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. 