Influenza Virus Can Enter and Infect Cells in the Absence of Clathrin-Mediated Endocytosis

Influenza virus has been described to enter host cells via clathrin-mediatedendocytosis. However, it has also been suggested that otherendocytic routes may provide additional entry pathways. Herewe show that influenza virus may enter and infect HeLa cellsthat are unable to take up ligands by clathrin-mediated endocytosis.By overexpressing a dominant-negative form of the Eps15 proteinto inhibit clathrin-mediated endocytosis, we demonstrate thatwhile transferrin uptake and Semliki Forest virus infectionwere prevented, influenza virus could enter and infect cellsexpressing Eps15 ${Delta}$ 95/295. This finding is supported by the successfulinfection of cells with influenza virus in the presence of chemicaltreatments that block endocytosis, namely, chlorpromazine andpotassium depletion. We show also that influenza virus may infectcells incapable of uptake by caveolae. Treatment with the inhibitorsnystatin, methyl-ß-cyclodextrin, and genistein, aswell as transfection of cells with dominant-negative caveolin-1,had no effect on influenza virus infection. By combining inhibitorymethods to block both clathrin-mediated endocytosis and uptakeby caveolae in the same cell, we demonstrate that influenzavirus may infect cells by an additional non-clathrin-dependent,non-caveola-dependent endocytic pathway. We believe this tobe the first conclusive analysis of virus entry via such a non-clathrin-dependentpathway, in addition to the traditional clathrin-dependent route.

INTRODUCTION

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Introduction

Clathrin-mediated endocytosis has been described as an internalizationpathway for many viruses (for reviews, see references 31, 46,and 52). Along with Semliki Forest virus (SFV) and vesicularstomatitis virus, influenza virus has been observed in clathrin-coatedvesicles upon entry into host cells based on analysis by transmissionelectron microscopy (30, 33, 34). However, influenza virionswere also observed in uncoated vesicles in these experiments(33). It has remained unclear whether the presence of theseuncoated vesicles was due to shedding of the clathrin coat orto the existence of a non-clathrin-mediated endocytic pathway.It is also unclear if virions within these uncoated vesiclescould proceed to cause a productive infection. More recent studieshave examined influenza virus entry by using the K44A dynaminmutant in Mv-1 lung cells (45). It was originally thought thatdynamin, a small GTPase which mediates the pinching of endocyticvesicles, was specific to clathrin-mediated endocytosis (20,50). However, it has become clear that dynamin functions inseveral endocytic vesicle scission events, including the formationof caveolae, budding of Golgi complex-derived vesicles, phagocytosis,and non-clathrin-mediated endocytosis (15, 18, 24, 26, 39).Here we have used more-specific inhibitors of clathrin-mediatedendocytosis to determine the specific action of this pathwayon influenza virus and to examine if additional, alternativevirus entry pathways might exist.

Several endocytic pathways have been implicated previously inpathogen entry (46). In the case of influenza virus, all ofthese appear to have the capacity to deposit the virus in alow-pH compartment, which is required for hemagglutinin-mediatedviral fusion and uncoating (11, 53, 58). Clathrin-mediated endocytosisis the best characterized of these pathways and is consideredthe primary route of endocytic entry into cells (10). Althoughimplicated as a route of entry for many viruses, clathrin-mediatedendocytosis has to date been defined in molecular terms onlyas the internalization pathway for SFV and Sindbis virus (12,13). As an alternative to clathrin, caveolae have recently beendescribed as mediating the entry of simian virus 40 (SV40),and in this case the virus enters so-called caveosomes beforebeing transported to the endoplasmic reticulum (41). Other routesof virus internalization include macropinocytosis, which hasbeen suggested for vaccinia virus and Epstein-Barr virus entry.Macropinocytosis is considered a nonspecific mechanism of internalizationmediated by the closure of plasma membrane ruffles to form largevesicles known as macropinosomes (29). A fourth route of entry,as yet ill defined, is a non-clathrin-mediated, non-caveola-mediatedinternalization pathway (8, 38). This pathway has been implicatedin the entry of many viruses, including picornaviruses and influenzavirus, primarily based on electron microscopic analysis (28,33).

Assembly of clathrin into a coated pit occurs at the insideface of the plasma membrane in response to a receptor-mediatedinternalization signal. Clathrin is recruited to the plasmamembrane by the AP-2 adapter complex, and while the assemblyprocess is known to require many other proteins, the functionof most is not yet clear (32). One of these regulatory proteinsis Eps15, which was originally identified as a substrate forthe receptor-activated epidermal growth factor tyrosine kinase(14). Eps15 has since been found to be ubiquitously and constitutivelyassociated with AP-2, suggesting a role in clathrin assemblyregulation (5, 55). This hypothesis has been supported by theinhibition of transferrin and epidermal growth factor internalizationin cells microinjected with antibodies against Eps15 or transientlytransfected with dominant-negative forms of the protein (6,12).

