key: cord-0430798-gsdv09ka
authors: Luteijn, Rutger D.; van Diemen, Ferdy; Blomen, Vincent A.; Boer, Ingrid G.J.; Sadasivam, Saran Manikam; van Kuppevelt, Toin H.; Drexler, Ingo; Brummelkamp, Thijn R.; Lebbink, Robert Jan; Wiertz, Emmanuel J.
title: A genome-wide haploid genetic screen for essential factors in vaccinia virus infection identifies TMED10 as regulator of macropinocytosis
date: 2018-12-11
journal: bioRxiv
DOI: 10.1101/493205
sha: 6fba26a85cc21b600807d523b4f94b646918e21b
doc_id: 430798
cord_uid: gsdv09ka

Vaccinia virus is a promising viral vaccine and gene delivery candidate, and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to Modified Vaccinia Virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2 and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical role of EXT1 in heparan sulfate synthesis and vaccinia virus infection was confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, suggesting that TMED10 regulates actin cytoskeleton remodelling necessary for virus infection. Importance Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells, and are used as vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knock-out library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, modulates the host cell membrane to facilitate virus uptake.

Vaccinia virus is a promising viral vaccine and gene delivery candidate, and has historically 25 been used as a model to study poxvirus-host cell interactions. We employed a genome-wide 26 insertional mutagenesis approach in human haploid cells to identify host factors crucial for 27 vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to Modified Upon binding, the viral envelope fuses with the host cell at either the plasma membrane or 77 the endosomal membrane after macropinocytotic uptake by the host (16). Release of the 78 virus core in the cytoplasm initiates transcription of more than 100 viral genes (17) Additionally, it contains a human U6 promoter which drives expression of a guideRNA 128 (gRNA) consisting of an 18-20bp target-specific CRISPR RNA (crRNA) fused to the trans-129 activating crRNA (tracrRNA) and a terminator sequence. This vector is called pSicoR-CRISPR-130 PuroR and has been described previously (22) . 131

The crRNA target sequences for each of the targeted genes were designed using an online 132 CRISPR design tool (crispr.mit.edu; Zhang lab, MIT). CRISPR gRNAs with the highest 133 specificity and lowest off-target rate for the human genome were selected and cloned into 134 the pSicoR-CRISPR-PuroR vector using Gibson assembly (NEB Lentivirus production and transduction 144

Third generation lentiviruses were produced in HEK-293T cells in a 24 well plate format 145 using standard lentivirus production protocols. MJS cells were transduced using spin 146 infection at 1,000 x g for 90 minutes at 33°C in the presence of 3.2 µg/ml polybrene. After 3 147 days, transduced cells were selected using zeocin (400 µg/ml). HepS for host cell attachment, including Lassa virus, RVFV, and adeno-associated virus (27, 411 42, 49) . In addition, a haploid genetic screen that directly assessed the role of host factors 412 involved in HepS biosynthesis revealed a similar enrichment of genes and also identified 413 EXT1 as the most significant hit (42). Other genes that were enriched in these screens 414 included TM9SF2 and prenyltransferase alpha subunit repeat containing 1 (PTAR1). The 415 involvement of PTAR1 in HepS surface expression has been confirmed recently (20, 21, 27) . 416 TM9SF2 is a member of the highly conserved nonaspanin proteins that have been 417 connected to diverse cellular processes, most notably receptor trafficking, and cellular 418 adhesion (50, 51). In addition, TM9SF2 affects the localization and stability of NDST1, which 419 is critical for N-sulfation of HepS (30). TM9SF2 single nucleotide polymorphisms have been 420 associated with several human diseases, including progression of AIDS(52). This association 421 may, in part, be explained by a TM9SF2-mediated decrease of HepS, thereby affecting 422 binding of HIV to the cell surface (53). 423

We identified TMED10 (also known as TMP21/p23/p24δ) as an important host factor for 425 VACV infection. TMED10 is a ubiquitously expressed type I transmembrane protein that 426 associates with coat complex protein I (COPI) vesicles through KKLIE motif in its cytosolic tail 427 (54, 55). As such, it is suggested to function as a cargo receptor in retrograde vesicular 428 trafficking from the Golgi to the ER (54, 56, 57). In addition, TMED10 localizes to the plasma 429 membrane independently of COPI (58). Plasma membrane-localized TMED10 controls the 430 activity of the presenilin/γ -secretase complex in neuronal tissue, thereby affecting the 431 formation of amyloid beta peptides implicated in Alzheimer's disease (59). Other binding 432 partners have also been described for TMED10, including ER-localized MHC class I (60), and 433 C1 domain-containing proteins such as the chimaerins (61, 62). These latter proteins are 434 critical regulators of the actin cytoskeleton modulator Rac1 (61-64). 435

The regulation of chimaerins by TMED10 may explain the effects on virus-induced 436 macropincotytosis observed in this study. Vaccinia-induced blebbing and subsequent 437 macropinocytosis critically depend on actin rearrangements regulated by the Rho GTPase 438 Rac1 (34, 35, 37, 65, 66) . Rac1 is deactivated by β2-chimaerin localized at the plasma 439 membrane (61). This localization is regulated by TMED10 that redistributes β2-chimaerin to 440 the perinucleus upon binding (61, 62) Conversely, depletion of TMED10 or disruption of the 441 β2-chimaerin/TMED10 complex relocates β2-chimaerin to the plasma membrane, and 442 thereby enhances Rac1 deactivation (61). Similarly, Rac1 deactivation in TMED10 KO cells 443 could explain the inhibitory effect on blebbing and macropinocytosis observed in this study. 444

