AXL-dependent infection of human fetal endothelialcells distinguishes Zika virus from otherpathogenic flavivirusesAudrey Stéphanie Richarda,1, Byoung-Shik Shima,1, Young-Chan Kwona, Rong Zhangb,c,d,e, Yuka Otsukaa,Kimberly Schmitta, Fatma Berria, Michael S. Diamondb,c,d,e, and Hyeryun Choea,2

aDepartment of Immunology and Microbial Science, The Scripps Research Institute, Jupiter, FL 33458; bDepartment of Medicine, Washington UniversitySchool of Medicine, St. Louis, MO 63110; cDepartment of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110;dDepartment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; and eCenter for Human Immunology andImmunotherapy Programs, Washington University School of Medicine, St. Louis, MO 63110

Edited by Eric O. Freed, National Cancer Institute, Frederick, MD, and accepted by Editorial Board Member Diane E. Griffin January 5, 2017 (received forreview December 14, 2016)

Although a causal relationship between Zika virus (ZIKV) andmicrocephaly has been established, it remains unclear why ZIKV,but not other pathogenic flaviviruses, causes congenital defects.Here we show that when viruses are produced in mammalian cells,ZIKV, but not the closely related dengue virus (DENV) or West Nilevirus (WNV), can efficiently infect key placental barrier cells thatdirectly contact the fetal bloodstream. We show that AXL, a receptortyrosine kinase, is the primary ZIKV entry cofactor on humanumbilical vein endothelial cells (HUVECs), and that ZIKV uses AXLwith much greater efficiency than does DENV or WNV. Consistentwith this observation, only ZIKV, but not WNV or DENV, bound theAXL ligand Gas6. In comparison, when DENV and WNV wereproduced in insect cells, they also infected HUVECs in an AXL-dependent manner. Our data suggest that ZIKV, when producedfrom mammalian cells, infects fetal endothelial cells much moreefficiently than other pathogenic flaviviruses because it binds Gas6more avidly, which in turn facilitates its interaction with AXL.

Zika virus | Flaviviruses | AXL | placental barrier | fetal endothelial cell

Zika (ZIKV), West Nile (WNV), and dengue (DENV) virusesare closely related, and belong to the Flavivirus genus in the

Flaviviridae family. Although a causal relation between ZIKV andmicrocephaly has been established by human and animal studies(1–7), it remains unclear why only ZIKV, but not other pathogenicflaviviruses, causes congenital diseases. Although WNV is knownto infect neuronal cells and results in encephalitis (8), it does notcause microcephaly. DENV is not generally neurotropic and is notlinked to congenital defects.To reach the fetal brain, a virus must be transported from the

maternal to the fetal circulation, which necessitates crossing of theplacental barrier. In the placenta, fetal blood in capillaries isseparated from maternal blood by placental barrier cells, namelytrophoblasts and fetal endothelial cells. Recent studies indicatethat the placenta and its barrier cells are infected by ZIKV, andfetal brain lesions develop in mice, pigtail macaques, and humans(1–6, 9). However, it remains unclear why only ZIKV, and notother neurotropic flaviviruses, results in microcephaly and othercongenital disorders.Although bona fide entry receptors for flaviviruses remain un-

known, many cell surface-expressed molecules contribute to in-fection, including C-type lectins dendritic cell-specific intercellularadhesion molecule-3-grabbing nonintegrin (DC-SIGN) and DC-SIGN–related protein (L-SIGN) (10, 11) and phosphatidylserine(PS) receptors (12–15). PS receptors, which serve as entry cofac-tors for flaviviruses, include members of the TIM (T-cell Ig mucin)family and the TAM (TYRO3, AXL, and MERTK) family. TIM-family receptors bind PS directly (14, 15), whereas TAM-familymembers bind PS indirectly, through the soluble intermediatesGas6 (growth arrest-specific 6) and protein S present in serum andother bodily fluids (16, 17). Whereas Gas6 binds to all three TAM