Eps15 consists of three structural domains. The N-terminal domaincontains three EH (Eps homology) domains which bind regulatoryproteins containing NPF amino acid motifs, including epsin,AP180, and synaptojanin (47). The central domain allows foroligomerization of Eps15 into homodimers (55). The C-terminaldomain contains an AP-2 binding site which, together with theEH domains, is required for targeting of Eps15 to clathrin-coatedpits (4, 7, 21). It has been reported previously that overexpressionof Eps15 lacking the second and third EH domains results ina dominant-negative protein that inhibits clathrin-mediatedendocytosis (3). Expression of this construct, Eps15 ${Delta}$ 95/295,results in the mislocalization of AP-2 and dynamin, suggestingthat Eps15 may play a role in AP-2 recruitment to the plasmamembrane.

Here we have used the green fluorescent protein (GFP)-Eps15 ${Delta}$ 95/295construct to determine the role of clathrin-mediated endocytosisin influenza virus entry. We demonstrate that cells expressingthis construct, and thus disabled in clathrin-mediated uptake,allow the entry and infection of influenza virus, while SFVinfection and transferrin uptake are prevented. These data aresupported by experiments using chemical inhibition and potassiumdepletion to prevent clathrin-mediated endocytosis. Additionally,using a series of chemical inhibitors and dominant-negativecaveolin-1 to disrupt caveolae, we show that influenza virusinfection occurs in the absence of uptake by caveolae. By combiningmethods to inhibit both clathrin-mediated endocytosis and uptakeby caveolae in the same cell, we demonstrate that influenzavirus may enter and infect cells via a non-clathrin-mediated,non-caveola-mediated alternative endocytic pathway, in additionto entry by clathrin-mediated endocytosis.

MATERIALS AND METHODS

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Materials and Methods

Cells, viruses, and infections. HeLa cells (American Type Culture Collection) were maintainedin ${alpha}$ -minimal essential medium ( ${alpha}$ -MEM) containing 10% calf serum,100 U of penicillin/ml, and 10 µg of streptomycin/ml andwere passaged twice-weekly. Influenza virus A/WSN/33 (H1N1)was grown in MDBK cells and plaque titered on MDCK cells. SFVstrain M1 was provided by Margaret Kielian, Albert EinsteinCollege of Medicine. Infections were performed essentially asdescribed previously (43). Briefly, viral stocks were dilutedin RPMI 1680 medium containing 0.2% bovine serum albumin andbuffered to pH 6.8 with HEPES. Influenza virus was adsorbedfor 90 min at 4°C, and cells were then maintained in growthmedium containing 2% serum at 37°C for either 60 min (at $~$ 100 to 200 PFU per cell) or 4 h (at $~$ 1 or <0.1 PFU per cell).SFV was bound as above (at $~$ 1 PFU per cell) and then incubatedat 37°C for 5 h. The chemical inhibitors bafilomycin A (25nM; Alexis Biochemicals), chlorpromazine (10 µg/ml; Alexis),nystatin (25 µg/ml; Calbiochem), methyl-ß-cyclodextrin(MßCD) (10 mM; Sigma), and genistein (100 µg/ml;Alexis) were added during both the virus adsorption and incubationperiods.

Transfections. The dominant-negative, GFP-tagged plasmid construct Eps15 ${Delta}$ 95/295(3) was provided by Jennifer Lippincott-Schwartz, National Institutesof Health (37). Plasmid constructs of wild-type and dominant-negativecaveolin-1 were provided by Ari Helenius, ETH-Zurich. Transfectionswere performed by using the Effectene transfection kit (Qiagen)according to the manufacturer's protocols and routinely exceeded70% efficiency. For transfection, HeLa cells were grown on coverslipsin 24-well plates and transfected with 0.5 µg of DNA.Transfections were typically allowed to proceed for 16 to 18h before infection or transferrin uptake.

Indirect immunofluorescence microscopy. Cells were prepared for indirect immunofluorescence microscopyas described previously (59). Influenza virus nucleoprotein(NP) was detected by using monoclonal antibody H10, L16-4R5(American Type Culture Collection). SFV was detected with monoclonalantibody E1-1 (provided by Margaret Kielian). Eps15 expressionwas detected by using an anti-Eps15 monoclonal antibody (BDTransduction Laboratories). Secondary antibodies used were Alexa488-labeled (green), Alexa 568-labeled (red), and Alexa 633-labeled(far red) goat anti-mouse immunoglobulin G (IgG) (MolecularProbes). Cells were viewed by using a Zeiss Axioskop 2 plusmicroscope with a 63x objective lens. Images were captured witha Zeiss Axiocam using Axiovision 3.0.6.1 software before beingtransferred into Adobe Photoshop. Confocal microscopy was performedusing an Olympus FluoView confocal station. Alexa 488 was excitedwith the 488-nm line of an argon laser, Alexa 594 was excitedwith the 568-nm line of a krypton laser, and Alexa 633 was excitedwith the 635-nm line of a He-Ne laser. Cells were viewed witha 60x objective lens, and images were captured with FluoViewsoftware (Olympus) before being transferred into Adobe Photoshop.

