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A Genome-Wide Haploid Genetic Screen for Essential Factors in Vaccinia Virus

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A Genome-Wide Haploid Genetic Screen for Essential Factors in Vaccinia Virus bioRxiv preprint doi: https://doi.org/10.1101/493205; this version posted December 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A genome-wide haploid genetic screen for essential factors in vaccinia virus 2 infection identifies TMED10 as regulator of macropinocytosis 3 4 Running title: Macropinocytosis of Vaccinia virus is regulated by TMED10 5 6 Rutger D. Luteijn1#a, Ferdy van Diemen1, Vincent A. Blomen2, Ingrid G.J. Boer1, Saran 7 Manikam Sadasivam3, Toin H. van Kuppevelt4, Ingo Drexler5, Thijn R. Brummelkamp2,, 8 Robert Jan Lebbink1¶, Emmanuel J. Wiertz1¶* 9 10 1Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, The 11 Netherlands 12 2Netherlands Cancer Institute, Amsterdam, The Netherlands 13 3Department of Membrane Biochemistry and Biophysics, Utrecht University, The 14 Netherlands 15 4Department of Biochemistry, Radboud University Medical Center, Nijmegen, The 16 Netherlands 17 5Institute for Virology, Universitätsklinikum Düsseldorf, Heinrich-Heine-University, 18 Düsseldorf, Germany 19 #aCurrent address: Department of Molecular and Cell Biology, University of California, 20 Berkeley, California, United States of America 21 22 *Corresponding author: [email protected] 23 ¶These authors contributed equally to this paper 1 bioRxiv preprint doi: https://doi.org/10.1101/493205; this version posted December 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Abstract 25 Vaccinia virus is a promising viral vaccine and gene delivery candidate, and has historically 26 been used as a model to study poxvirus-host cell interactions. We employed a genome-wide 27 insertional mutagenesis approach in human haploid cells to identify host factors crucial for 28 vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to Modified 29 Vaccinia Virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified 30 host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein 31 trafficking. We validated EXT1, TM9SF2 and TMED10 (TMP21/p23/p24δ) as important host 32 factors for vaccinia virus infection. The critical role of EXT1 in heparan sulfate synthesis and 33 vaccinia virus infection was confirmed. TM9SF2 was validated as a player mediating heparan 34 sulfate expression, explaining its contribution to vaccinia virus infection. In addition, 35 TMED10 was found to be crucial for virus-induced plasma membrane blebbing and 36 phosphatidylserine-induced macropinocytosis, suggesting that TMED10 regulates actin 37 cytoskeleton remodelling necessary for virus infection. 38 39 Importance 40 Poxviruses are large DNA viruses that can infect a wide range of host species. A number of 41 these viruses are clinically important to humans, including variola virus (smallpox) and 42 vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus 43 and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically 44 target cancer cells, and are used as vaccine vector against tuberculosis, influenza, and 45 coronaviruses. 2 bioRxiv preprint doi: https://doi.org/10.1101/493205; this version posted December 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 46 Poxviruses rely on host factors for most stages of their life cycle, including attachment to 47 the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. 48 We used a genome-wide knock-out library of host cells to identify host factors necessary for 49 vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus 50 attachment. Additionally, we show that TMED10, previously not implicated in virus 51 infections, modulates the host cell membrane to facilitate virus uptake. 52 53 Keywords 54 poxvirus, vaccinia virus, heparan sulfate, genome-wide screen, macropinocytosis, TMED10, 55 phosphatidylserine 56 3 bioRxiv preprint doi: https://doi.org/10.1101/493205; this version posted December 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 57 Introduction 58 59 The poxvirus family represents a group of large enveloped DNA viruses that infect a wide 60 variety of hosts. Poxvirus species capable of infecting humans include variola virus, which 61 causes smallpox and is one of the most destructive pathogens in human history. Since its 62 eradication through successful vaccination using vaccinia virus, poxvirus outbreaks in 63 humans are