family members with high affinity, protein S binds to TYRO3 andMERTK, but not to AXL (17). The TAM receptor AXL wasrecently shown to support ZIKV infection of human foreskin fi-broblasts (12), and its expression was noted in the brain andneuroprogenitor cells (18–21). However, its deletion had no effecton ZIKV infection of induced pluripotent human stem cell-derivedneuroprogenitor cells or cerebral organoids (22) or on virus accu-mulation of the eye, brain, or testis in Axl−/−mice (23, 24). Expressionof other flavivirus entry cofactors (genatlas.medecine.univ-paris5.fr/fiche.php?onglet=4&n=26146, genatlas.medecine.univ-paris5.fr/fiche.php?onglet=4&n=1364, and refs. 22 and 25) in addition to AXL,however, might have compensated for the absence of AXL in thesecells and tissues.In contrast, we show here that AXL is the only relevant ZIKV

entry cofactor expressed on fetal endothelial cells, and that whenproduced in mammalian cells, only ZIKV, but not WNV orDENV, can use AXL, because it more efficiently binds Gas6.These differences may help explain why only ZIKV, and notother flaviviruses, can access the fetal bloodstream to infect fetaltissues and cause microcephaly.

Significance

Zika virus (ZIKV) causes microcephaly, whereas other relatedpathogenic flaviviruses do not. To reach the fetal brain, a virusmust be transported from the maternal to the fetal circulation,which requires crossing of the placental barrier. Our studiesdemonstrate that mammalian cell-derived ZIKV, but not twoother globally relevant flaviviruses, efficiently infects fetal en-dothelial cells, a key component of the placental barrier, becauseonly ZIKV can efficiently use the cell-surface receptor AXL. Thesedata suggest that use of AXL allows ZIKV to enter the fetalbloodstream to gain access to other fetal tissues. Thus, this studyprovides insight into the unique properties of ZIKV that con-tribute to its ability to cause microcephaly and other congenitalinfections and diseases.

Author contributions: A.S.R., B.-S.S., and H.C. designed research; A.S.R., B.-S.S., Y.-C.K.,R.Z., Y.O., K.S., and F.B. performed research; R.Z. and M.S.D. contributed new reagents/analytic tools; and A.S.R. and H.C. wrote the paper.

Conflict of interest statement: M.S.D. is a consultant for Inbios, Visterra, Sanofi, andTakeda Pharmaceuticals, on the scientific advisory boards of Moderna and OraGene,and a recipient of research grants from Moderna, Sanofi, and Visterra.

This article is a PNAS Direct Submission. E.O.F. is a Guest Editor invited by the EditorialBoard.

Freely available online through the PNAS open access option.1A.S.R. and B.-S.S. contributed equally to this work.2To whom correspondence should be addressed. Email: hchoe@scripps.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1620558114/-/DCSupplemental.

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ResultsHuman Umbilical Vein Endothelial Cells Are More Susceptible to ZIKVthan to DENV or WNV. To investigate why ZIKV, and not closelyrelated flaviviruses (e.g., WNV and DENV), causes microcephaly,we initially evaluated the susceptibility to ZIKV, DENV, andWNV of fetal endothelial cells, which serve as a barrier betweenthe placenta and fetal tissues. We propagated viruses in a mam-malian cell line (Vero 76), rather than a commonly used insect cellline, to more accurately reflect the virus population generatedduring maternal infection. Human umbilical vein endothelial cells(HUVECs) from three independent donors (donors 1 to 3) orpooled HUVECs from additional donors (donor 4) were infectedat various multiplicities of infection (MOIs of 0.1 to 1.0) withZIKV (FSS13025, Cambodia), DENV (serotype 2, NewGuinea C), or WNV (lineage I, New York 1999). At all MOIsand in all four HUVEC preparations, ZIKV infection wasmuch more efficient than WNV or DENV (Fig. 1 A–C and Fig.S1 A–C). To confirm that the higher level of ZIKV infectionwas not an artifact of preferential recognition of ZIKV Eprotein by the pan-flavivirus antibody 4G2, we compared 4G2staining with that by virus-specific antibodies and observed noqualitative difference (Fig. S1D and Table S1). ZIKV progenyviruses in the culture supernatants, as measured by plaqueassays, also showed ∼100- and 1,000-fold higher titers thanthose from WNV- and DENV-infected cells, respectively (Fig.1D). Thus, ZIKV infects primary fetal endothelial cells to asubstantially greater level than does DENV or WNV.