Fluorescence-activated cell sorter (FACS) analysis. For flow cytometry preparation, cells were trypsinized, washedin phosphate-buffered saline (PBS), fixed in 3% paraformaldehyde-PBS,and permeabilized in 0.075% saponin in 10% goat serum-PBS. Cellswere incubated with a monoclonal antibody to influenza virusNP or Eps15 for 30 min, followed by Alexa 488-labeled or Alexa568-labeled goat anti-mouse IgG for 30 min. Cells were analyzedon a FACSCalibur cytometer using CellQuest 3.1f software (BectonDickinson Immunocytometry Systems). At least 10,000 cells wereanalyzed for each sample.

Transferrin uptake. Transferrin uptake assays were performed using Alexa 594-labeledhuman transferrin (kindly provided by Colin Parrish, CornellUniversity). HeLa cells were serum starved for 30 min, incubatedwith 50 µg of Alexa 594-labeled transferrin/ml for 20min at 4°C for binding, washed, and transferred to 37°Cfor 15 min. Cells were washed with 0.1 M glycine-0.1 M NaCl,pH 3.0, to remove any uninternalized ligand, unless stated otherwise.

K⁺ depletion. Potassium depletion was performed as described previously (27).Briefly, HeLa cells were shocked in a hypotonic medium for 5min. Virus or transferrin was bound and internalized in thepresence of an isotonic K⁺-free medium ( ${alpha}$ -MEM with 50 mM Na-HEPESand 100 mM NaCl [pH 7.4]).

RESULTS

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Results

Overexpression of GFP-Eps15 ${Delta}$ 95/295 prevents transferrin uptake. To determine the overexpression efficiency of the Eps15 ${Delta}$ 95/295plasmid in our cell culture and transfection system, we comparedEps15 expression in transfected and untransfected cells by usinga monoclonal antibody against Eps15. Analysis by flow cytometryindicated that Eps15 expression in transfected cells increasedapproximately 1.8 times over that in untransfected cells, basedon the median signal expression determined after adjustmentfor 70% transfection efficiency (Fig. 1A).

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FIG. 1. GFP-Eps15 ${Delta}$ 95/295 is overexpressed and prevents transferrin uptake. (A) HeLa cells were either transiently transfected with GFP-Eps15 ${Delta}$ 95/295 or left as untransfected controls. Cells were prepared for FACS analysis by using the monoclonal antibody against Eps15 and an Alexa 568-labeled secondary antibody. Shaded curve, untransfected cells; solid curve, transfected cells. Numbers indicate median fluorescence expression as determined by FluoView software. At least 10,000 cells were counted. (B) HeLa cells were transiently transfected with either GFP-Eps15 ${Delta}$ 95/295 or GFP alone. Alexa 594-labeled transferrin (Tfn) was bound to serum-starved cells for 20 min at 4°C. Cells were washed, transferred to 37°C for 15 min, washed with low-pH glycine to remove uninternalized ligand, and fixed for viewing. (C) Transferrin uptake quantitated by indirect immunofluorescence. One hundred cells from each category were counted, and residual surface transferrin was distinguished from a strong perinuclear signal.

To further test the efficacy of the plasmid in our system, wetransiently transfected HeLa cells, using GFP alone as a positivecontrol, and then performed a transferrin uptake assay. It hasbeen reported previously that expression of Eps15 ${Delta}$ 95/295 preventstransferrin internalization (3). Our results agreed with thisfinding, since cells transfected with GFP allowed normal transferrinuptake (Fig. 1B, panel d), whereas cells expressing GFP-Eps15 ${Delta}$ 95/295were unable to internalize the ligand (panel c). Quantificationof transferrin uptake indicated that among GFP-transfected oruntransfected control cells, at least 90% displayed strong perinuclearsignals, indicative of transferrin in recycling endosomes (Fig.1C). In contrast, in approximately 90% of cells transfectedwith Eps15 ${Delta}$ 95/295, transferrin was undetectable or remained asa residual signal on the surface after the low-pH wash. Thesedata indicate that the Eps15 ${Delta}$ 95/295 construct is well expressedin our transfection system, resulting in inhibition of liganduptake by clathrin-mediated endocytosis.

