Flavivirus internalization is regulated by asize-dependent endocytic pathwayBrent A. Hacketta and Sara Cherrya,1

aDepartment of Microbiology, University of Pennsylvania, Philadelphia, PA 19104

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved March 1, 2018 (received for review November 16, 2017)

Flaviviruses enter host cells through the process of clathrin-mediated endocytosis, and the spectrum of host factors requiredfor this process are incompletely understood. Here we found thatlymphocyte antigen 6 locus E (LY6E) promotes the internalizationof multiple flaviviruses, including West Nile virus, Zika virus, anddengue virus. Perhaps surprisingly, LY6E is dispensable for theinternalization of the endogenous cargo transferrin, which is alsodependent on clathrin-mediated endocytosis for uptake. Sinceviruses are substantially larger than transferrin, we reasoned thatLY6E may be required for uptake of larger cargoes and tested thisusing transferrin-coated beads of similar size as flaviviruses. LY6Ewas indeed required for the internalization of transferrin-coatedbeads, suggesting that LY6E is selectively required for large cargo.Cell biological studies found that LY6E forms tubules upon viralinfection and bead internalization, and we found that tubuleformation was dependent on RNASEK, which is also required forflavivirus internalization, but not transferrin uptake. Indeed, wefound that RNASEK is also required for the internalization oftransferrin-coated beads, suggesting it functions upstream ofLY6E. These LY6E tubules resembled microtubules, and we foundthat microtubule assembly was required for their formation andflavivirus uptake. Since microtubule end-binding proteins linkmicrotubules to downstream activities, we screened the threeend-binding proteins and found that EB3 promotes virus uptakeand LY6E tubularization. Taken together, these results highlight aspecialized pathway required for the uptake of large clathrin-dependent endocytosis cargoes, including flaviviruses.

endocytosis | flavivirus | entry | virus | LY6E

Clathrin-mediated endocytosis is a major endocytic route bywhich cells sample their environment and take up nutrients.

Thus, many viruses, including flaviviruses, hijack this endocyticpathway to gain access to internal compartments of host cells (1–3). Flaviviruses are a large group of reemerging viruses trans-mitted to humans by mosquito species of global significance (4).West Nile virus (WNV) is widespread in the Americas, neuro-tropic, and can cause encephalitis (5, 6). Zika virus (ZIKV) isnewly emerging in the Americas and can also be neurotropic (7–9). Dengue virus (DENV) is reemerging globally, infecting350 million people annually (10). Presently, there are no specificantiviral treatments or vaccines approved to treat these viruses.Clathrin-mediated endocytosis is a tightly orchestrated path-

way whereby cargoes bound to surface receptors are internalizedusing a number of well-characterized cellular factors (reviewedin ref. 11). Indeed, for those viruses that depend on clathrin-mediated endocytosis for entry, the classically known canonicalcomponents are required to facilitate this process (12, 13).However, most of our understanding of this cellular pathwaycomes from studies on endogenous cargoes, such as transferrin,which are an order of magnitude smaller than viral particles.Studies on vesicular stomatitis virus found that actin, which isdispensable for transferrin uptake, is required for internalizationof this virus (14). Therefore, it is likely that there are additionalrequirements for the internalization of flavivirus particles. Indeed,RNASEK, a recently identified ∼100-aa transmembrane protein,was found to be required for the internalization of flaviviruses, but

was dispensable for transferrin, suggesting that RNASEK mayrepresent a protein required for particular cargo (15, 16). How-ever, it is unknown how RNASEK facilitates the internalization ofthese viruses and whether there are other factors that selectivelypromote viral endocytosis.Here we report that lymphocyte antigen locus 6 E (LY6E) is

