Adenoviruses Use Lactoferrin as a Bridge for CAR-Independent Binding to and Infection of Epithelial Cells

JOURNAL OF VIROLOGY, Jan. 2007, p. 954–963 Vol. 81, No. 20022-538X/07/$08.00�0 doi:10.1128/JVI.01995-06Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Adenoviruses Use Lactoferrin as a Bridge for CAR-IndependentBinding to and Infection of Epithelial Cells�

Cecilia Johansson,1 Mari Jonsson,1 Marko Marttila,1 David Persson,1 Xiao-Long Fan,2 Johan Skog,1Lars Frangsmyr,3,4 Goran Wadell,1 and Niklas Arnberg1*

Departments of Virology,1 Anatomy,3 and Sports Medicine,4 Umeå University, Umeå, Sweden, and Department ofMolecular Medicine and Gene Therapy, Lund University, Lund, Sweden2

Received 13 September 2006/Accepted 24 October 2006

Most adenoviruses bind to the coxsackie- and adenovirus receptor (CAR). Surprisingly, CAR is not ex-pressed apically on polarized cells and is thus not easily available to viruses. Consequently, alternativemechanisms for entry of coxsackievirus and adenovirus into cells have been suggested. We have found that tearfluid promotes adenovirus infection, and we have identified human lactoferrin (HLf) as the tear fluid compo-nent responsible for this effect. HLf alone was found to promote binding of adenovirus to epithelial cells in adose-dependent manner and also infection of epithelial cells by adenovirus. HLf was also found to promotegene delivery from an adenovirus-based vector. The mechanism takes place at the binding stage and functionsindependently of CAR. Thus, we have identified a novel binding mechanism whereby adenovirus hijacks HLf,a component of the innate immune system, and uses it as a bridge for attachment to host cells.

Human adenoviruses (Ads) cause disease of the respiratorytract, intestine, eyes, liver, and urinary tract and in lymphoidtissue (23). The most commonly isolated adenovirus serotypesbelong to species C (Ad1, Ad2, Ad5, and Ad6) and causeroughly 5% of all symptomatic upper respiratory tract infec-tions (8) and 15% of all lower respiratory tract infections (4) inchildren younger than 5 years. Early studies documented theisolation of species C adenoviruses following explant of humantonsils and adenoid tissue for culture (46), and it has beenshown that species C adenoviruses are latent in T lymphocytesisolated from human tonsils (17).

With few exceptions, adenoviruses bind to target cells bymeans of an interaction between the viral fiber and its cellularreceptor (34), whereas an interaction between the viral pentonbase protein and cellular integrins promotes internalization(52). It has been concluded that selected members of all ade-novirus species except species B use the coxsackievirus andadenovirus receptor (CAR) as a cellular receptor (6, 35, 43).Most, but not all, species B adenoviruses use CD46 as a cel-lular receptor instead of CAR (15, 25, 36).

When CAR was initially identified as a cellular receptor forhuman adenoviruses, the experiments were performed usingnonpolarized cell lines in vitro. In these model systems, CARis distributed equally over the cell surface and is available toviruses. In vivo, however, epithelial cells are polarized andCAR is not readily available to virions (50). Moreover, inpolarized epithelium, CAR forms lateral homodimers intercel-lularly, regulates cell-to-cell adhesion, and facilitates viraltransport between cells in order to promote escape from thesite of replication, rather then being involved in virus bindingto target cells (49).

The poor ability of adenoviruses to reach their cellular re-

ceptor on polarized epithelial cells has become evident fromwork aimed at developing vectors for human gene therapy.Adenovirus vectors proved to be inefficient due to the lowaccessibility of CAR on polarized airway cells (31). Thus, it hasbeen suggested that, alternative, apically expressed moleculesare used as cellular receptors by adenovirus and coxsackievirus(11, 49, 50). In agreement with this, the roles of decay accel-erating factor (DAF), which was first identified as a cellularreceptor for coxsackie B viruses in 1995 (38), and CAR wasrecently investigated in detail during the early steps of cox-sackie B virus replication. DAF was found to serve as the mainattachment receptor for coxsackie B virus, whereas CAR wasfound to be involved in a subsequent step of the coxsackie Bvirus life cycle (12).

Before finding a suitable receptor, every virus has to avoid orovercome the innate immune defense present in the skin, mu-cosal layers, and body fluids. Human lactoferrin (HLf) ispresent in mucosa and most body fluids (51) and plays a role inthe first line of defense against microbial infections (45). Theantiviral effects of HLf are mainly related to inhibition of virusentry into host cells, either by binding to viral ligands, such asgp120 of human immunodeficiency virus (41), or by binding tocellular receptors, such as heparan sulfate glycosaminoglycans(24). In contrast, HLf has also been demonstrated to transac-tivate the long terminal repeat promoter of human T-lympho-tropic leukemia virus 1, which is transmitted vertically throughbreast milk (28). Thus, in addition to having antiviral proper-ties, HLf may also have the capacity to promote infection bycertain viruses.

In this study, we set out to investigate the role of tear fluidin ocular adenovirus infections. We found an unexpected effectof tear fluid during infections by respiratory adenoviruses anddecided to investigate this effect further.

MATERIALS AND METHODS

Cells and viruses. Human epithelial lung carcinoma A549 cells and epitheliallarynx carcinoma Hep2 cells were grown in Dulbecco’s modified Eagle’s medium

* Corresponding author. Mailing address: Department of Virology,Umeå University, Umeå SE-90185, Sweden. Phone: 46 90 7858440.Fax: 46 90 129905. E-mail: [email protected].

� Published ahead of print on 1 November 2006.