Overexpression of GFP-Eps15 ${Delta}$ 95/295 prevents SFV infection. Entry of SFV has been demonstrated to be dependent on clathrin-mediatedendocytosis by injection of anti-clathrin antibodies, whichprevented infection (12). Therefore, we employed SFV as a clathrin-dependentvirus with which to compare our subsequent influenza virus results.After transfection with GFP or GFP-Eps15 ${Delta}$ 95/295, cells were infectedwith SFV for 5 h before examination by indirect immunofluorescenceor FACS analysis. As with infection in untransfected controlcells, cells transfected with GFP were infected with SFV, asindicated by the strong cytoplasmic antibody signal (Fig. 2A,panel b). However, cells expressing Eps15 ${Delta}$ 95/295 were not infectedwith SFV (Fig. 2A, panel a), due to inhibition of clathrin-mediatedentry. Rarely, a cell appeared to be both transfected and infected(Fig. 2A, panel a). We attribute this to the long infectiontime required and unavoidable residual wild-type Eps15 protein.The percentage of these double-labeled cells viewed under themicrocope correlates with the data obtained by FACS analysis.The immunofluorescence data were quantified by flow cytometry,which allows distinctions between infected versus noninfectedcells and transfected versus nontransfected cells. When nontransfectedcells were infected with SFV, 80% of the cell population expressedan E1-1 signal above background, indicating a large infected-cellpopulation (Fig. 2B). As a monitor of transfection efficiency,transfection with GFP alone resulted in more than 66% of thecells expressing the fluorescent tag. Cells transfected withGFP and infected with SFV showed a major shift (more than 45%of the cell population) to the upper right-hand quadrant, representingcells that were both transfected and infected. Conversely, cellsexpressing Eps15 ${Delta}$ 95/295 did not demonstrate this population shift,as indicated by the fact that only 6% of the cell populationwas located in the upper right-hand quadrant, confirming thatSFV infection is inhibited.

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FIG. 2. SFV infection is prevented in GFP-Eps15 ${Delta}$ 95/295-expressing cells. HeLa cells either were transiently transfected with either GFP or GFP-Eps15 ${Delta}$ 95/295 or were left as untransfected controls before 5 h of infection. Infection was monitored using monoclonal antibody E1-1. (A) Indirect immunofluorescence monitoring of infection. Arrows indicate uninfected cells; arrowhead indicates a transfected, infected cell. (B) FACS analysis to quantitate immunofluorescence results. Infection is monitored on the y axis, and transfection is monitored on the x axis. The percentage of cells within each quadrant is given.

Influenza virus infection is not inhibited by GFP-Eps15 ${Delta}$ 95/295 expression. Entry of influenza virus into MDCK, BHK, and Mv-1 lung cellsby clathrin-mediated endocytosis has been well described. However,we wished to determine whether additional entry pathways couldexist. To examine this, we blocked clathrin-mediated uptakewith Eps15 ${Delta}$ 95/295 and monitored influenza virus entry and infection.As in transiently transfected cells expressing GFP alone, influenzavirus was capable of entering cells expressing GFP-Eps15 ${Delta}$ 95/295and of releasing viral RNPs (vRNPs) into the nucleus, as indicatedby the strong nuclear vRNP signal (Fig. 3A). To examine productiveinfection, cells were allowed to be infected at a low multiplicityof infection (MOI) of approximately 1 PFU/cell for 4 h aftertransfection. Cells expressing Eps15 ${Delta}$ 95/295 were capable of beinginfected by influenza virus, as indicated by the similar vRNPsignals in GFP-expressing and control cells (Fig. 3B). To guardagainst the possibility that Eps15 ${Delta}$ 95/295 was providing onlypartial inhibition of clathrin-mediated endocytosis, and thatextraneous virions were eluding this block, the 4-h infectionwas repeated at a very low MOI (<0.1 PFU per cell). As withthe above assays, Eps15 ${Delta}$ 95/295-expressing cells were infectedwith influenza virus (Fig. 3C). The data for the 4-h low-MOIinfection were quantified by flow cytometry and are summarizedin Fig. 3D. In GFP-transfected cells, 36% of the cell populationwas both transfected and infected, and such a cell populationrepresented 44% of the Eps15 ${Delta}$ 95/295-transfected cells (Fig. 3D).It is not clear at present why Eps15 ${Delta}$ 95/295 would result in thisapparent increase in influenza virus infectivity (36% versus44%). These data show that influenza virus is capable of enteringand causing a productive infection in cells lacking a functionalclathrin-mediated endocytic pathway.