AXL Is the Primary ZIKV Entry Cofactor in HUVECs. To identify themechanism by which ZIKV infected HUVECs, we measured thesurface expression of several established flavivirus entry cofactors:TIM- and TAM-family receptors, and DC-SIGN and L-SIGN. AllHUVECs expressed AXL, as previously reported (26), and, atlower levels, MERTK (Fig. 2A and Fig. S2A). No expression wasobserved for other entry cofactors, indicating they do not con-tribute to the infection of HUVECs by these viruses. The speci-ficity of the antibodies was confirmed by staining relevant moleculesectopically expressed in HEK293T cells (Fig. S2B).To determine whether AXL expression on HUVECs con-

tributed to ZIKV infection, cells were preincubated with an anti-AXL antibody, which blocks Gas6 binding but induces AXLphosphorylation (27, 28). ZIKV infection in HUVECs was ef-fectively inhibited by the anti-AXL antibody, but not by controlIgG (Fig. 2B). The same antibody did not inhibit the infection ofinfluenza A virus (IAV), which does not use any TIM or TAM

family members (13, 15). We similarly assessed the role ofMERTK in ZIKV infection of HUVECs. An anti-MERTK an-tibody, which also blocks Gas6 binding (29), did not inhibitZIKV infection (Fig. 2C). To corroborate these findings, we usedtwo genetic approaches: CRISPR/Cas9 gene editing and siRNAgene silencing. Both methods reduced AXL expression to un-detectable levels, and abolished ZIKV infection as judged byintracellular staining of E protein (Fig. 2 D and E). Collectively,these data indicate that AXL is the primary cofactor for ZIKVinfection of fetal endothelial cells.

ZIKV, but Not DENV or WNV, Efficiently Uses AXL. To investigatewhether the differential ability of ZIKV, WNV, and DENV toinfect HUVECs was due to distinct AXL-use patterns, we evalu-ated the effect of the AXL antibody on infection of HUVECs bythese viruses produced from Vero 76 cells. We used a highMOI of20 to obtain measurable intracellular staining levels of WNV andDENV infection (Fig. S3A) and an MOI of 1 for progeny virustitering (Fig. 3A). Infection of WNV and DENV was not affectedby the anti-AXL antibody, whereas ZIKV infection was markedlyreduced. We then conducted similar experiments in Vero 76 cells,which support efficient infection of all three viruses, likely becausemultiple flavivirus entry cofactors are expressed on these cells. Weobtained similar results from these cells, using reporter virus-likeparticles (RVPs), which are capable of single-round infection (Fig.S3B). Again, only ZIKV RVP infection was inhibited by the anti-AXL antibody. We then assessed AXL use by these viruses bytitering their progeny viruses produced from AXL-KO and controlHUVECs (Fig. 3B). Whereas ZIKV titer was substantially de-creased, WNV and DENV titers were only marginally reduced.We confirmed these AXL-use patterns using HEK293T cells ec-topically expressing AXL. Note that parental HEK293T cells lackendogenous expression of DC-SIGN and AXL, and DC-SIGNwas included as a control, because it is used by many flaviviruses toenter cells (10, 11). Only ZIKV infection was enhanced in AXL-expressing cells, whereas infection by all three flaviviruses wasincreased in DC-SIGN–expressing cells, compared with the pa-rental HEK293T cells (Fig. 3C). Although the basal infection ofthe parental HEK293T cells by WNV and DENV is much higherthan that by ZIKV, AXL use by WNV or DENV was not apparenteven at low MOIs, in contrast to that seen with DC-SIGN. Thesedata verify that the low level of WNV and DENV infection ob-served in HUVECs was not mediated by AXL.