required for flavivirus infection at the level of entry. LY6E is asmall (101 aa) GPI-anchored protein of the Ly6/uPAR super-family of proteins that can be IFN-inducible (17, 18). Ectopicexpression of LY6E can promote replication of the flavivirusyellow fever virus (19, 20). Additionally, a genome-wide siRNAscreen revealed that knocking down LY6E reduces susceptibilityto West Nile virus infection (21). However, the mechanism bywhich LY6E promotes flavivirus infection was unknown. Wefound that LY6E facilitates infection of flaviviruses but notparainfluenza virus 5 (PIV5), which fuses at the plasma mem-brane. Further, we find that LY6E is required for the in-ternalization of flavivirus virions, but is dispensable for theinternalization of canonical clathrin cargo, transferrin. To di-rectly test the role of size in this process, we coated beads ofsimilar size of flaviviruses with transferrin and found that LY6Eis specifically required for the internalization of this large cargo.Since we had previously found that RNASEK promoted in-ternalization of virions, but not transferrin, we also tested therequirement for this factor in transferrin-bead uptake. We foundthat RNASEK is also necessary for the uptake of these largecargoes. Morphological studies revealed that virus infection in-duces tubularization of LY6E, which is dependent on RNASEKand microtubules. Moreover, disruption of microtubules, or theplus-end–binding, microtubule-associated protein EB3 abrogatesLY6E tubule formation and attenuates viral entry. Taken to-gether, these data demonstrate that large cargoes, includingviruses, are dependent on additional cellular factors for their

Significance

Flaviviruses are a globally important group of human viralpathogens. These viruses enter cells by hijacking an endocyticpathway, clathrin-mediated endocytosis, used by cells to takeup growth factors and nutrients. While most cargo is small,virions are large, and we identified host factors specificallyrequired for the internalization of large cargo including theseviruses. These studies define a set of requirements for viralinternalization and which may be amenable to therapeuticinterventions.

Author contributions: B.A.H. and S.C. designed research; B.A.H. performed research;B.A.H. contributed new reagents/analytic tools; B.A.H. and S.C. analyzed data; andB.A.H. and S.C. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).1To whom correspondence should be addressed. Email: cherrys@pennmedicine.upenn.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1720032115/-/DCSupplemental.

Published online April 2, 2018.

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uptake and that elucidation of this process may aid in thefuture development of new classes of broad-spectrum antiviraltherapies.

ResultsLY6E Promotes Flavivirus Infection. Previous studies suggested thatLY6E promotes infection of multiple flaviviruses (20, 21). LY6Eis ubiquitously expressed and in some studies has been shown tobe IFN-inducible (17, 18). However, in human osteosarcomacells (U2OS) we found that LY6E is basally expressed andlargely insensitive to IFN stimulation (Fig. S1). We next testedthe role of LY6E in WNV infection. First, we transfected eithernontargeting control or LY6E-directed siRNAs and found thatwe could deplete LY6E mRNA (Fig. 1A). We then challengedthese cells with WNV–Kunjin, as it is closely related to circu-lating strains of WNV in the United States (98% identical) andcan be used under BSL2 conditions (22). We found that there isa significant decrease in WNV-KUN RNA at 8 hours post-infection (hpi) as measured by RT-qPCR (Fig. 1B) and proteinat 24 hpi as measured by immunoblot (Fig. 1C). We next tookadvantage of cells that express a WNV replicon with a GFP re-porter in place of the structural proteins (23). We found thatwhile targeting GFP with siRNAs significantly reduces the re-porter signal, LY6E knockdown has no apparent impact on viralreplication in this system (Fig. S1 B and C). This result suggestedthat LY6E may be required upstream, at the level of entry.Since many flaviviruses use a similar pathway for entry, we

next tested whether other flaviviruses were dependent on LY6Eand found that dengue virus [Serotype 2, New Guinea C(DENV2-NGC)] and ZIKV (MR766) are also dependent onLY6E for efficient infection as measured by RT-qPCR (Fig. 1D).In contrast, the paramyxovirus PIV5, which enters at the plasmamembrane, does not require LY6E, and there is a trend toward