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FIG. 3. GFP-Eps15 ${Delta}$ 95/295 expression does not prevent influenza virus infection. HeLa cells either were transiently transfected with either GFP or GFP-Eps15 ${Delta}$ 95/295 or were left as untransfected controls before infection with influenza virus. Infection was monitored using monoclonal antibody H10, L16-4R5 against NP. (A) Entry assay. Cells were infected with $~$ 100 to 200 PFU per cell, and the virus was allowed to internalize for 60 min at 37°C. (B) Infection. Cells were infected with $~$ 1 to 5 PFU per cell and then incubated at 37°C for 4 h. (C) Low-MOI infection. Cells were infected with $~$ 0.1 PFU per cell and incubated at 37°C for 4 h. (D) FACS analysis of infection assay. Infection is monitored on the y axis, and transfection is monitored on the x axis. The percentage of cells within each quadrant is given.

Confocal microscopy of Eps15 ${Delta}$ 95/295-transfected cells. To demonstrate the inhibitory effects of Eps15 ${Delta}$ 95/295 expressionon transferrin uptake and the ability of influenza virus toovercome this inhibition, we performed confocal microscopy oncells that were transfected, infected, and then exposed to transferrin(Fig. 4). In nontransfected cells, the influenza virus vRNPsignal was present primarily in the nucleus (Fig. 4c). Transferrinwas internalized and concentrated in the perinuclear regionof the cell (Fig. 4b). In transfected cells, vRNPs were againpresent in the nucleus; however, transferrin remained on thesurface of the cell, unable to be internalized (Fig. 4b). Asthe plane of view is through the nuclear region of the cell,the plasma membrane surface above the nucleus has been eliminated,accounting for the lack of transferrin in this area. It is interestingthat much of the uninternalized transferrin appears to colocalizewith Eps15 ${Delta}$ 95/295 (Fig. 4d), as though it has bound to nonfunctioningsites of clathrin-mediated endocytosis at the plasma membrane.

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FIG. 4. Confocal microscopy of GFP-Eps15 ${Delta}$ 95/295-expressing cells with influenza virus and transferrin (Tfn). HeLa cells were transiently transfected with GFP-Eps15 ${Delta}$ 95/295 before high-MOI infection with influenza virus. The transferrin uptake assay was then performed; note that the cells were not acid washed after incubation. Influenza virus localization was determined by indirect immunofluorescence with an anti-NP monoclonal antibody. Images were created using Fluoview software and were transferred into Adobe Photoshop. The short arrow in panel b and the long arrow in panel c indicate transferrin and influenza vRNP signal localization, respectively, in untransfected cells.

Influenza virus infects in the presence of chlorpromazine. To provide additional support for our Eps15 data, we employedchemical and physiological inhibitors of clathrin-mediated endocytosis.We first inhibited clathrin by treating cells with chlorpromazine,which induces the misassembly of clathrin-coated pits at theplasma membrane (57). Cells were treated with chlorpromazineor bafilomycin A, a vacuolar ATPase inhibitor that blocks endosomalacidification and prevents endocytic trafficking during entryof pH-dependent viruses such as influenza virus or SFV (9, 16),and then were analyzed by flow cytometry. The control SFV infection(without any drug) showed more than 54% of the cells expressinga signal above background (mock) (Fig. 5A). The percent infectedcells dropped to approximately 1% with the addition of bafilomycinA and to 2% upon chlorpromazine treatment. The control influenzavirus infection resulted in 77% of the cells being infected(Fig. 5B). Treatment with bafilomycin A caused the infectionrate to drop to 1%, while chlorpromazine treatment allowed 41%of the cells to be infected with the influenza virus. The discrepancybetween the numbers of infected cells in the control and chlorpromazinetreatment infections is likely due to the high toxicity of thedrug.

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FIG. 5. Chlorpromazine treatment does not inhibit influenza virus infection. (A) HeLa cells were infected with SFV for 5 h, and infection was monitored using monoclonal antibody E1-1. (B) HeLa cells were infected with influenza virus for 4 h, and infection was monitored using an anti-NP monoclonal antibody. Cells were treated with either 25 nM bafilomycin A or 10 µg of chlorpromazine/ml during both the adsorption and incubation periods. Infection was monitored by FACS analysis. Key indicates treatment of cells. Background was determined by residual antibody staining on uninfected cells.

Influenza virus infects despite K⁺ depletion. As an alternative to chlorpromazine, potassium depletion combinedwith hypotonic shock has been used as a possibly more specificand less toxic means of inhibiting clathrin-mediated endocytosis.Such treatment has been well established to reversibly arrestclathrin-coated-pit formation (27). For examination of virusentry, cells underwent a brief hypotonic shock before infectionor transferrin uptake in an isotonic K⁺-free medium. This treatmentprevented transferrin uptake (Fig. 6A, panel b) but had no effecton influenza vRNP nuclear entry (panel c) or productive virusinfection at either 1 or <0.1 PFU/cell (panels e and g).FACS analysis of potassium-depleted cells confirmed the immunofluorescencedata: 81% of K⁺-depleted cells were infected in an entry assay,compared to 86% of untreated cells (Fig. 6B). A control experimentwith SFV demonstrated that potassium depletion prevents theentry of clathrin-dependent viruses, since SFV infection droppedfrom 39 to 9% upon treatment (Fig. 6C). These data, along withthe chlorpromazine results, support our findings from Eps15 ${Delta}$ 95/295transfection experiments that influenza virus may enter andinfect cells in the absence of clathrin-mediated endocytosis.