Fig. 1. HUVECs are more susceptible to ZIKV infection than to DENV or WNV. (A) HUVECs were infected with Vero 76-produced ZIKV, DENV, or WNV at an MOI of 1.Infection levels, assessed at 24 h postinfection by staining permeabilized cells with the pan-flavivirus antibody 4G2, were normalized to that of ZIKV within each donor.Averages± SD of three experiments performed in duplicate are shown. ***P< 0.0001. See also Fig. S1. (B) Infection profiles of a representative experiment fromA are shownas histograms, where infected cells (colored lines) are compared with mock-infected cells (gray lines). (C) Similar to A, except that cells were fixed and stained with theantibody 4G2 on multiwell plates. (D) The progeny viruses in the supernatants from the experiments in A were quantified by plaque assays in Vero cells. The average titersbased on three independent experiments are presented as plaque-forming units per milliliter.

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Gas6 Binds to ZIKV, but Not to DENV or WNV. To assess whetherAXL utilization by ZIKV was mediated by the AXL ligand Gas6,we first attempted to inhibit virus–AXL interaction with C-Gas6-Ig, an Ig-fusion form of a human Gas6 variant that contains onlythe C-terminal half (AXL-binding domain) of the molecule. Thisconstruct thus lacks the PS-binding domain. TIM1(AA)-Ig doesnot bind PS (13), and was used as a negative control. At 0.4 μg/mL,C-Gas6-Ig substantially inhibited the infection of HUVECs byZIKV (Fig. 4 A and B), indicating that ZIKV associates with AXLat the same site where Gas6 binds, and suggesting that AXL use byZIKV is mediated by Gas6. Note that serum concentrations ofGas6 in healthy humans range from 13 to 100 ng/mL (30–32),and that Gas6 in medium containing 10% (vol/vol) FBS is suf-ficient for maximal transduction by various viruses mediated byAXL (33). Although various tissues and cells produce Gas6,the scale of this production is unknown.To investigate whether ZIKV, but not WNV or DENV, inter-

acts with Gas6, we performed a modified immunoprecipitationassay. Viruses were preincubated with Gas6-Ig, C-Gas6-Ig, orTIM1-Ig, and bound to protein A-Sepharose beads. Capturedviruses were quantified by RT-quantitative (q)PCR of the viral

RNA or visualized by Western blot (Fig. 4 C and D and Fig. S4).C-Gas6-Ig was used as a negative control, and TIM1-Ig was usedas a positive control, because all three viruses use TIM1 to infectcells (Fig. S5) (13, 15). ZIKV, but not DENV or WNV, wascaptured by Gas6-Ig immobilized on protein A beads. As expected,no virus was captured by C-Gas6-Ig, and all three viruses werecaptured by TIM1-Ig. Together, these data demonstrate that ZIKV,but not WNV or DENV, can efficiently use AXL, because onlyZIKV is able to bind Gas6 efficiently.

Insect Cell-Derived DENV and WNV also Use AXL. Although our dataindicate that Vero 76-produced DENV and WNV do not useAXL, AXL-dependent infection by DENV and WNV has beenreported by others (14, 34). We investigated whether differences invirus producer cells could influence infection outcomes, andtherefore repeated our infection studies with virus produced inC6/36 insect cells. Note that viruses were produced in insect cellsin the studies by Meertens et al. (14) (DENV) and Bhattacharyyaet al. (34) (WNV) but in Vero 76 cells in our study. Infection ofHUVECs by C6/36-produced DENV and WNV was much moreefficient than that produced in Vero 76 cells, when infected at the