increased permissivity (Fig. 1D). These data suggest that LY6Epromotes flavivirus entry.To directly assay entry we implemented a RT-qPCR–based

assay we previously developed that allows us to quantitativelyassess viral binding and uptake (15). Briefly, we prebind virus tocells at 4 °C, wash off unbound virus, and monitor the levels ofbound virus by RT-qPCR. Using this assay, we found that WNV-KUN binding to cells is not affected by RNAi against LY6Eor clathrin light chain (CLTC), which is known to be required forinternalization (Fig. 1E). Next, we monitored virus internaliza-tion in cells prebound with virus by subsequently incubating thecells at 37 °C for 4 h. We remove the surface-bound virus withtrypsin and quantify the internalized virus. Under these condi-tions we found that depletion of either LY6E or the controlCLTC leads to significant reduction in virus internalization(Fig. 1F).

LY6E and RNASEK Facilitate Internalization of Large Endocytic Cargo.Since LY6E promotes WNV internalization, which is dependenton clathrin-mediated endocytosis, we reasoned that LY6E mayalso play a role in canonical clathrin-mediated endocytosis.Therefore, we tested whether LY6E is required for uptake oftransferrin—a well-characterized endogenous cargo for clathrin-mediated endocytosis. While depletion of CLTC attenuatedtransferrin uptake, as expected, loss of LY6E had no effect (Fig.2 A and B).One clear difference between transferrin and viral cargoes is

the significantly larger size of virions. Flaviviruses are ∼50 nm,while transferrin is ∼6 nm. We thus developed an assay to di-rectly test whether cargo size alters LY6E dependence, takingadvantage of the fact that transferrin uptake is not dependent onLY6E. For these studies we conjugated transferrin to streptavidin-labeled 40-nm microspheres that are green fluorescent (schema-tized in Fig. 2C). We developed a confocal microscopy-based assaywhereby extracellular beads could be detected using red fluores-cent streptavidin, while internalized particles were not bound bythe red fluorophore since we did not permeabilize the cells. Giventhe fact that the beads are constitutively green, they are observedboth inside and outside of the cells. This assay results in extra-cellular yellow beads and green intracellular beads. Using thisassay, we assessed whether LY6E plays a role in internalizing thesame cargo (transferrin) that differs in size. We added transferrin-bound beads to cells and allowed uptake for 45 min where weobserved ∼50% internalization in control cells (Fig. 2 D and E).As a positive control we depleted CLTC and found that there wasalmost a complete block in internalization (Fig. 2 D and E).Likewise, LY6E was required for the internalization of transferrin-bound beads (Fig. 2 D and E). Since globular transferrin uptakewas independent of LY6E, our data suggest that LY6E is selec-tively required for clathrin-mediated endocytosis of this largerform of cargo.RNASEK is another small protein that is required for WNV

internalization but dispensable for transferrin uptake (refs. 15and 16 and Fig. 2 A and B). We next tested whether RNASEKwas required for the internalization of this transferrin-boundbead cargo and found that, indeed, depletion of RNASEK alsoblocked transferrin-bound bead uptake (Fig. 2 B and C), dem-onstrating a requirement for both LY6E and RNASEK in theinternalization of large cargo. We also tested ∼20 nm beadscoupled to transferrin and found that uptake of these beadswas not dependent on LY6E or RNASEK (Fig. S2). Thesedata suggest that cargo of the size of virions is dependent onthis pathway.