954

(DMEM; Sigma-Aldrich) containing NaHCO3 (0.75 g/liter), 10% fetal calf se-rum (FCS; Sigma-Aldrich), 20 mM HEPES (Euroclone), and penicillin-strepto-mycin (Invitrogen) at 37°C. Human corneal epithelial (HCE) cells were grown insteroid-supplemented hormone medium (SHEM) as described previously (1).Acute lymphoblastic T-cell leukemia CEM and KE37 cells and CAR cDNA-transfected CEM and KE37 cells (CEM-CARhi and KE37-CARhi) were grownas described previously (26).

Adenovirus type 31 (Ad31; strain 13-15/63), Ad7 (Gomen), Ad11 (Slobitski),Ad1 (strain Ad71), Ad2 (strain Ad6), Ad5 (strain Ad75), Ad6 (Tonsil 99), Ad37(1477), Ad4 (RI-67), and Ad41 (Tak) were propagated in A549 or Hep2 (Ad41)cells and purified as described elsewhere (27), with the following exception:instead of treating the cells with sonication and Arclone extraction, the cells werebroken by three cycles of freeze-thawing.

Fluorescent focus assay. Human tear fluid was induced with freshly mincedonions and collected from volunteers in the laboratory. Five or sometimes 10 �lof tear fluid was mixed with virus (Ad37, 1.4 � 109 virions; Ad5, 1.8 � 108

virions), together with 500 �l SHEM (containing 1% FCS). In other experiments,tear fluid was replaced by various concentrations of HLf (Sigma), lysozyme(Sigma), or lipophilin (a kind gift of Robert Lehrer), as indicated in the figurelegends. After incubation for 1 hour on ice, the mixtures were added to subcon-fluent cells in 24-well plates (Nunc) and incubated for another hour on ice.Unbound virions were removed by a two-step wash with SHEM (1% FCS).When experiments were performed with A549 or Hep2 cells, DMEM (including1% FCS, penicillin-streptomycin, and 20 mM HEPES) was used instead ofSHEM in all steps. The amount of virions that was added to each well wasadapted to obtain approximately 10 to 20 infected cells in each viewfield (in theabsence of effectors) and ranged from 4.2 � 106 virions/well (Ad5 and Ad6; A549and Hep2 cells) to 2.6 � 109 virions/well (Ad41; A549). After 44 h of incubationat 37°C, the cells were fixed with methanol (400 �l/well) and stained first withhomotypic rabbit antiadenovirus serum, produced as described elsewhere (48)and diluted 200 to 1,000 times in phosphate-buffered saline (PBS; Medicago),and then with fluorescein isothiocyanate (FITC)-conjugated swine anti-rabbitantibodies (Dako Cytomation) diluted 1:50 in PBS. Incubations with antibodieswere performed in a final volume of 400 �l for 1 h at room temperature, and allwashes were done in PBS twice for 15 min. When Ad5CMVeGFP vectors(Baylor College of Medicine) or enhanced green fluorescent protein (eGFP)-expressing Ad5 vectors pseudotyped with the Ad35 fiber (Ad5F35-GFP) (30)were used instead of virions, 104 vector particles were added per cell and 2%paraformaldehyde (J.T. Baker) was used for fixation. Fluorescing cells wereexamined and quantified using a fluorescence microscope (Axioskop2; Zeiss)at �10 magnification and linked to a digital camera (AxioCam MRm; Zeiss) andAxiovision AC software (Zeiss).

Western blotting. Tear fluid proteins, and also HLf, lysozyme, and lipophilin,were separated on a 4 to 20% Criterion precast gradient gel (Bio-Rad) underdenaturing conditions and transferred to a polyvinylidene difluoride membrane(Hybond-P; Amersham) using a Trans-Blot SD semidry transfer cell (Bio-Rad).The membrane was blocked with PBS in 5% nonfat dry milk (Semper) overnightat 4°C. After washing three times for 5 min in PBS-Tween (0.05% Tween;Medicago), the membrane was incubated with 9 � 109 Ad5 virions diluted in 10ml PBS-Tween containing 1% nonfat dry milk (Bio-Rad). After 1 hour ofincubation at room temperature with constant agitation, rabbit anti-Ad5 serum(diluted 1:2,000) in PBS-Tween and 1% nonfat dry milk was added to themembrane. The membrane was then incubated and washed as before and furtherincubated with horseradish peroxidase-conjugated swine anti-rabbit antibodies(Dako Cytomation), diluted 1:5,000 in PBS-Tween with 1% nonfat dry milk.After another round of incubation and subsequent washing, enhanced chemilu-minescence Western blotting detection reagents and Hyperfilm TM enhancedchemiluminescence (both from Amersham) were used according to the instruc-tions of the manufacturer.

Binding assay. Cells (2 � 105) were harvested using PBS-EDTA and recov-ered in binding buffer (BB) consisting of DMEM, penicillin-streptomycin, 10mM HEPES, 1% bovine serum albumin (BSA; Roche) with a 1-hour incubationat 37°C with constant agitation. Simultaneously, 35S-labeled Ad5 virions (104

particles/cell) were incubated with or without 10 �g HLf in BB (100 �l) on ice.One hour later, the virion mixtures were transferred to recovered, pelleted cellsand incubated for another hour on ice (final volume, 100 �l). Unbound virionswere removed by washing, and the cell-associated radioactivity was measuredusing a Wallac 1409 liquid scintillation counter (Perkin-Elmer). In one type ofbinding experiment, 35S-labeled Ad5 virions were preincubated in 100 �l of BBwith or without tear fluid (1:10 dilution) before incubation with cells (see Fig. 1D,below). In another type of binding experiment (see Fig. 2B, below), the data werenormalized with respect to HLf-dependent aggregation of virions: from parallelsamples containing either virions only or virions and HLf, but not cells, radio-

activity in the supernatant (top 95 �l) or “pellet” (remaining 5 �l) was measuredwith respect to radioactivity. Approximately twice as many more virions werefound in the pellet fraction when virions were preincubated with HLf. In a thirdtype of binding experiment (see Fig. 4B, below), cells were preincubated in 100�l BB with or without 1 �g anti-CAR monoclonal antibodies E1-1 (a kind gift ofSilvio Hemmi) and/or RmcB (Upstate), prior to incubation with HLf and 35S-labeled Ad5 virions (1 hour on ice).

