human pathogens such as dengue, Zika, West Nile, and yellow fever viruses, which pose a serious threat for global health. Recent genetic screens identified endoplasmic reticulum (ER)-membrane multiprotein complexes, including the oligosaccharyltransferase (OST) complex, as critical flavivirus host factors. Here, we show that a chemical modulator of the OST complex termed NGI-1 has promising antiviral activity against flavivirus infections. We demonstrate that NGI-1 blocks viral RNA replication and that antiviral activity does not depend on inhibition of the N-glycosylation function of the OST. Viral mutants adapted to replicate in cells deficient of the OST complex showed resistance to NGI-1 treatment, reinforcing the on-target activity of NGI-1. Lastly, we show that NGI-1 also has strong antiviral activity in primary and disease-relevant cell types. This study provides an example for advancing from the identification of genetic determinants of infection to a host-directed antiviral compound with broad activity against flaviviruses.

    View details for DOI 10.1016/j.celrep.2017.11.054

    View details for PubMedID 29241533

    View details for PubMedCentralID PMC5734657

  • DDX6 Represses Aberrant Activation of Interferon-Stimulated Genes. Cell reports Lumb, J. H., Li, Q., Popov, L. M., Ding, S., Keith, M. T., Merrill, B. D., Greenberg, H. B., Li, J. B., Carette, J. E. 2017; 20 (4): 819–31

    Abstract

    The innate immune system tightly regulates activation of interferon-stimulated genes (ISGs) to avoid inappropriate expression. Pathological ISG activation resulting from aberrant nucleic acid metabolism has been implicated in autoimmune disease; however, the mechanisms governing ISG suppression are unknown. Through a genome-wide genetic screen, we identified DEAD-box helicase 6 (DDX6) as a suppressor of ISGs. Genetic ablation of DDX6 induced global upregulation of ISGs and other immune genes. ISG upregulation proved cell intrinsic, imposing an antiviral state and making cells refractory to divergent families of RNA viruses. Epistatic analysis revealed that ISG activation could not be overcome by deletion of canonical RNA sensors. However, DDX6 deficiency was suppressed by disrupting LSM1, a core component of mRNA degradation machinery, suggesting that dysregulation of RNA processing underlies ISG activation in the DDX6 mutant. DDX6 is distinct among DExD/H helicases that regulate the antiviral response in its singular ability to negatively regulate immunity.

    View details for DOI 10.1016/j.celrep.2017.06.085

    View details for PubMedID 28746868

    View details for PubMedCentralID PMC5551412

  • Genetic dissection of Flaviviridae host factors through genome-scale CRISPR screens NATURE Marceau, C. D., Puschnik, A. S., Majzoub, K., Ooi, Y. S., Brewer, S. M., Fuchs, G., Swaminathan, K., Mata, M. A., Elias, J. E., Sarnow, P., Carette, J. E. 2016; 535 (7610): 159-?

    Abstract

    The Flaviviridae are a family of viruses that cause severe human diseases. For example, dengue virus (DENV) is a rapidly emerging pathogen causing an estimated 100 million symptomatic infections annually worldwide. No approved antivirals are available to date and clinical trials with a tetravalent dengue vaccine showed disappointingly low protection rates. Hepatitis C virus (HCV) also remains a major medical problem, with 160 million chronically infected patients worldwide and only expensive treatments available. Despite distinct differences in their pathogenesis and modes of transmission, the two viruses share common replication strategies. A detailed understanding of the host functions that determine viral infection is lacking. Here we use a pooled CRISPR genetic screening strategy to comprehensively dissect host factors required for these two highly important Flaviviridae members. For DENV, we identified endoplasmic-reticulum (ER)-associated multi-protein complexes involved in signal sequence recognition, N-linked glycosylation and ER-associated degradation. DENV replication was nearly completely abrogated in cells deficient in the oligosaccharyltransferase (OST) complex. Mechanistic studies pinpointed viral RNA replication and not entry or translation as the crucial step requiring the OST complex. Moreover, we show that viral non-structural proteins bind to the OST complex. The identified ER-associated protein complexes were also important for infection by other mosquito-borne flaviviruses including Zika virus, an emerging pathogen causing severe birth defects. By contrast, the most significant genes identified in the HCV screen were distinct and included viral receptors, RNA-binding proteins and enzymes involved in metabolism. We found an unexpected link between intracellular flavin adenine dinucleotide (FAD) levels and HCV replication. This study shows notable divergence in host-depenency factors between DENV and HCV, and illuminates new host targets for antiviral therapy.

    View details for DOI 10.1038/nature18631

    View details for Web of Science ID 000379015600044

    View details for PubMedID 27383987

    View details for PubMedCentralID PMC4964798

  • An essential receptor for adeno-associated virus infection. Nature Pillay, S., Meyer, N. L., Puschnik, A. S., Davulcu, O., Diep, J., Ishikawa, Y., Jae, L. T., Wosen, J. E., Nagamine, C. M., Chapman, M. S., Carette, J. E. 2016; 530 (7588): 108-112

    Abstract

    Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr(-/-) (also known as Au040320(-/-) and Kiaa0319l(-/-)) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.