LY6E Adopts a Microtubule-Like Organization upon Flavivirus Infection.We next examined the subcellular localization of LY6E dur-ing infection. We found that LY6E is diffuse in uninfectedcells and becomes tubular within 20 min of infection with

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Fig. 1. LY6E promotes flavivirus entry. U2OS cells were transfected withsiRNAs against LY6E or control before infection with WNV-KUN (MOI 1, 8 h).Total cellular RNA was collected, and RT-qPCR was performed to measure(A) LY6E or (B) WNV-KUN RNA levels; n = 3, mean ± SEM, *P < 0.05.(C) siRNA-treated U2OS cells were infected with WNV-KUN (MOI 1, 24 h) andprocessed for immunoblot against WNV NS1. Representative blot of threeindependent experiments shown. (D) Total RNA was collected for RT-qPCRanalysis of U2OS cells following 8 h infection with the indicated viruses (MOI 1);n = 3, mean ± SEM, *P < 0.05. (E and F) siRNA-transfected U2OS cells wereinfected with WNV-KUN (MOI 15), and total RNA was collected at (E) 0 hpi or(F) 4 hpi for RT-qPCR analysis; n = 3, mean ± SEM, *P < 0.05.

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either WNV-KUN or ZIKV infection (Fig. 3A and Fig. S3A).Furthermore, tubularization of LY6E is also induced bytransferrin-coated beads (Fig. S4). Moreover, in cells ectopi-cally expressing RNASEK-Flag (RK-F), LY6E is more tubularand no longer relocalizes upon infection (Fig. 3B and Fig. S3B),suggesting a potential link between these proteins. Because thetubular pattern of LY6E resembles microtubules, we alsotested whether this tubularization is dependent on microtubuleintegrity. Pretreatment of U2OS cells with the microtubuleinhibitor nocodazole drastically reduces WNV-induced LY6Etubularization and prevents virus-induced relocalization (Fig.3C and Fig. S3C). These data suggest that microtubules mayplay a role in WNV entry.

Flaviviral Uptake and Large-Cargo Internalization Are Dependentupon Microtubules. It has been previously established that flavi-viruses remodel the cytoskeleton at late stages postinfection andthat treatment with microtubule inhibitors can impact multiplesteps in the viral lifecycle (24–27). We specifically set out todetermine whether flavivirus uptake is dependent on microtu-bules. We first monitored viral uptake using a microscopy-basedassay we previously developed (15). After binding the virus tocells for 1 h at 4 °C (0 hpi) and washing off unbound virus,samples are either fixed to monitor virus binding or subsequently

shifted to 37 °C for 4 h (4 hpi) to allow for uptake before fixation.We performed the immunostaining in the absence of permeablizationto specifically monitor extracellular virions using an antibody to

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Fig. 2. LY6E and RNASEK facilitate size-dependent endocytosis. (A and B) Biotin-conjugated transferrin (25 μg/mL) was added to siRNA-treated U2OS cells for theindicated times. (A) Images are representative of three independent experiments. (B) Quantification of transferrin (Tf) uptake; n = 3, mean ± SEM, **P < 0.01.(C) Schematic of bead uptake assay. (D and E) Transferrin-conjugated GFP beads were added to siRNA-transfected U2OS cells for 45 min before fixation. Non-permeabilized cells were then treated with streptavidin-594. (D) Images are representative of three independent experiments. Arrows show internalized beads (greenonly). (E) Quantification of transferrin-conjugated bead uptake (Mander’s colocalization analysis); n= 3, mean± SEM, *P< 0.05. n.s., not significant. (Magnification: 63×.)

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Fig. 3. Virus-induced LY6E reorganization into tubules. (A) U2OS or (B) RK-Fexpressing U2OS cells were infected with the indicated viruses for 20 min andimmunostained for LY6E. Representative images shown from three independentexperiments. (C) U2OS cells were pretreated for 45 min with 50 μM nocodazolebefore infection with WNV for 20 min and immunostained for LY6E. Represen-tative images of three independent experiments shown. (Magnification: 63×.)