Fig. 2. AXL is the primary ZIKV entry factor in HUVECs. (A) The cell-surface expression of the indicated proteins (red) or isotypes (gray) was assessed in HUVECs. Seealso Fig. S2. (B) HUVECs were preincubated with an anti-AXL antibody (AF154) or control IgG, and infected with ZIKV or IAV. Infection levels were normalized to thoseof cells infected without antibody. (C, Left) Similar to A, except that an anti-MERTK antibody (AF891) was compared with the anti-AXL antibody. Infection levels werenormalized as in A. (C, Right) The ability of the antibody to bind MERTK on HUVECs is shown. (D, Left) The efficiency of AXL gene editing via the CRISPR/Cas9 method,directed by an AXL-specific sgRNA in HUVECs (AXLKO cells; red) and an untargeted sgRNA (control cells; blue), is shown. Cells were stained with anti-AXL antibody(clone 108724). (D, Right) These cells were infected with ZIKV or IAV and infection levels were normalized to those of control cells for each virus. (E, Left) AXL ex-pressionwas analyzed, using the anti-AXL antibody (clone 108724), in HUVECs transfectedwith the indicated siRNA. (E, Right) These cells were infectedwith ZIKV at anMOI of 1, and infection levels were normalized to those of cells transfected without any siRNA. ZIKV was produced in Vero 76 cells and IAV in Madin–Darby caninekidney (MDCK) cells. (B–E) Averages ± SD of three (B and C) or five (D and E) experiments performed in duplicate are shown. **P < 0.001, ***P < 0.0001.

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same MOI, and was efficiently inhibited by the anti-AXL antibody,judged by both intracellular staining and plaque assays of progenyviruses (Fig. 5 A and B). Moreover, infection of C6/36-producedWNV and DENV was substantially reduced in AXLKO

HUVECs relative to the parental HUVECs (Fig. 5 C and D).To verify that mammalian cell-derived WNV and DENV donot use AXL, we produced these viruses in human cell linesA549 and Huh7. As with Vero 76-produced virus, ZIKV

Fig. 3. ZIKV, but not DENV or WNV, uses AXL efficiently. (A and B) HUVECs, preincubated with 50 nM anti-AXL antibody (AF154) or control IgG (A), or HUVECAXLKO and control cells (B), were infected with Vero 76-produced ZIKV, DENV, or WNV at an MOI of 1. The progeny viruses at 24 h postinfection werequantified by plaque assays in Vero cells. Results are expressed as plaque-forming units per milliliter. (C, Left) Expression levels of AXL or DC-SIGN intransduced HEK293T cells are shown. (C, Right) Parental HEK293T-, AXL-, or DC-SIGN–transduced cells were infected with ZIKV, DENV, or WNV at the in-dicated MOI. Results are presented as percent infected cells. Averages ± SD of three (A and C) or five (B) experiments performed in duplicate are shown. *P <0.01, **P < 0.001, ***P < 0.0001.

Fig. 4. Gas6 binds to ZIKV but not to DENV or WNV. (A) HUVECs were preincubated with C-Gas6-Ig or TIM1(AA)-Ig, and infected with Vero 76 cell-producedZIKV, DENV, or WNV at an MOI of 1. Infection levels were normalized to those of cells infected in the absence of any Ig-fusion protein within each virus.(B) Progeny viruses in the culture supernatants from the experiments in A were quantified by plaque assays in Vero cells. Results are expressed as plaque-forming units per milliliter. (C and D) ZIKV, DENV, or WNV, produced in Vero 76 cells, was incubated with Gas6-Ig, C-Gas6-Ig, or TIM1-Ig and immunopre-cipitated using protein A-Sepharose beads. (C) The RNA of the bound viruses was extracted and quantified by RT-qPCR. Binding is represented as foldincreases normalized within each virus to that of virus incubated with C-Gas6-Ig. See also Fig. S6. (A–C) Averages ± SD of three experiments performed induplicate (A and B) or quadruplicate (C) are shown. ***P < 0.0001. (D) Bound viruses were analyzed by Western blot (WB) using antibodies against E protein.A fraction (15%) of the input virus was loaded as a quantity control. A representative experiment of three performed is shown. IP, immunoprecipitation.

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derived from these cell lines efficiently used AXL but DENVand WNV did not (Fig. S6). Thus, DENV and WNV can useAXL to infect HUVECs when produced from insect cells butnot when produced from mammalian cells.