Flow cytometry. Cells were harvested using PBS-EDTA and recovered in BB,followed by a 1-hour incubation at 37°C with constant agitation. The cells(5 �105 cells/sample) were then further incubated on ice for 1 hour in 100 �lDMEM containing 1% BSA and 0.01% NaN3, with or without HLf (4 �g) ormouse anti-CAR (RmcB ascites; a kind gift of Jeffrey M. Bergelson) diluted1:1,000. Cells that had been preincubated with HLf were washed and resus-pended in 100 �l DMEM containing 1% BSA, 0.01% NaN3, and rabbit anti-hLf(Biodesign) diluted 1:30 and incubated on ice. Thirty minutes later, these cells(and also cells preincubated with RmcB) were washed and incubated with swineanti-rabbit antibodies (in the case of cells with HLf) or rabbit anti-mouse anti-bodies (in the case of cells with RmcB). Both conjugates (Dako Cytomation)were FITC conjugated and diluted 1:50 in PBS with 1% BSA. After 30 min ofincubation on ice, the cells were resuspended in 300 �l PBS containing pro-pidium iodine (1 �g/ml; Sigma-Aldrich). Each incubation was followed by twowashing steps. The samples were then examined using a FACScan flow cytometerand LYSIS II software (Becton Dickinson).

RESULTS

Tear fluid promotes Ad5 infection of corneal cells. We havepreviously shown that Ad37 uses sialic acid as a cellular receptor(2). Unlike most other adenoviruses, the tropism of Ad37 islargely restricted to the eye (14). With this in mind, we set out toinvestigate the effect of tear fluid on Ad37 infection of cornealcells. As a control we used Ad5, which, like other members ofspecies C, mainly causes tonsillitis and upper respiratory tractinfections and, much less often, ocular infections (19). Tear fluiddid not affect the infectivity of Ad37, but to our surprise it effi-ciently enhanced the infectivity of Ad5 in HCE cells (Fig. 1A). Inorder to investigate this unexpected finding, we first sought toidentify the step in the adenovirus life cycle in which tear fluidexerted this effect. Tear fluid promoted infection when preincu-bated with Ad5 virions or when incubated with Ad5 virions to-gether with cells, but not when added directly after Ad5 hadbound to cells (Fig. 1B and C), suggesting that tear fluid promotesan early step in the life cycle of Ad5. In agreement with this, a 1:10dilution of tear fluid promoted binding of 35S-labeled Ad5 toHCE cells approximately 13-fold (Fig. 1D), which led us to be-lieve that one or more tear fluid components promote binding ofAd5 to cells through a direct interaction with virions. To test this,we performed a virus protein overlay blotting assay and foundthat Ad5 virions interacted with at least three different tear fluidproteins (Fig. 1E), which, according to their size and relativeamounts, appeared to correspond to HLf (80 kDa), lysozyme (14kDa), and lipophilin (5 to 8 kDa) (Fig. 1F). When blotted tolarger amounts of tear fluid proteins, Ad5 virions also interactedwith lipocalin, secreted immunoglobulin A, and phospholipase A2

(data not shown).HLf promotes dose-dependent binding of Ad5 to and infec-

tion of cells that correspond to the natural tropism of Ad5. Toelucidate the role of specific tear fluid proteins during Ad5infection, we preincubated Ad5 virions with purified HLf, ly-sozyme, or lipophilin before allowing Ad5 to infect HCE cells.Whereas lysozyme and lipophilin had no effect at 6 �g/ml, HLfcaused an increase in the number of infected cells (Fig. 2A).Also, HLf was found to mimic the effect observed with tearfluid, in that HLf promoted infection when preincubated with

VOL. 81, 2007 HLf ROLE IN ADENOVIRUS BINDING AND INFECTION 955

Ad5 virions or when coincubated with Ad5 virions and cells,but not when added to cells directly after the virus binding step(data not shown). We also found that HLf promoted binding ofAd5 to cells only when preincubated with virions, but not when

HLf was preincubated with cells (Fig. 2B), indicating that HLfmust be in suspension and first interact with virions in order tobe able to promote the binding of Ad5 to cells. Taken together,these findings suggest that HLf promotes Ad5 binding to and

FIG. 1. Effects of tear fluid on adenovirus infection and characterization of tear fluid components that interact with virions. (A) Tear fluid promotesinfection of Ad5 virions in HCE cells. Virions were mixed with or without tear fluid (diluted 1:100) and allowed to infect cells. Forty-four hourspostinfection, the cells were fixed, stained, and analyzed in a fluorescence microscope. (B and C) Tear fluid promotes infection of Ad5 in HCE cellsthrough a preentry mechanism. Panel C is a quantification of the findings shown in panel B. Tear fluid (final dilution, 1:50) was added at different timepoints of the Ad5 infectious cycle. At 44 h postinfection, the cells were fixed, stained, and analyzed in a fluorescence microscope. CTRL, no tear fluidwas included; “before,” tear fluid was preincubated with virions; “during,” tear fluids were incubated with cells and virions; “after,” tear fluid was addedto cells after binding of virions to cells. (D) Tear fluid promotes binding of Ad5 to HCE cells. Tear fluid (diluted 1:10) was preincubated with 35S-labeledvirions and then incubated with cells. After removal of unbound virions by washing, the cell-associated radioactivity was quantified with a beta counter.For panels C and D, the data shown are the results of three independent experiments, and each experiment was performed in duplicate. (E) Ad5 virionsinteract with different tear fluid proteins. Tear fluid (5 �l), HLf (5 �g), lysozyme (5 �g), and lipophilin (2 �g) were loaded separately onto a gel, separated,and transferred to a polyvinylidene difluoride membrane. The membrane was then treated consecutively with Ad5 virions, anti-Ad5 antibodies, and horseradishperoxidase-conjugated anti-antibodies as described in Materials and Methods. (F) Visualization of tear fluid (5 �l) and individual tear fluid proteins (HLf, 5 �g;lysozyme, 5 �g; lipophilin, 2 �g). Note that the sizes of these three proteins correspond to the sizes of the proteins detected by Ad5 virions in panel E.