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the glycoprotein (4G2). In vehicle-treated cells (DMSO), we ob-serve WNV-KUN virions bound at 0 hpi and internalization at4 hpi since no surface-bound virus is detected at this time point(Fig. 4A). As a control, we treated cells with the flavivirus entryinhibitor nanchangmycin, which robustly blocks internalization(28). As expected, WNV remains on the surface of nanchangmycin-treated cells at 4 hpi. Similarly, we found that the microtubule in-hibitor nocodazole blocks uptake of WNV (Fig. 4A). We found thatnocodazole and nanchangmycin also inhibit uptake of ZIKV (Fig.S5). To confirm that nocodazole impacts virus uptake, we alsomonitored internalization of WNV-KUN using our RT-qPCR assayand found that treatment with either nanchangmycin or nocodazoleattenuates WNV internalization (Fig. 4B). This effect was specificfor flavivirus entry, as PIV5, which fuses at the plasma membrane,was insensitive to either nanchangmycin or nocodazole treatments(Fig. 4B).Next we set out to determine if microtubules play a role in

canonical clathrin-mediated endocytosis. As has been previouslyshown, we found that microtubule polymerization is not requiredfor transferrin uptake but rather for downstream trafficking asthe intracellular pattern of staining is distinct in nocodazoletreated cells, while the dynamin inhibitor dynasore completelyabrogated uptake (Fig. S6 and ref. 29). This led us to test ifmicrotubules are required for large-cargo uptake. Using transferrin-bound beads as the cargo, we found that both nocodazole andnanchangmycin block this internalization equivalently to dynasore,

an inhibitor of dynamin (Fig. 4 C and D).This requirement is spe-cific, as treatment with latrunculin A, which leads to actin de-polymerization, does not block viral uptake as measured by RT-qPCR (Fig. S7A) or the internalization of transferrin-coated beads(Fig. S7B).

EB3, a Microtubule-Associated Protein, Is Essential for FlavivirusInfection. Because microtubules are necessary for virus-inducedLY6E reorganization and the internalization of viral cargo, wereasoned that microtubule end-binding proteins, particularlyplus-end–tracking proteins (+TIPs), which influence the rate ofmicrotubule growth, may link virus internalization to the mi-crotubule network. The core components of +TIP complexes arethe end-binding (EB) family, EB1, EB2, and EB3 (30, 31). Re-cent studies found that EB1 plays roles in the early stages of viralreplication of several viruses, whereas roles for EB2 or EB3 havenot been described (4, 32). We used RNAi to deplete these EBsand validated mRNA knockdown (Fig. S8A). We screened theseEB-depleted cells for their impact on WNV infection and foundthat depletion of EB3, but not EB1 or EB2, significantly atten-uates WNV-KUN infection (Fig. 5A). Furthermore, we foundthat EB3, but not EB1 or EB2, promotes DENV and ZIKVinfection, while all three EBs were dispensable for PIV5 in-fection (Fig. 5B and Fig. S8 B and C). Interestingly, depletion ofEB2 exacerbated infection of WNV-KUN and trended toward

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Fig. 4. Flavivirus entry and uptake are dependent on microtubule polymerization. (A) U2OS cells were pretreated with the indicated drug 45 min beforeinfection with WNV-KUN (MOI 15) and fixed at 0 hpi or 4 hpi before processing for microscopy in the absence of permeablization and stained for the viralglycoprotein (anti-E, 4G2). Representative image of three experiments shown. (B) U2OS cells were pretreated with the indicated drug for 45 min, and WNV-KUN or PIV5 internalization at 4 hpi was measured by RT-qPCR; n = 3, mean ± SEM, *P < 0.05. (C and D) U2OS cells were pretreated with the indicated drug for45 min and subsequently treated with transferrin-conjugated beads for 45 min. Nonpermeabilized cells were then probed with streptavidin-594. Arrowsshow internalized beads (green only). (C ) Representative images shown; n = 3. (D) Quantification of transferrin-conjugated bead uptake (Mander’scolocalization analysis); n = 3, mean ± SEM, *P < 0.05. n.s., not significant. (Magnification: 63×.)