DiscussionOur current studies show that mammalian cell-derived ZIKV effi-ciently infects one of the two major placental barrier cells, namelyfetal endothelial cells, whereas WNV and DENV do not under thesame experimental conditions. This implies that fetal endothelial cellsserve as a barrier to WNV and DENV, but not to ZIKV, and maycontribute to the ability of ZIKV to disseminate to other fetal tissues.Our studies also show that AXL is the primary cofactor for ZIKVinfection in these cells, and suggest that the differential infection ofHUVECs by ZIKV, WNV, and DENV is due in large part to thedifferent efficiencies with which they bind Gas6 and use AXL.Tabata et al. recently reported that trophoblasts, the first layer

of the placental barrier, expressed TIM1 and were infected byZIKV (9). Although it is not yet established, WNV and DENVlikely infect primary trophoblasts because they both efficiently useTIM1 (Fig. S5) (13, 15). This implies that the trophoblast layermight not be an effective barrier for WNV, DENV, or ZIKV. Incomparison, our studies show that only ZIKV efficiently infectsfetal endothelial cells, another key placental barrier cell. A recentstudy by Miner et al. showed that both trophoblasts and fetalcapillaries were infected and injured by ZIKV infection in apregnancy model in mice (3). Accordingly, it will be of interest todetermine whether fetal endothelial cells in the mouse modelpresent a barrier to DENV and WNV as suggested by our studies.TAM receptors mediate phagocytosis of apoptotic cells in var-

ious tissues, including blood–brain barrier endothelial cells, retinalpigment epithelial cells of the eye, and Sertoli cells of the testes(25, 35, 36). This expression pattern is consistent with ZIKVpathogenesis observed in the brain and eye and its ability totransmit sexually (4, 37, 38). However, recent studies did not ob-serve differences in ZIKV infection of the eye, brain, or testesbetween wild-type and Axl−/− mice (23, 24). Similarly, Wells et al.observed no difference in ZIKV infection between wild-type and

AXL-KO stem cell-derived neuroprogenitor cells and organoids(22). Of note, Axl−/− mice exhibited elevated blood–brain barrierpermeability (39, 40). In addition, TIM1 is expressed at high levelsin retinal pigment epithelium and uroepithelium (genatlas.medecine.univ-paris5.fr/fiche.php?onglet=4&n=26146), DC-SIGN and L-SIGNare expressed in brain microvascular cells (25), and TYRO3 isexpressed in the brain and neuroprogenitor cells (genatlas.medecine.univ-paris5.fr/fiche.php?onglet=4&n=1364 and ref. 22). Thus, it re-mains possible that increased endothelium permeability in Axl−/−

mice could have contributed to the observed outcomes, and theabsence of AXL in those tissues of Axl−/− mice was compensatedfor by other entry cofactors. In contrast, no functional flavivirusentry cofactor other than AXL is expressed in HUVECs (Fig. 2A),explaining its greater contribution to ZIKV infection in these cellsthan in those tissues.One outstanding question in the field is how PS receptors pro-

mote flavivirus infection, because structural studies show that theE-protein shell occludes most of the virion membrane (41–43).However, flavivirus particles assume many asymmetric states (44–46)and are in continuous dynamic motion (47, 48), which likely exposespatches of the virion membrane. In addition, mosaic virions—thosethat are both mature and immature in patches—have been observedfunctionally and by cryo-EM (47, 49–52). At present, it is unclearwhy only ZIKV preferentially binds Gas6 whereas all three virusesbind TIM1 (Fig. 4 and Fig. S4). One possible explanation is thatbecause Gas6 is a larger protein, relative to TIM1, it requires largerpatches of exposed virion membrane, which might be more availableon ZIKV than onWNV or DENV. Greater membrane exposure onZIKV could be achieved if its structural proteins facilitate increaseddynamic motion, or if more ZIKV particles are in a mosaic state. Italso remains uncertain why insect cell-derived DENV and WNV,but not those produced in mammalian cells, can use AXL. Differentlipid composition in insect cells compared with mammalian cellsand the lower temperature used for virus production (e.g., 28 °C)could affect assembly and thus the ensembles of conformationalstates adopted. Clearly, the detailed mechanism by which ZIKVbinds Gas6 and uses AXL warrants further investigation.In summary, our data suggest that the unique ability of ZIKV