nowadays mainly caused by zoonotic infections of cowpox virus, monkeypox 64 virus, and recently discovered poxvirus species (1-3). The number of zoonotic infections is 65 predicted to rise, due to waning population immunity (4). In part, the decreased immunity is 66 caused by concerns about vaccinia virus safety, as a minority of vaccinated individuals show 67 adverse side effects (5). These safety concerns have led to the development of safer, 68 attenuated vaccinia virus strains, including the Modified Vaccinia Ankara virus (MVA). By 69 passaging vaccinia virus over 500 times on chicken embryo fibroblasts, the resulting 70 attenuated MVA lost 10% of the parental vaccinia genome, and displays an abortive 71 replication cycle in most cell lines (6). MVA is potent vaccine against poxviruses and serves 72 as a vaccine vector against a variety of other diseases (7-13). 73 Due to their low pathogenicity and wide range of applications, vaccinia virus strains are used 74 as model viruses to study the unique life cycle of poxviruses (14). The vaccinia virus life cycle 75 starts with binding to heparan sulfate (HepS) and other glycosaminoglycans expressed on 76 the host cell, although laminin and unidentified cellular proteins may also play a role (15). 77 Upon binding, the viral envelope fuses with the host cell at either the plasma membrane or 78 the endosomal membrane after macropinocytotic uptake by the host (16). Release of the 79 virus core in the cytoplasm initiates transcription of more than 100 viral genes (17). Early 80 gene transcripts mediate virus core uncoating and DNA replication (18). Replicated viral 4 bioRxiv preprint doi: https://doi.org/10.1101/493205; this version posted December 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 81 genomes serve as template for transcription of intermediate and late genes, many of which 82 are involved in the assembly of virus progeny (18). 83 In contrast to most DNA viruses, poxvirus replication is located outside the cellular nucleus 84 in specialized compartments known as viral factories (14). Due to their independence from 85 the host nucleus, poxviruses are required to encode most of the genes involved in DNA 86 replication and transcription. Consequently, poxviruses are considered to be less dependent 87 on host factors compared to other DNA viruses. Nevertheless, interaction with the host cell 88 are required during most stages of virus infection. Here, we employed a genome-wide 89 haploid genetic screen to identify host factors involved in MVA infection. Deep-sequencing 90 of gene trap insertion sites in the virus-resistant population identified genes involved in 91 HepS biosynthesis, Golgi organization, vesicular protein trafficking and ubiquitination. 92 Besides confirming TM9SF2 as a player in heparan sulfate biosynthesis, we identified 93 TMED10 as a crucial factor for vaccinia virus-induced macropinocytosis. 94 95 Materials and Methods 96 Cells and viruses 97 The human melanoma cell line MelJuSo (MJS) and T2 cells were cultured in RPMI 1640 98 supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin and 2mM L- 99 glutamine (complete medium). HEK-293T and Hela cells were cultured in DMEM 100 supplemented with 10% FCS, 100U/ml penicillin, 100 µg/ml streptomycin and 2mM ʟ- 101 glutamine. 102 VACV strain Western Reserve (WR) encoding eGFP under control of the early/late P7.5 103 promoter (VACV-eGFP) was a generous gift from Dr. Jon Yewdell (NIH, Bethesda, USA). 104 VACV-eGFP was propagated and titrated on Vero cells. 5 bioRxiv preprint doi: https://doi.org/10.1101/493205; this version posted December 11, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 105 Recombinant modified vaccinia virus Ankara (MVA) expressing eGFP under the early/late 106 promoter P7.5 (MVA-eGFP) or P11 late promoter (MVA-eGFPlate) was propagated and 107 titrated in chicken embryonic fibroblasts (CEFs). All viruses have been amplified, purified 108 (sucrose cushion), and titrated according to standard methodology(19). 109 110 Insertional mutagenesis 111 Mutagenesis of HAP1 cells using a retroviral gene trap vector was performed as described 112 previously (20). For the screen, 1x108 cells were infected with MVA-eGFP (MOI 50), and 113 surviving cells were expanded and harvested for genomic DNA isolation. Retroviral insertion 114 sites were amplified using a linear amplification-mediated PCR and deep-sequenced 115
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