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increased infection for the other flaviviruses (Fig. 5A andFig. S8C).We next explored whether EB3 promotes entry using our RT-

qPCR assay. We found that EB3 is dispensable for WNV bindingto cells but required for uptake (Fig. 5 C and D). We then set outto link LY6E to EB3. We find that depletion of EB3 reduces theLY6E tubularization upon WNV infection (Fig. 5 E–H). Further,we found that silencing of RNASEK also attenuates LY6Etubularization (Fig. 5 E–H). We explored the possibility thatEB2 may impact LY6E tubularization and found that depletionof EB2 led to increased LY6E tubules at baseline (Fig. S9). Thismay suggest a negative regulatory role of EB2, which may ex-plain the increased infection.Our data suggest a link between LY6E, RNASEK, and EB3.

Furthermore, the small-molecule entry inhibitor nanchangmycinphenocopies the depletion of these genes. Therefore, we ex-plored whether there was an interaction between these entryfactors and the inhibitor. To test this, we determined if depletionof these entry factors impacted the antiviral activity of nan-changmycin against WNV-KUN. The IC50 of nanchangmycin incontrol cells was 158 nM, and that was significantly reduced upondepletion of LY6E, RNASEK, or EB3 (Fig. 5I, P < 0.05, t test onpIC50s). Altogether, these data functionally link the microtubule-associated protein EB3 to LY6E- and RNASEK-dependent viraluptake, providing evidence for microtubules in viral endocytosisand the uptake of large cargoes.

DiscussionMany viruses gain entry into cells using clathrin-mediated en-docytosis. This pathway is important for several aspects of cellbiology, including nutrient uptake, and thus, clathrin-mediatedendocytosis has been extensively studied. Indeed, many of thefactors required for cargo internalization have been identified.

However, it is clear that numerous unknowns remain, includingcellular factors that may be required for specific cargoes and, inparticular, large cargo. As viruses are typically much larger thanmost endogenous cargoes, it remains unclear whether there areparticular proteins that facilitate this process. Here we foundthat LY6E promotes flavivirus entry at the level of internaliza-tion. Further study found that this requirement for internaliza-tion was not general for all clathrin-mediated endocytosiscargoes as LY6E was dispensable for the uptake of the endog-enous cargo transferrin. This led us to test if this difference wasdue to the size of the cargo. We developed an assay where wecompared internalization of transferrin to that of transferrin-coated beads, which are similar in size to virions, and found thatLY6E was specifically required for the larger cargo.To our knowledge, the only other cellular gene known to

promote internalization of flaviviruses yet dispensable for trans-ferrin uptake is RNASEK, another small-membrane–associatedprotein (15). Using the transferrin bead uptake assay, we foundthat RNASEK also facilitated internalization of this large cargo,suggesting that RNASEK may work with LY6E to promote viralentry. Cell biological studies revealed that LY6E relocalizes dur-ing flavivirus uptake to form tubular structures. Furthermore, thisrelocalization is dependent on RNASEK. Loss of RNASEK at-tenuates virus-induced tubularization of LY6E, suggesting thatRNASEK is upstream of LY6E.This tubularization of LY6E resembled microtubules, and we

found that microtubule assembly is required for this virus-induced change in LY6E morphology. This led us to explore therole of microtubules in flavivirus uptake. Microtubules have beenshown to play important roles in downstream aspects of flavivirusinfection, including trafficking of endosomes and viral release(24–27, 33). Therefore, we tested whether flavivirus uptake wasdependent on microtubules and found that indeed flavivirus