to infect fetuses and cause congenital malformations may derive,

Fig. 5. DENV and WNV produced in C6/36 cells use AXL, whereas those produced in Vero 76 cells do not. HUVECs preincubated with 50 nM anti-AXL (AF154) orcontrol antibody (A and B), or HUVEC AXLKO or control cells (C and D), were infected at an MOI of 1 with DENV or WNV produced either in Vero 76 or in C6/36cells. (A and C) Results are presented as percent infected HUVECs. (B and D) Progeny viruses at 24 h postinfection were quantified by plaque assays in Vero cells.Results are expressed as plaque-forming units per milliliter. Averages ± SD of three experiments performed in duplicate are shown. **P < 0.001, ***P < 0.0001.

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at least in part, from its capacity to bind Gas6 and use AXL,which may enable it to infect fetal endothelial cells and cross theplacental barrier more efficiently than other flaviviruses.

Materials and MethodsSee SI Materials and Methods for detailed methods.

Virus Infection and Inhibition Assays. For infection assays, cells were incubated at37 °C with viruses at the indicatedMOI for 6 h (HUVECs) or 1 h (HEK293T and Vero76 cells), and then further grown in fresh medium for 24 h (HUVECs and Vero76 cells) or 48 h (HEK293T cells). Cells were stained with the pan anti-flavivirusantibody 4G2, unless otherwise stated, or with the anti-IAV antibody (clone C179;Takara), and analyzed by flow cytometry. For inhibition assays, the indicatedcells were preincubated with an anti-AXL (AF154; R&D Systems) or anti-MERTK (AF891; R&D Systems) antibody for 20 min at room temperature,infected with replication-competent viruses or reporter virus-like particles,and analyzed as described above.

CRISPR/Cas9- and siRNA-Mediated Silencing of AXL Expression. To generate theAXL gene-edited HUVECs, an AXL-specific single-guide (sg)RNA was clonedinto the lentiCRISPR v2 plasmid (Addgene; 52961). An untargeted sgRNA wasused as a control. HUVECs were transduced with lentiviruses coexpressing Cas9and sgRNA, and selected with 1.5 μg/mL puromycin at 24 h posttransduction

for 4 d. To silence AXL expression, HUVECs (at 80 to 85% confluence) weretransfected with 25 nM untargeted or AXL-specific siRNA using DharmaFECT 4reagent (Dharmacon). The day after transfection, cells were detached and platedfor further infection assays and the analysis of AXL cell-surface expression.

Gas6-Ig Binding to Viruses. ZIKV, DENV, andWNV were quantified by RT-qPCRof their respective NS3 gene, mixed with Gas6-Ig, C-Gas6-Ig, or TIM1-Ig, andincubated at 37 °C with 5% CO2. After 1 h, protein A-Sepharose beads,preblocked with 2% (vol/vol) BSA, were added and further incubated for 1 hat room temperature on a rocking platform. After washing, bound viruseswere quantified by RT-qPCR of viral RNA or visualized byWestern blot analysesusing anti-E protein antibodies 4G2 (ZIKV and DENV) or 3.91D (WNV).

Data and Statistics. The difference between groups was tested using anunpaired two-tailed t test with Welch’s correction. The null hypothesis wasrejected when P < 0.01.

ACKNOWLEDGMENTS. We thank Dr. Robert Tesh at the University of TexasMedical Branch at Galveston, and the World Reference Center for EmergingViruses and Arboviruses, for providing us with WNV, ZIKV, and hyperim-mune ascites against ZIKV. This work was supported by startup funds fromThe Scripps Research Institute and NIH Grants R01 AI110692 (to H.C.) and R01AI073755 and R01 AI101400 (to M.S.D.).

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