We identified TMED10 as an important factor in PS-induced macropinocytosis. This pathway 445 is not only used by vaccinia viruses to enter cells; many other viruses expose PS to allow 446 entry by macropinocytosis (reviewed in (34)). In contrast to plasma membrane fusion, 447 macropinocytosis allows viruses to bypass the dense cortical actin layer to enter the cytosol. 4D ) were incubated with VACV-WR (MOI 100) (+VACV) or left untreated (-VACV) for one hour on ice. Subsequently, the cells were incubated at 37C for 45 min to allow bleb formation. Blebs were visualized by staining the cells for actin (green) using phalloidin iFluor 488; nuclei were counterstained (red) using TO-PRO3. A representative image field of 20 different image fields is presented. Bar size: 7.5 µm. 

Human monkeypox: an emerging zoonosis

Human infection with a zoonotic vaccine affords protection in the influenza A(H7N9) pneumonia ferret model

Recombinant MVA vaccines: dispelling the 507 myths

Immunogenicity of 511 a highly attenuated MVA smallpox vaccine and protection against monkeypox

Identification of 515 vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative 516 analysis of smallpox vaccines

Cold Spring Harb Perspect Biol 5. 518 15. Moss B. 2012. Poxvirus cell entry: how many proteins does it take?

Simultaneous high-523 resolution analysis of vaccinia virus and host cell transcriptomes by deep RNA 524 sequencing

Expression 526 profiling of the intermediate and late stages of poxvirus replication

Construction and isolation of recombinant MVA

Deciphering the glycosylome of 533 dystroglycanopathies using haploid screens for lassa virus entry

Gene essentiality and synthetic 538 lethality in haploid human cells

Quantitative analysis of brain 545 and spinach leaf lipids employing silicic acid column chromatography and acetone 546 for elution of glycolipids

Heparan sulfate biosynthesis: regulation and variability

A27L protein mediates vaccinia virus 600 interaction with cell surface heparan sulfate

Vaccinia virus envelope H3L protein 604 binds to cell surface heparan sulfate and is important for intracellular mature virion 605 morphogenesis and virus infection in vitro and in vivo

Entry of the vaccinia virus 609 intracellular mature virion and its interactions with glycosaminoglycans

Vaccinia virus 4c (A26L) protein 612 on intracellular mature virus binds to the extracellular cellular matrix laminin

An essential receptor for adeno-616 associated virus infection

Synergistic control of cellular adhesion by transmembrane 9 proteins

Genetic associations of variants in genes encoding HIV-dependency factors 626 required for HIV-1 infection

Human immunodeficiency virus and heparan 628 sulfate: from attachment to entry inhibition

A major transmembrane protein of Golgi-derived COPI-coated 631 vesicles involved in coatomer binding

Tmp21 and p24A, two type I proteins enriched in pancreatic microsomal 634 membranes, are members of a protein family involved in vesicular trafficking

p23, a major COPI-vesicle 640 membrane protein, constitutively cycles through the early secretory pathway

The luminal domain of p23 (Tmp21) plays a critical role in 643 p23 cell surface trafficking

647 TMP21 is a presenilin complex component that modulates gamma-secretase but not 648 epsilon-secretase activity

Tmp21, a novel MHC-I interacting protein, preferentially binds to 650 Beta2-microglobulin-free MHC-I heavy chains

Chimaerins, novel non-protein kinase C phorbol ester 652 receptors, associate with Tmp21-I (p23): evidence for a novel anchoring mechanism 653 involving the chimaerin C1 domain

2010. p23/Tmp21 differentially targets the Rac-GAP beta2-655 chimaerin and protein kinase C via their C1 domains

Expression of alpha 1-chimaerin (rac-1 GAP) alters the 657 cytoskeletal and adhesive properties of fibroblasts

Phospholipase Cgamma/diacylglycerol-dependent activation of beta2-chimaerin 660 restricts EGF-induced Rac signaling

Dissecting the roles of Rac1 activation and 662 deactivation in macropinocytosis using microscopic photo-manipulation

Membrane ruffling and macropinocytosis 665 in A431 cells require cholesterol

Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory 668 cytokine production through autocrine/paracrine mechanisms involving TGF-beta, 669 PGE2, and PAF

Immunosuppressive effects of apoptotic cells

After five hours of infection, cells were harvested and the amount of infected cells (GFP-positive) was quantified by flow cytometry. S.E.M. of three independent infections is indicated. Three representative clones of five are shown for EXT1, four of these clones were protected from MVA infection . Three representative clones of eleven are shown for TM9SF2. (B) Clonal lines indicated in (A) were analyzed for HepS expression levels by flow cytometry. (C) Eleven clonal lines obtained from MJS cells transfected with gRNA TM9SF2#1 or TM9SF2#2 were infected with MVA-eGFP (MOI 10). In addition, the cells were stained for HepS surface expression and the mean fluorescent intensity (MFI) was quantified by flow cytometry. (3 µM) in the absence (dashed line) or presence or the specific macropinocytosis inhibitor EIPA (black line). Panel 3: LUV uptake (3 µM) in the presence (dashed line) or absence (black line) of serum

We thank Ronny Tao for technical assistance. RJL was supported by Marie Curie Career 463Integration Grant PCIG-GA-2011-294196. SMS was supported by the seventh framework 464 program of the European Union (Initial Training Network "ManiFold," Grant 317371), and ID 465