Publications

Associate Professor of Microbiology and Immunology

Publications

  • An RNA-centric dissection of host complexes controlling flavivirus infection. Nature microbiology Ooi, Y. S., Majzoub, K., Flynn, R. A., Mata, M. A., Diep, J., Li, J. K., van Buuren, N., Rumachik, N., Johnson, A. G., Puschnik, A. S., Marceau, C. D., Mlera, L., Grabowski, J. M., Kirkegaard, K., Bloom, M. E., Sarnow, P., Bertozzi, C. R., Carette, J. E. 2019

    Abstract

    Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.

    View details for DOI 10.1038/s41564-019-0518-2

    View details for PubMedID 31384002

  • 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 PubMedID 29883610

  • 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 PubMedID 29241533

    View details for PubMedCentralID PMC5734657

  • 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

  • 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

  • GluA4-Targeted AAV Vectors Deliver Genes Selectively to Interneurons while Relying on the AAV Receptor for Entry. Molecular therapy. Methods & clinical development Hartmann, J., Thalheimer, F. B., Hopfner, F., Kerzel, T., Khodosevich, K., Garcia-Gonzalez, D., Monyer, H., Diester, I., Buning, H., Carette, J. E., Fries, P., Buchholz, C. J. 2019; 14: 252–60

    Abstract

    Selective gene delivery into subtypes of interneurons remains an important challenge in vector development. Adeno-associated virus (AAV) vector particles are especially promising for intracerebral injections. For cell entry, AAV2 particles are supposed to attach to heparan-sulfate proteoglycans (HSPGs) followed by endocytosis via the AAV receptor (AAVR). Here, we assessed engineered AAV particles deficient in HSPG attachment but competent in recognizing the glutamate receptor 4 (GluA4, also known as GluRD or GRIA4) through a displayed GluA4-specific DARPin (designed ankyrin repeat protein). When injected into the mouse brain, histological evaluation revealed that in various regions, more than 90% of the transduced cells were interneurons, mainly of the parvalbumin-positive subtype. Although part of the selectivity was mediated by the DARPin, the chosen spleen focus-forming virus (SFFV) promoter had contributed as well. Further analysis revealed that the DARPin mediated selective attachment to GluA4-positive cells, whereas gene delivery required expression of AAVR. Our data suggest that cell selectivity of AAV particles can be modified rationally and efficiently through DARPins, but expression of the AAV entry receptor remains essential.

    View details for DOI 10.1016/j.omtm.2019.07.004

    View details for PubMedID 31463334

  • Identification of the Cell-Surface Protease ADAM9 as an Entry Factor for Encephalomyocarditis Virus. mBio Baggen, J., Thibaut, H. J., Hurdiss, D. L., Wahedi, M., Marceau, C. D., van Vliet, A. L., Carette, J. E., van Kuppeveld, F. J. 2019; 10 (4)

    Abstract

    Encephalomyocarditis virus (EMCV) is an animal pathogen and an important model organism, whose receptor requirements are poorly understood. Here, we employed a genome-wide haploid genetic screen to identify novel EMCV host factors. In addition to the previously described picornavirus receptors sialic acid and glycosaminoglycans, this screen unveiled important new host factors for EMCV. These factors include components of the fibroblast growth factor (FGF) signaling pathway, such as the potential receptors FGFR1 and ADAM9, a cell-surface metalloproteinase. By employing various knockout cells, we confirmed the importance of the identified host factors for EMCV infection. The largest reduction in infection efficiency was observed in cells lacking ADAM9. Pharmacological inhibition of the metalloproteinase activity of ADAM9 did not affect virus infection. Moreover, reconstitution of inactive ADAM9 in knockout cells restored susceptibility to EMCV, pointing to a proteinase-independent role of ADAM9 in mediating EMCV infection. Using neutralization assays with ADAM9-specific antiserum and soluble receptor proteins, we provided evidence for a role of ADAM9 in EMCV entry. Finally, binding assays showed that ADAM9 facilitates attachment of EMCV to the cell surface. Together, our findings reveal a role for ADAM9 as a novel receptor or cofactor for EMCV.IMPORTANCE EMCV is an animal pathogen that causes acute viral infections, usually myocarditis or encephalitis. It is thought to circulate mainly among rodents, from which it is occasionally transmitted to other animal species, including humans. EMCV causes fatal outbreaks of myocarditis and encephalitis in pig farms and zoos, making it an important veterinary pathogen. Although EMCV has been widely used as a model to study mechanisms of viral disease in mice, little is known about its entry mechanism. Here, we employ a haploid genetic screen for EMCV host factors and identify an essential role for ADAM9 in EMCV entry.

    View details for DOI 10.1128/mBio.01780-19

    View details for PubMedID 31409686

  • Capsid engineering overcomes barriers toward Adeno-associated viral (AAV) vector-mediated transduction of endothelial cells. Human gene therapy Zhang, L., Rossi, A., Lange, L., Meumann, N., Koitzsch, U., Christie, K., Nesbit, A., Moore, T., Hacker, U., Morgan, M. A., Hoffmann, D., Zengel, J. R., Carette, J. E., Schambach, A., Salvetti, A., Odenthal, M., Buning, H. 2019

    Abstract

    Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but are inefficiently transduced with Adeno-associated viral (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable already few hours post transduction. Notably, AAV-VEC transduced not only proliferating EC, but also quiescent EC although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid-engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.

    View details for DOI 10.1089/hum.2019.027

    View details for PubMedID 31407607