956 JOHANSSON ET AL. J. VIROL.

infection of target cells by serving as a bridge between thevirion and the cells.

Since Ad5 causes respiratory infections more frequentlythan ocular infections, we hypothesized that HLf would pro-mote the binding of Ad5 to respiratory cells also. In agreementwith this, HLf alone was sufficient for promotion of Ad5 bind-ing not only to HCE cells (5-fold), but also to the A549 cell line(2.5-fold) (Fig. 3A), which is derived from respiratory cells.Moreover, when preincubated with Ad5 virions, HLf efficientlypromoted infections in ocular (HCE), respiratory (A549), andlarynx (Hep2) epithelial cells (Fig. 3B). Thus, HLf promotedinfection by Ad5 in cells that corresponded to the normaltropism of this virus. From previous work by others, it has beensuggested that HLf inhibits Ad2 from infecting Hep2 cells (3,13). In these studies, however, the concentrations of HLf thatwere inhibitory (0.5 mg/ml abolished 50% of the cytopathiceffect) were higher than those we found to promote Ad5 in-fection in HCE, A549, and Hep2 cells. To investigate whetherdifferent concentrations exert different effects, we performeddose-dependent studies of HLf-mediated Ad5 binding to HCEcells. At concentrations lower than 0.01 mg/ml, HLf did notexert any effect on Ad5 binding to HCE cells, but at 0.1 and 1mg/ml HLf promoted binding 7.1- and 3.3-fold, respectively(Fig. 3D). At the highest concentration tested (10 mg/ml), HLfreduced binding to a level that was lower than in its absence.

One possible explanation for this is that at very high con-centrations, individual HLf proteins bind to either virions orcells, but not to both, and thus HLf may be prevented fromforming the bridge that is required for linking virions tocells, giving results similar to those described previously (3,13). Assuming 2 mg/ml HLf in tear fluid (51), the dilutionused here (1:10) corresponds to 0.2 mg/ml, which is withinthe range in which HLf was found to act as a promoter ofvirus binding.

FIG. 2. HLf promotes infection and binding of Ad5 to HCE cells.(A) HLf, but not lysozyme or lipophilin, promotes infection by Ad5 inHCE cells. Ad5 virions were preincubated with or without HLf, ly-sozyme, or lipophilin (6 �g/ml) and allowed to infect cells. Forty-fourhours postinfection, the cells were fixed, stained, and analyzed in afluorescence microscope. (B) Free, but not cell-associated, HLf pro-motes binding of Ad5 virions to HCE cells. HLf (100 �g/ml) was firstpreincubated with 35S-labeled virions and then incubated with cells(Ad5 � HLf to cells), or HLf (100 �g/ml) was first preincubated withcells and then with virions (Ad5 to HLf � cells). As a control, 35S-labeled virions was incubated directly with cells in the absence of HLf[Ad5 to cells (no HLf)]. After removal of unbound virions from cells bywashing, the cell-associated radioactivity was quantified with a betacounter. The data are normalized with respect to the possible effect ofaggregation (see Materials and Methods). For panel B, the data shownare the results of three independent experiments, and each experimentwas performed in duplicate.

FIG. 3. HLf promotes infection and dose-dependent binding ofAd5 in respiratory and laryngeal epithelial cells. (A) HLf promotesbinding of Ad5 virions to A549 and HCE cells. 35S-labeled virions werepreincubated with or without HLf (100 �g/ml) and then incubated withcells. After removal of unbound virions by washing, the cell-associatedradioactivity was quantified with a beta counter. Data shown are theresults of three independent experiments, and each experiment wasperformed in duplicate. (B) HLf promotes infection of Ad5 virions inHCE, A549, and Hep2 cells. Virions were mixed with or without HLf(100 �g/ml) and allowed to infect cells. At 44 h postinfection, the cellswere fixed, stained, and analyzed in a fluorescence microscope. (C) In-creasing concentrations of HLf were preincubated with 35S-labeledvirions and then incubated with cells. After removal of unbound virionsby washing, the cell-associated radioactivity was quantified with a betacounter. For panels A to C, three independent experiments wereperformed, each in duplicate. Representative figures were selected.


CAR is not involved in HLf-mediated binding of Ad5 virionsto cells. Since CAR is distributed equally over the surface ofnonpolarized epithelial cells in vitro and is thereby able tofunction efficiently as a cellular receptor for human adenovi-

ruses, we hypothesized that the efficiency of HLf-mediatedadenovirus binding to cells in vitro may depend on a combi-nation of the presence of CAR and suitable receptors for HLf.To investigate this, we first compared the expression of CAR