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Fig. 5. EB3 is required for flavivirus uptake and LY6E tubularization. (A) siRNA-transfected U2OS cells were infected with WNV-KUN (MOI 1, 8 h) before RT-qPCR analysis; n = 3, mean ± SEM, *P < 0.05, ***P < 0.001. (B) siRNA-transfected U2OS cells were infected with the indicated viruses (MOI = 1, 8 h), andinfection was monitored by RT-qPCR analysis; n = 3, mean ± SEM, *P < 0.05, **P < 0.01. (C) WNV-KUN binding and (D) internalization was measured in siRNA-transfected U2OS cells (MOI 15, 4 h) by RT-qPCR; n = 3, mean ± SEM, **P < 0.01. (E–H) siRNA-transfected U2OS cells were uninfected or infected with WNV-KUN (MOI 50, 20 min) and processed for microscopy. (E–G) Images shown are representative of three independent experiments. (H) Quantification of LY6Etubularization 20 min postinfection shown; n = 3, mean ± SEM, *P < 0.05. (I) siRNA-transfected U2OS cells were pretreated with the indicated concentrationsof nanchangmycin before WNV-KUN infection (MOI 2, 24 h). Quantification of percent infection was used to calculate IC50s (n = 3, mean ± SEM plotted) andIC50 values shown. n.s., not significant. (Magnification: 63×.)

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internalization is blocked by microtubule inhibitors, while trans-ferrin internalization is not. Furthermore, we found that nocodazoleblocked transferrin-coated bead uptake. These data suggest that thislarge-cargo internalization requires changes in the microtubulecompartment.Microtubule plus-ends polymerize rapidly and are the sites

where microtubule elongation occurs. Microtubule stabilizationis controlled by plus-end–tracking proteins whose accumulationis facilitated by microtubule end-binding proteins (EBs), whichare the core components of these complexes (30, 31). Wescreened the three mammalian EBs and found a role for EB3 inflavivirus infection. EB3 is required for WNV uptake as well asthe tubularization of LY6E, suggesting a role for EB3 in large-cargo uptake. While EB1 and EB3 are thought to play similarroles and can heterodimerize, some processes are selectivelydependent on EB3 (34, 35). We found that depletion of EB1 didnot impact infection suggesting a specific requirement for EB3.Interestingly, EB2, which is not known to directly interact withEB1 or EB3, may play a negative role in this process. Silencing ofEB2 leads to increased WNV infection concomitant with alteredLY6E localization. It is unclear how EB2 could negatively reg-ulate this process; however, previous studies have shown that lossof EB2 leads to more stable microtubule ends (36). Futurestudies will explore potential interactions between the EBs andvirus-induced structures.Our findings suggest an additional level of regulation on the

internalization of larger cargo. LY6E, RNASEK, and EB3 are

defining a pathway required for the endocytosis of viruses as wellas our model cargo, transferrin-coated beads, but dispensable forcanonical transferrin cargo. Additionally, our discovery that de-pletion of these factors increases the potency of the entry in-hibitor nanchangmycin suggests that nanchangmycin targets thissize-dependent pathway. The full spectrum of cargoes that aredependent on these proteins is unclear. Future studies will definethe rules of engagement. Nevertheless, given the potentiallynarrow list of cargoes dependent on these genes, the develop-ment of inhibitors active against this pathway, such as nan-changmycin, may represent a class of antiviral compounds, whichmay be effective against flaviviruses or other viruses that useclathrin-mediated endocytosis for entry.

Materials and MethodsPlease see SI Appendix, SI Materials and Methods for detailed descriptionof cell culture, viral infections, siRNA transfections, immunoblotting, RT-qPCR, microscopy, internalization assays, transferrin uptake, and IC50

analysis. siRNA and primer sequences are found in SI Appendix, TablesS1 and S2.

ACKNOWLEDGMENTS. We thank members of the S.C. laboratory fortechnical support and advice; Julie To for IC50 replicates, and Drs. DavidSchultz and Matthew Tudor for advice and IC50 analysis. We thank Dr. DerekWalsh for advice on EBs. This work was supported by NIH Grants R01AI074951,R01AI122749, and R01AI095500, the Linda Montague Investigator Award,ITMAT Pilot, and the Burroughs Wellcome Investigators in the Pathogenesisof Infectious Disease Award (to S.C.).

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