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FIG. 6. K⁺ depletion does not inhibit influenza virus infection. (A) HeLa cells were shocked in a hyptonic medium for 5 min before either an influenza vRNP entry assay, a 4-h infection (1 PFU/cell), a low-MOI infection (<0.1 PFU/cell), or a transferrin (Tfn) uptake assay in a K⁺-free medium. Influenza virus infection was detected by indirect immunofluorescence with a monoclonal antibody against NP. Note that cells were not acid washed after transferrin uptake. (B) FACS analysis. HeLa cells were shocked in a hyptonic medium for 5 min before an influenza virus entry assay in a K⁺-free medium. Influenza virus was detected by a monoclonal antibody against NP. (C) FACS analysis of SFV infection in cells depleted of potassium. HeLa cells were shocked in a hypotonic medium for 5 min before 5 h of infection with SFV in a K⁺-free medium. Virus infection was detected by monoclonal antibody E1-1.

Influenza virus infection is independent of caveolae. Although caveolae have recently been described as an internalizationpathway for several pathogens (51), their role in influenzavirus entry has not been addressed. To examine the role of thispathway, cells were treated with caveola-inhibiting drugs duringinfection. Nystatin and MßCD are sterol-binding drugsthat sequester cholesterol and are not believed to affect clathrin-mediatedendocytosis (36, 44). We treated cells with concentrations ofthese drugs known to deplete the cholesterol content of HeLacell plasma membranes by 60% (54). Genistein is a tyrosine kinaseinhibitor which is thought to inhibit the receptor-induced formationof caveolae (40). Drug-treated cells were monitored for infectionboth by FACS analysis after an entry assay infection and byindirect immunofluorescence after both an entry assay (datanot shown) and a 4-h infection. Immunofluorescence analysisshows that drug-treated cells contain a strong nuclear vRNPstaining pattern, characteristic of influenza virus-infectedcells (Fig. 7A). The vRNP staining pattern in genistein-treatedcells is slightly different, containing some additional cytoplasmicstaining. Although the cells are clearly infected, we do notyet know if this difference is relevant or is due to toxic effectsof the drug. Quantitative FACS analysis shows that the percentageof infected cells is not reduced by drug treatment (Fig. 7B),since at least 96% of cells treated with nystatin or genisteinexpressed vRNP signals above background and 70% of cells treatedwith MßCD were infected with influenza virus. Thisinfection rate is comparable to that of untreated control cellsand indicates that influenza virus does not require uptake bycaveolae for infection. As a more specific way to examine uptakeby caveolae, cells were transfected with GFP-tagged wild-typeor dominant-negative forms of caveolin-1, the major structuralprotein of caveolae (kindly provided by Ari Helenius). In bothcases, cells displayed normal influenza virus infection whenanalyzed by indirect immunofluorescence (Fig. 7C) and flow cytometry(data not shown). To confirm the inhibition of caveolar endocytosis,we analyzed HeLa cells for the ability to be infected by SV40,which is known to use caveolae for productive entry. SV40 infectionwas prevented by transfection with the dominant-negative caveolin-1construct but was unaffected by overexpression of wild-typecaveolin (data not shown), confirming that our protocol forinhibiting caveolar uptake is functioning properly.

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FIG. 7. Treatment with caveola-inhibiting drugs does not prevent influenza virus infection. HeLa cells were treated with either 25 µg of nystatin/ml, 10 mM MßCD, or 100 µg of genistein/ml during both the adsorption and incubation periods of influenza virus infection. Influenza virus infection was detected with a monoclonal antibody against NP. (A) Indirect immunofluorescence analysis of 4-h influenza virus infection in drug-treated cells. (B) FACS analysis of entry assay infection in drug-treated cells. Key indicates treatment of cells. Background was determined by residual antibody staining on uninfected cells. (C) HeLa cells were transiently transfected with wild-type or dominant-negative caveolin-1 constructs before entry assay infection with influenza virus. Transfected cells are indicated by GFP expression; infection was monitored via indirect immunofluorescence using a monoclonal antibody against NP.