FIG. 4. HLf promotes interaction of Ad5 with target cells independently of CAR. (A) Relative expression levels of cell surface CAR and HLfreceptors differ between cells. Hep2, A549, or HCE cells were incubated with RmcB ascites (anti-CAR antibodies) followed by FITC-conjugatedanti-antibodies, or with HLf followed by rabbit anti-HLf antibodies and FITC-conjugated anti-antibodies, and analyzed by flow cytometry. Datawere normalized to 100% (A549), and HCE and Hep2 data were normalized accordingly. (B) Blocking of cell surface CAR improves the efficiencyby which HLf promotes binding of Ad5 to A549 cells. 35S-labeled virions were preincubated with or without HLf (100 �g/ml) at the same time ascells were preincubated with or without RmcB and/or E1-1 anti-CAR antibodies (10 �g/ml) Thereafter, virions (with or without HLf) wereincubated with cells (with or without anti-CAR antibodies). After removal of unbound virions by washing, the cell-associated radioactivity wasquantified with a beta counter. (C) Lack of cell surface CAR improves the efficiency by which HLf promotes binding of Ad5 to lymphoblastic T-cellleukemia cells. 35S-labeled virions were preincubated with or without HLf (100 �g/ml) and thereafter with cells. After removal of unbound virionsby washing, the cell-associated radioactivity was quantified with a beta counter. (D and E) HLf promotes GFP expression in A549 cells fromadenoviruses or adenovirus vectors equipped with the Ad5 fiber, but not with the Ad35 fiber. Panel E is a quantification of the results shown inpanel D. Ad5 or Ad35 virions, or Ad5 vector with Ad5 fiber (Ad5CMVeGFP) or Ad35 fiber (Ad5F35-GFP), were preincubated with or withoutHLf (60 �g/ml) and allowed to infect cells. At 44 h postinfection, the cells were fixed and analyzed in a fluorescence microscope. For panels A toC and E, the data shown are the results of three independent experiments, and each experiment was performed in duplicate.


and HLf receptors on A549, HCE, and Hep2 cells. HLf boundto all three cell lines with similar efficiency, indicating thatthere are similar numbers of receptors for HLf on these epi-thelial cells (Fig. 4A). Anti-CAR monoclonal antibody RmcB,on the other hand, bound with highest efficiency to Hep2 cellsand with least efficiency to HCE cells. To investigate the role ofCAR during HLf-mediated Ad5 infection of A549 cells ingreater depth, we blocked cell surface CAR with two mono-clonal antibodies, RmcB and E1-1, during the adsorption step.Neither of the antibodies inhibited HLf-mediated infection(Fig. 4B). On the contrary, the HLf-mediated infectivity ofAd5 was enhanced in the presence of anti-CAR antibodies,indicating that CAR is not involved in HLf-mediated bindingof Ad5 to cells. Moreover, HLf promoted Ad5 binding toCAR-negative T cells (CEM and KE37) more efficiently thanto corresponding T cells transfected with the cDNA encodinghuman CAR (CEM-CARhi and KE37-CARhi) (Fig. 4C).Based on these experiments, we concluded that in the absenceof CAR, HLf efficiently promotes binding of Ad5 to targetcells.

The outer surface of the adenovirus capsid is composed ofthree well-exposed proteins (the hexon, the penton base, andthe fiber) and two less-exposed proteins (IIIa and IX). To testwhether the fiber is involved in the HLf-mediated interactionbetween the adenovirus capsid and cells, we used two differentAd5-based vectors that express GFP: one vector (Ad5F35-GFP) containing the Ad5 tail and the shaft and knob domainof Ad35 (a CD46 binding adenovirus, i.e., non-CAR-bindingadenovirus), and another vector containing the wild-type Ad5fiber (Ad5CMVeGFP). We observed a 5-fold increase in thenumber of GFP-expressing A549 cells upon preincubation ofthe Ad5CMVeGFP vector with HLF compared to control (noHLf) (Fig. 4D and E), whereas preincubation of the Ad5F35-GFP vector with HLf resulted only in 1.5-fold more GFP-expressing cells. Ad5 virions behaved similarly to theAd5CMVeGFP vector in that a 5-fold increase in the numberof infected cells was observed upon preincubation with HLf,whereas preincubation with HLf had only a minor effect onAd35 infection of A549 cells (1.1-fold increase). Thus, eventhough it is too early to exclude other capsid proteins frombeing involved in the HLf-mediated interaction of Ad5 withcells, it seems likely that the fiber plays an important role inthis mechanism.

HLf promotes species C-specific adenovirus infection ofA549 cells. Since HLf did not promote infection of Ad35,which is one of the nine members of species B, we hypothe-sized that the effect of HLf could be specific for certain ade-novirus species. In order to investigate this, we infected A549cells with at least one serotype from each species and foundthat HLf promoted infection by all species C adenoviruses(Ad1, Ad2, Ad5, and Ad6) but not by representative adenovi-ruses of species A (Ad31), B (Ad7 and Ad11), D (Ad37), E(Ad4), or F (Ad41) (Fig. 5). Thus, the ability of HLf to pro-mote adenovirus infection in A549 cells appears to be specificfor adenoviruses of species C.

DISCUSSION

Species C adenoviruses are a relatively frequent cause oftonsillitis and adenoiditis in young children, and DNA of spe-

cies C adenoviruses but not DNA from other species has beendetected in T cells isolated from tonsils and adenoids (17).Thus, we speculated that usage of HLf as a bridge may be animportant mechanism by which species C adenoviruses cancause latent/persistent infections of T cells localized in tonsilsand adenoids. This idea is supported by the lack of CAR onprimary T cells (10), the lack of Ad5 binding to and replicationin T cells (reference 37 and this paper), and the efficient bind-ing of Ad5 to CAR-negative T cells in the presence of HLf(this paper). Human herpesvirus type 8 (HHV8) is anothervirus that infects tonsils and adenoids in young children (9). Aninteresting parallel between Ad5 and HHV8 is that the latterhas been suggested to require salivary lactoferrin for infectionof target cells (20).

Species C adenoviruses are one of the most frequently usedviral vectors for human gene therapy, but the in vivo resultshave not lived up to expectations derived from in vitro work.There are at least two possible reasons for this. First, most ofthe human population carries neutralizing antibodies to theAd5-based vectors, which are used more frequently than vec-tors based on other serotypes. In order to circumvent this,vectors have been developed based on Ad11 (22, 40), Ad19(42), and Ad35 (16), as their seroprevalence is considerablylower (22, 40, 47). Another recent approach to circumventimmunity, but still benefit from the characteristics of the well-studied Ad5 serotype, is to exchange the immunogenic do-mains on the surface of the hexon protein for correspondingdomains chosen from a less common serotype (e.g., Ad48)(33). A second major reason as to why Ad5-based vectorsfunction poorly in vivo is likely to be the absence (or lowpresence) of CAR on polarized epithelial cells and other cells,such as primary T cells. We predict that it might be possible toimprove the efficiency of gene therapy based on Ad5 vectorssimply by including HLf in gene therapy protocols.