    View details for DOI 10.1038/nature16465

    View details for PubMedID 26814968

    View details for PubMedCentralID PMC4962915

  • The adherens junctions control susceptibility to Staphylococcus aureus a-toxin. Proceedings of the National Academy of Sciences of the United States of America Popov, L. M., Marceau, C. D., Starkl, P. M., Lumb, J. H., Shah, J., Guerrera, D., Cooper, R. L., Merakou, C., Bouley, D. M., Meng, W., Kiyonari, H., Takeichi, M., Galli, S. J., Bagnoli, F., Citi, S., Carette, J. E., Amieva, M. R. 2015; 112 (46): 14337-14342

    Abstract

    Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

    View details for DOI 10.1073/pnas.1510265112

    View details for PubMedID 26489655

  • Ebola virus entry requires the cholesterol transporter Niemann-Pick C1 NATURE Carette, J. E., Raaben, M., Wong, A. C., Herbert, A. S., Obernosterer, G., Mulherkar, N., Kuehne, A. I., Kranzusch, P. J., Griffin, A. M., Ruthel, G., Dal Cin, P., Dye, J. M., Whelan, S. P., Chandran, K., Brummelkamp, T. R. 2011; 477 (7364): 340-U115

    Abstract

    Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

    View details for DOI 10.1038/nature10348

    View details for Web of Science ID 000294852400033

    View details for PubMedID 21866103

  • Editing N-Glycan Site Occupancy with Small-Molecule Oligosaccharyltransferase Inhibitors CELL CHEMICAL BIOLOGY Rinis, N., Golden, J. E., Marceau, C. D., Carette, J. E., Van Zandt, M. C., Gilmore, R., Contessa, J. N. 2018; 25 (10): 1231-+

    Abstract

    The oligosaccharyltransferase (OST) is a multisubunit enzyme complex that N-glycosylates proteins in the secretory pathway and is considered to be constitutive and unregulated. However, small-molecule OST inhibitors such as NGI-1 provide a pharmacological approach for regulating N-linked glycosylation. Herein we design cell models with knockout of each OST catalytic subunit (STT3A or STT3B) to screen the activity of NGI-1 and its analogs. We show that NGI-1 targets the function of both STT3A and STT3B and use structure-activity relationships to guide synthesis of catalytic subunit-specific inhibitors. Using this approach, pharmacophores that increase STT3B selectivity are characterized and an STT3B-specific inhibitor is identified. This inhibitor has discrete biological effects on endogenous STT3B target proteins such as COX2 but does not activate the cellular unfolded protein response. Together this work demonstrates that subsets of glycoproteins can be regulated through pharmacologic inhibition of N-linked glycosylation.

    View details for DOI 10.1016/j.chembiol.2018.07.005

    View details for Web of Science ID 000447868100007

    View details for PubMedID 30078634

  • Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1 CELL DEATH & DISEASE Guo, H., Gilley, R. P., Fisher, A., Lane, R., Landsteiner, V. J., Ragan, K. B., Dovey, C. M., Carette, J. E., Upton, J. W., Mocarski, E. S., Kaiser, W. J. 2018; 9: 816

    Abstract

    Necroptosis complements apoptosis as a host defense pathway to stop virus infection. Herpes simplex virus shows a propensity to trigger necroptosis of mouse cells and mice even though cell death is blocked in human cells through UL39-encoded ICP6. This ribonucleotide reductase large subunit (R1) nucleates RHIM-dependent oligomerization of RIP3 kinase (RIPK3, also known as RIP3) in mouse cells but inhibits activation in cells from the natural human host. By interrogating the comparative behavior of ICP6-deficient viruses in mouse and human cells, here we unveil virus-induced necroptosis mediated by Z-DNA-binding protein 1 (ZBP1, also known as DAI). ZBP1 acts as a pathogen sensor to detect nascent RNA transcripts rather than input viral DNA or viral DNA generated through replication. Consistent with the implicated role of virus-induced necroptosis in restricting infection, viral pathogenesis is restored in Zbp1-/-, Ripk3-/- and Mlkl-/- mice. Thus, in addition to direct activation of RIPK3 via ICP6, HSV1 infection in mice and mouse cells triggers virus-induced necroptosis through ZBP1. Importantly, virus-induced necroptosis is also induced in human HT-29 cells by ICP6 mutant viruses; however, ZBP1 levels must be elevated for this pathway to be active. Thus, our studies reveal a common, species-independent role of this nucleic acid sensor to detect the presence of this virus. HSV1 ICP6 functions as a bona fide RHIM signaling inhibitor to block virus-induced necroptosis in its natural host. Altogether, ZBP1-dependent restriction of herpesvirus infection emerges as a potent antiviral armament of the innate immune system.

    View details for DOI 10.1038/s41419-018-0868-3

    View details for Web of Science ID 000440242800003

    View details for PubMedID 30050136

    View details for PubMedCentralID PMC6062522