Influenza virus infects cells in which both clathrin-mediated endocytosis and uptake by caveolae are inhibited. Our data suggested that influenza virus was unaffected by inhibitionof either the clathrin or the caveolar route of internalization.However, to determine if influenza virus could truly employa non-clathrin-mediated, non-caveola-mediated endocytic pathway,both of these routes needed to be inhibited within the samecell. To do this, inhibitory protocols were combined so thatcells were first transiently transfected with either GFP orGFP-Eps15 ${Delta}$ 95/295 and then treated with either nystatin, MßCD,or genistein. Influenza virus infection was monitored by flowcytometry (Fig. 8). As with each individual inhibition experiment,influenza virus was able to infect cells in which both clathrin-mediatedendocytosis and uptake by caveolae were disabled. Transfectionwith Eps15 ${Delta}$ 95/295 followed by treatment with 25 µg of nystatin/mlresulted in 40% of cells expressing both GFP-Eps15 ${Delta}$ 95/295 andan influenza virus vRNP signal. Eps15 ${Delta}$ 95/295-transfected cellstreated with 100 µg of genistein/ml were infected at therate of 23% of the cell population. These percentages are comparableto that for transfection with GFP alone before drug treatment.Similar results were observed after treatment with MßCD(data not shown). It is also significant that in all cases oftransfection and drug-treatment, fewer than 4% of cells remainedtransfected alone, indicating that almost all available transfectedcells were being infected by the influenza virus. Also, theresults of the reciprocal experiment, in which cells were transientlytransfected with dominant-negative caveolin-1 and treated withchlorpromazine during infection, support the above data (datanot shown). These results indicate that influenza virus is capableof using a non-clathrin-mediated, non-caveola-mediated endocyticpathway for entry and infection of host cells, in addition tothe traditional clathrin-mediated endocytic route.

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FIG. 8. Inhibition of both clathrin-mediated endocytosis and uptake by caveolae does not prevent influenza virus infection. HeLa cells were transiently transfected with either GFP or GFP-Eps15 ${Delta}$ 95/295. Cells were treated with either 25 µg of nystatin/ml or 100 µg of genistein/ml during a high-MOI infection with influenza virus. Infection was monitored by FACS analysis using a monoclonal antibody against NP. Infection is monitored on the y axis, and transfection is monitored on the x axis. The percentage of cells within each quadrant is given.

DISCUSSION

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Discussion

We demonstrate here that in addition to clathrin-mediated endocytosis,influenza virus may enter and infect HeLa cells by a non-clathrin-mediated,non-caveola-mediated internalization pathway. Through transienttransfection with a dominant-negative acting Eps15 construct,we demonstrate that while clathrin-dependent transferrin andSFV are prohibited from entering cells, internalization of influenzavirus is unaffected. This finding is supported by treatmentof cells with chlorpromazine and by exposure to potassium depletion.Potassium depletion has been used to investigate the role ofclathrin-mediated endocytosis in rhinovirus infection (2, 28),and chlorpromazine has been used previously to demonstrate clathrin-mediateduptake for the polyomavirus JC virus, parechovirus 1, and Epstein-Barrvirus (23, 35, 42). Although chlorpromazine has been shown toreduce influenza virus yields by 2 to 3 log PFU in chicken embryofibroblasts (25), this may be due to the severe toxic effectsof the drug itself or disruptions in viral replication ratherthan a direct effect on entry. Also, by employing dominant-negativeacting caveolin-1 and a tyrosine kinase inhibitor, we demonstratethat influenza virus does not require caveolae for entry, andby use of selective sterol-binding agents, we demonstrate thatinfluenza virus does not seem to depend on specific cholesterol-richlipid microdomains. By combining inhibitory protocols, we showthat influenza virus may infect cells deficient in both clathrin-mediatedendocytosis and uptake by caveolae. Again, we believe that thesedata demonstrate an alternative entry pathway for influenzavirus in the form of a non-clathrin-dependent route, in additionto clathrin-mediated endocytosis, as described previously (33).

Such a non-clathrin-dependent pathway has been implicated invirus entry before, but its nature is still ill defined (8,38). As yet, no specific means to inhibit this pathway exists,and therefore it must remain as a default route when all otherendocytic routes have been disabled. However, several recentreports have described entry by this non-clathrin-dependent,non-caveola-dependent alternative pathway. Expression of Eps15 ${Delta}$ 95/295in caveola-deficient T lymphocytes has been shown not to preventuptake and delivery to lysosomes of interleukin-2 (IL-2) (26).The IL-2 entry pathway has been characterized as requiring dynamin,like clathrin-mediated endocytosis and caveolae, and occurringon detergent-resistant membranes (DRMs). Other studies haveexamined the uptake of glycosylphosphatidylinositol (GPI)-anchoredproteins via a pathway that may bypass the endosomal systemto deliver contents to the Golgi apparatus (37). The GPI anchorhas also been examined in the context of diphtheria toxin. ThisGPI-linked toxin can enter epithelial cells deficient in bothclathrin-mediated and caveolar uptake (54). In contrast to IL-2,entry of diphtheria toxin occurs independently of dynamin, andsince sterol-binding drugs do not affect diphtheria toxin entry,it appears to be independent of DRMs.