Based on our results, and with the knowledge that CAR isabsent from the apical side of polarized cells, we conclude thateither CAR or HLf alone is sufficient for efficient binding ofAd5 to and infection of nonpolarized cells (i.e., in vitro) (Fig.6). In CAR-negative cells, on the other hand (thus mimickingthe in vivo situation, where epithelial cells are polarized), theinteraction with HLf may be an important mechanism used byAd5 and other species C adenoviruses for binding to andinfection of cells. Because CAR is normally inaccessible fromthe apical surface, it has been suggested that the initial ade-novirus infection may occur when a transient break in theepithelium allows luminal virus to reach its receptor or duringrepair of injured epithelium when CAR might be accessible(49). To the best of our knowledge, such breaks are poorlycharacterized, and gaining access to CAR via this pathway maynot explain how adenoviruses, which are a common cause ofrespiratory disease, bind to and infect epithelial cells in vivo.

Confusingly, HLf has already been suggested to inhibit Ad2from infecting Hep2 cells through a mechanism that takesplace during an early step in the infection (3, 13). The contra-dictory results may simply be explained by the different con-centrations of HLf used and the different number of HLfbinding sites on virions and cells in the two different types ofexperiments. We speculate that in the presence of low concen-trations of HLf in relation to the number of HLf binding siteson virions and cells, which was the case in most experiments of


this study, HLf is allowed to bind both virions and cells at thesame time, but at higher concentrations of HLf there is anexcess of HLf in relation to the number of HLf binding sites onvirions and cells (3, 13), resulting in binding to either virions orcells, but not both simultaneously, thus not allowing a bridge tobe formed. The cutoff value at which HLf inhibits rather thanpromotes binding probably varies between cell types, as differ-ent cell types express different numbers of HLf receptors.When we increased the concentration of HLf above 1 mg/ml,we found that binding of Ad5 to HCE cells was reduced, thusmimicking the results obtained with Ad2 infection of Hep2cells (3, 13). In saliva, the concentration is rather low andwithin the same range as we used here, indicating that use of

HLf as a bridge to cells may be a functional pathway used byAd5 during infections in vivo. We also speculate that the rel-ative expression of HLf receptors and CAR, as well as thedifferent concentrations of HLf found in different body fluids,may at least partially explain the tropism of adenoviruses: intear fluid the concentration is high and HLf may thereforeinhibit (or at least fail to promote) adenovirus infection. Onthe other hand, in saliva the concentration is much lower, andHLf may therefore promote adenovirus infection.

At low pH, the N-terminal domain of HLf, which is knownas lactoferricin, is cleaved from HLf by pepsin. We did not findany difference in the activity between pepsin-cleaved andmock-treated HLf on Ad5 infection of HCE cells (data not

FIG. 5. HLf promotes infection of A549 cells by species C adenoviruses. Panel B shows a quantification of the results in panel A. Virions ofdifferent serotypes representing all species (as indicated) were mixed with HLf (60 �g/ml) or untreated, and allowed to infect cells. At 44 hpostinfection, the cells were fixed, stained, and analyzed in a fluorescence microscope. Data shown are the result of three independent experiments,and each experiment was performed in duplicate.


shown). Besides being cleaved by pepsin and other proteases,HLf itself may act as a protease. The proteolytic activity of HLfthat has been described previously takes place at serine resi-dues surrounded by arginines (RSRR or RRSR) (21). How-ever, these sites are not found in the three major Ad2 or Ad5capsid proteins (hexon, fiber, and penton base). We thereforedo not expect that HLf-mediated binding and infection ofspecies C involves the proteolytic capacity of HLf. Moreover,HLf has very high affinity for Fe3� ions. We compared holo-HLf (iron saturated) with apo-HLf and found that holo-HLf isa more effective promoter of Ad5 binding to HCE cells thanapo-HLf (data not shown). We speculate that the ability ofFe3� ions to affect the overall structure of HLf may affect theability of HLf to promote binding of Ad5 to HCE cells.

Bovine lactoferrin (BLf) has been demonstrated to inhibitAd2 infection of Hep2 cells (13). We found BLf to exert asmall promoting effect of Ad5 infection of A549 cells, but notas much as HLf (data not shown). The previously describedinhibitory effect of BLf has been suggested to involve theheparin binding site of BLf. We have tested whether heparansulfate might serve as the HLf receptor responsible for HLf-mediated Ad5 binding to and infection of target cells, usingheparan sulfate-deficient CHO-2241 cells (ATCC pgsB-618),but HLf promoted Ad5 infection in CHO cells with an effi-ciency that was similar to that in CHO-2241 cells (data notshown), thus indicating that the cell surface component that isresponsible for this effect remains to be identified. However, itis obvious that there are receptors for HLf on both humanepithelial cells and T cells (this paper and references 7 and 18),suggesting that HLf may indeed interact with these cells andalso promote Ad5 binding to and infection of them in vivo,even in the absence of CAR.

The most commonly described mechanism used by virusesfor binding to target cells usually involves a direct interactionbetween virions and their cell surface receptor. In addition,antibody-dependent enhancement of infection mediated by in-teractions between the Fc region of virus-specific immunoglob-

ulin G and Fc receptors on certain cells has been described fora number of viruses (29). There have only been a few reportsindicating that other components serve as a bridge betweenviruses and target cells: (i) C4BP and coagulation factor IXhave been reported to promote Ad5 interactions with targetcells when the CAR binding site of the Ad5 fiber has beenablated (39); (ii) dipalmitoyl phosphatidylcholine may be in-volved in Ad2 entry into alveolar epithelial cells (5); (iii) po-lymerized human serum albumin is necessary and sufficient forbinding of hepatitis B virus-like particles to human liver plasmamembranes (32, 44); (iv) BLf has been suggested to enhanceinfection of gC-negative herpes simplex viruses in mousefibroblasts (24); and (v) HLf has been proposed to generateformation of an HHV8-lactoferrin-glycosaminoglycan-epithe-lial cell receptor complex, which may increase the initial infec-tive viral dose in the vicinity of potential target cells and in-crease the risk of infection (20). This report further potentiatesthe role of lactoferrin as a soluble component with the abilityto promote binding to and infection of target cells, by servingas a bridge between the virion and the surface of the target cell.

HLf has previously been shown to serve as an antimicrobialcomponent of the innate immune system. Numerous bacteria,fungi, and viruses have been found to be inhibited or inacti-vated by HLf, by various mechanisms (45, 51). With few ex-ceptions, the mechanism by which HLf inhibits viruses appearsto be at the level of binding to target cells, either by blockingthe cellular receptor or the viral ligand. Since binding of Ad5to cells is promoted by free HLf, but not by cell-associatedHLf, we suggest that Ad5 hijacks HLf and thereafter links thevirions to the cell surface. Thus, in this case the activity of HLfis promicrobial rather than antimicrobial.

ACKNOWLEDGMENTS

We thank Linda Gooding for the generous gift of CEM, CEM-CARhi, KE37, and KE37-CARhi cells, Jeffrey M. Bergelson for thegenerous gift of RmcB ascites, Silvio Hemmi for the generous gift of

FIG. 6. Model of adenovirus hijacking HLf for infection of cells in the absence and presence of apical CAR. In the presence of CAR, mimickingthe in vitro situation with nonpolarized epithelial cells, species C adenoviruses can bind to and infect cells either via CAR or via the HLf-mediatedpathway. In the absence of CAR, mimicking the in vivo situation with polarized epithelial cells, species C adenoviruses can bind to and infect cellsvia the HLf-mediated pathway alone.


the monoclonal antibody E1-1, and Robert Lehrer for the generousgift of lipophilin.

This work was supported by grants from the Swedish ResearchCouncil (grants no. 2003-6008 and 2004-6174), Umeå University (bio-technology grant), the Swedish Society for Medical Research, theJeansson Foundation, and the Stiftelsen Clas Groschinskys Founda-tion.

REFERENCES

1. Araki-Sasaki, K., K. Y. Ohasi, T. Sasabe, K. Hayashi, H. Watanabe, Y. Tano,and H. Handa. 1995. An SV-40-immortalized human corneal epithelial cellline and its characterization. Investig. Ophthalmol. 36:614–621.

2. Arnberg, N., K. Edlund, A. H. Kidd, and G. Wadell. 2000. Adenovirus type37 uses sialic acid as a cellular receptor. J. Virol. 74:42–48.

3. Arnold, D., A. M. Di Biase, M. Marchetti, A. Pietrantoni, P. Valenti, L.Seganti, and F. Superti. 2002. Antiadenovirus activity of milk proteins:lactoferrin prevents viral infection. Antivir. Res. 53:153–158.

4. Avila, M. M., G. Carballal, H. Rovaletti, B. Ebekian, M. Cusminsky, and M.Weissenbacher. 1989. Viral etiology in acute lower respiratory infections inchildren from a closed community. Am. Rev. Respir. Dis. 140:634–637.

5. Balakireva, L., G. Schoehn, E. Thouvenin, and J. Chroboczek. 2003. Bindingof adenovirus capsid to dipalmitoyl phosphatidylcholine provides a novelpathway for virus entry. J. Virol. 77:4858–4866.

6. Bergelson, J. M., J. A. Cunningham, G. Droguett, E. A. Kurt-Jones, A.Krithivas, J. S. Hong, M. S. Horwitz, R. L. Crowell, and R. W. Finberg. 1997.Isolation of a common receptor for coxsackie B viruses and adenoviruses 2and 5. Science 275:1320–1323.

7. Bi, B. Y., B. Leveugle, J. L. Liu, A. Collard, P. Coppe, A. C. Roche, N.Nillesse, M. Capron, G. Spik, and J. Mazurier. 1994. Immunolocalization ofthe lactotransferrin receptor on the human T lymphoblastic cell line Jurkat.Eur. J. Cell Biol. 65:164–171.

8. Brandt, C. D., H. W. Kim, A. J. Vargosko, B. C. Jeffries, J. O. Arrobio, B.Rindge, R. H. Parrott, and R. M. Chanock. 1969. Infections in 18,000 infantsand children in a controlled study of respiratory tract disease. I. Adenoviruspathogenicity in relation to serologic type and illness syndrome. Am. J.Epidemiol. 90:484–500.

9. Chagas, C. A., L. H. Endo, E. Sakano, G. A. Pinto, P. Brousset, and J.Vassallo. 2006. Detection of herpesvirus type 8 (HHV8) in children’s tonsilsand adenoids by immunohistochemistry and in situ hybridization. Int. J. Pe-diatr. Otorhinolaryngol. 70:65–72.

10. Chen, Z., M. Ahonen, H. Hamalainen, J. M. Bergelson, V. M. Kahari, and R.Lahesmaa. 2002. High-efficiency gene transfer to primary T lymphocytes byrecombinant adenovirus vectors. J. Immunol. Methods 260:79–89.

11. Coyne, C. B., and J. M. Bergelson. 2005. CAR: a virus receptor within thetight junction. Adv. Drug Deliv. Rev. 57:869–882.

12. Coyne, C. B., and J. M. Bergelson. 2006. Virus-induced Abl and Fyn kinasesignals permit coxsackievirus entry through epithelial tight junctions. Cell124:119–131.