That influenza virus infects in the presence of the sterol-bindingdrugs nystatin and MßCD suggests that virus entryis independent of DRMs, and we are currently investigating thisfurther. This possibility is supported by recent data demonstratingthat the influenza virus hemagglutinin fusion peptide does notassociate with DRMs (1). However, it remains possible that anovel and to date uncharacterized type of DRM, which is insensitiveto the drug treatments used here, is involved in uptake. Inaddition, it is clear that use of pharmocological treatmentsalone makes it difficult to draw conclusions on microdomainfunction at the plasma membrane (22). The use of dominant-negativecaveolin allows a firm conclusion to be drawn about the roleof caveolin in influenza virus entry, but the role of otherlipid microdomains remains an open question at present. Thetransmembrane domain of hemagglutinin does, however, appearto have a known affinity for DRMs with regard to virus buddingfrom the host cell plasma membrane (48, 49), and such DRMs withinthe virus envelope are likely to affect the fusion propertiesof the virus.

We have demonstrated previously, by using the K44A dynamin mutantin Mv-1 lung cells, that influenza virus entry is dependenton dynamin. Although dynamin activity was previously thoughtto be specific to clathrin-mediated endocytosis, it is now clearthat this small GTPase functions in a variety of endocytic vesiclescission events (15, 18, 24, 39), and it has been describedas a essential component of non-clathrin-mediated endocytosis(26). Although we have not yet differentiated the roles of dynaminin influenza virus entry via clathrin- versus non-clathrin-mediatedendocytosis, it is quite likely that dynamin plays a crucialrole in both endocytic pathways. Although we have yet to examinethe role of dynamin in influenza virus entry in a HeLa cellsystem, we would expect that entry is similarly inhibited bydominant-negative dynamin. A non-clathrin- and non-caveola-dependent,dynamin-dependent pathway characterized for influenza virusmay represent a novel alternative internalization pathway. Togetherwith data for other ligands, this leads to the suggestion thatthere may be multiple non-clathrin-mediated endocytic routes,which may vary in requirements for entry and trafficking withinthe cell. Additionally, there may be cell type differences inthe relative action or abundance of the endocytic pathways discussedhere, and such differences may have effects on the preferredroute of entry for influenza virus. The next step will be todetermine which endocytic vesicles the influenza virus is traffickedthrough when it enters cells by clathrin-mediated endocytosisand by its alternative route of entry. It can be hypothesizedthat the trafficking events from both entry pathways will besimilar to that already proposed for influenza virus—passagethrough early or sorting endosomes followed by fusion from lateendosomes in a pH-dependent manner.

As yet, it is not clear why and how influenza virus has thecapacity to enter cells through multiple pathways. One obviouspossibility relates to the nonspecific influenza virus receptorsialic acid, which can take the form of either a glycoproteinor a glycolipid (19). Other viruses that have been describedas entering cells solely by clathrin-mediated endocytosis havealso been determined to have specific receptors for binding.For instance, SFV is known to bind to major histocompatibilitycomplex class I molecules (17), while Sindbis virus binds tolaminin receptor (56). Binding to these specific receptors maytarget the virus to a particular pathway of entry, in this caseclathrin-mediated endocytosis. Influenza virus, due to its nonspecificbinding, may lack this targeting, and this may, in turn, leadto multiple pathways of entry. In this regard, influenza virusmay share common properties with Shiga toxin, which binds theglycosphingolipid receptor globotriaosyl ceramide and has beenshown to enter cells by both clathrin-dependent and -independentpathways (for a review, see reference 22).

Taken together, our data would indicate that influenza virusmay enter cells by a non-clathrin-mediated, non-caveola-mediatedendocytic pathway, in addition to entry by clathrin-mediatedendocytosis. It is therefore possible that other viruses mayhave multiple entry pathways that could be dependent on receptorbinding or cell type.

ACKNOWLEDGMENTS

We thank Benjamin Briggs for preliminary work on caveolin constructsand SV40 control experiments. We thank Ari Helenius for helpfuldiscussions and for providing reagents. We also thank JenniferLippincott-Schwartz, Colin Parrish, and Margaret Kielian fortheir kind contributions of reagents.

S.B.S. was supported by a Training Grant from the National Institutesof Health (T32AI07618). This work was supported by a ScientistDevelopment Grant from the American Heart Association and bygrant R01AI48678 from the National Institutes of Health (toG.R.W.).

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Veterinary Medical Center, Cornell University, Ithaca, NY 14853. Phone: (607) 253-4019. Fax: (607) 253-3384. E-mail: grw7{at}cornell.edu.

REFERENCES

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