13. Di Biase, A. M., A. Pietrantoni, A. Tinari, R. Siciliano, P. Valenti, G.Antonini, L. Seganti, and F. Superti. 2003. Heparin-interacting sites ofbovine lactoferrin are involved in anti-adenovirus activity. J. Med. Virol.69:495–502.

14. Ford, E., K. E. Nelson, and D. Warren. 1987. Epidemiology of epidemickeratoconjunctivitis. Epidemiol. Rev. 9:244–261.

15. Gaggar, A., D. M. Shayakhmetov, and A. Lieber. 2003. CD46 is a cellularreceptor for group B adenoviruses. Nat. Med. 9:1408–1412.

16. Gao, W., P. D. Robbins, and A. Gambotto. 2003. Human adenovirus type 35:nucleotide sequence and vector development. Gene Ther. 10:1941–1949.

17. Garnett, C. T., D. Erdman, W. Xu, and L. R. Gooding. 2002. Prevalence andquantitation of species C adenovirus DNA in human mucosal lymphocytes.J. Virol. 76:10608–10616.

18. Ghio, A. J., J. D. Carter, J. M. Samet, W. Reed, J. Quay, L. A. Dailey, J. H.Richards, and R. B. Devlin. 1998. Metal-dependent expression of ferritin andlactoferrin by respiratory epithelial cells. Am. J. Physiol. 274:28–36.

19. Gordon, Y. J., K. Aoki, and P. R. Kinchington. 1996. Adenovirus keratocon-junctivitis, p. 877–894. In J. S. Pepose, G. N. Holland, and K. R. Wilhelmus(ed.), Ocular infection and immunity. Mosby, St. Louis, Mo.

20. Grange, P. A., A. G. Marcelin, V. Calvez, C. Chauvel, J. P. Escande, and N.Dupin. 2005. Salivary lactoferrin is recognized by the human herpesvirus-8.J. Investig. Dermatol. 124:1249–1258.

21. Hendrixson, D. R., J. Qiu, S. C. Shewry, D. L. Fink, S. Petty, E. N. Baker,A. G. Plaut, and J. W. R. St. Geme. 2003. Human milk lactoferrin is a serineprotease that cleaves Haemophilus surface proteins at arginine-rich sites.Mol. Microbiol. 47:607–617.

22. Holterman, L., R. Vogels, R. van der Vlugt, M. Sieuwerts, J. Grimbergen,J. Kaspers, E. Geelen, E. van der Helm, A. Lemckert, G. Gillissen, S.Verhaagh, J. Custers, D. Zuijdgeest, B. Berkhout, M. Bakker, P. Quax, J.Goudsmit, and M. Havenga. 2004. Novel replication-incompetent vectorderived from adenovirus type 11 (Ad11) for vaccination and gene therapy:low seroprevalence and non-cross-reactivity with Ad5. J. Virol. 78:3207–3215.

23. Horwitz, M. S. 2001. Adenoviruses, p. 2301–2326. In D. M. Knipe and P. M.Howley (ed.), Fields virology, 4th ed., vol. 2. Lippincott Williams & Wilkins,Philadelphia, Pa.

24. Marchetti, M., E. Trybala, F. Superti, M. Johansson, and T. Bergstrom.2004. Inhibition of herpes simplex virus infection by lactoferrin is dependenton interference with the virus binding to glycosaminoglycans. Virology 318:405–413.

25. Marttila, M., D. Persson, D. Gustafsson, M. K. Liszewski, J. P. Atkinson, G.Wadell, and N. Arnberg. 2005. CD46 is a cellular receptor for all species Badenoviruses except types 3 and 7. J. Virol. 79:14429–14436.

26. McNees, A. L., J. A. Mahr, D. Ornelles, and L. R. Gooding. 2004. Post-internalization inhibition of adenovirus gene expression and infectious virusproduction in human T-cell lines. J. Virol. 78:6955–6966.

27. Mei, Y. F., K. Lindman, and G. Wadell. 1998. Two closely related adenovirusgenome types with kidney or respiratory tract tropism differ in their bindingto epithelial cells of various origins. Virology 240:254–266.

28. Moriuchi, M., and H. Moriuchi. 2001. A milk protein lactoferrin enhanceshuman T cell leukemia virus type I and suppresses HIV-1 infection. J. Im-munol. 166:4231–4236.

29. Munakata, Y., I. Kato, T. Saito, T. Kodera, K. K. Ishii, and T. Sasaki. 2006.Human parvovirus B19 infection of monocytic cell line U937 and antibody-dependent enhancement. Virology 345:251–257.

30. Nilsson, M., J. Ljungberg, J. Richter, T. Kiefer, M. Magnusson, A. Lieber, B.Widegren, S. Karlsson, and X. Fan. 2004. Development of an adenoviralvector system with adenovirus serotype 35 tropism; efficient transient genetransfer into primary malignant hematopoietic cells. J. Gene Med. 6:631–641.

31. Pickles, R. J., D. McCarty, H. Matsui, P. J. Hart, S. H. Randell, and R. C.Boucher. 1998. Limited entry of adenovirus vectors into well-differentiatedairway epithelium is responsible for inefficient gene transfer. J. Virol. 72:6014–6023.

32. Pontisso, P., M. A. Petit, M. J. Bankowski, and M. E. Peeples. 1989. Humanliver plasma membranes contain receptors for the hepatitis B virus pre-S1region and, via polymerized human serum albumin, for the pre-S2 region.J. Virol. 63:1981–1988.

33. Roberts, D. M., A. Nanda, M. J. Havenga, P. Abbink, D. M. Lynch, B. A.Ewald, J. Liu, A. R. Thorner, P. E. Swanson, D. A. Gorgone, M. A. Lifton,A. A. Lemckert, L. Holterman, B. Chen, A. Dilraj, A. Carville, K. G. Mans-field, J. Goudsmit, and D. H. Barouch. 2006. Hexon-chimaeric adenovirusserotype 5 vectors circumvent pre-existing anti-vector immunity. Nature 441:239–243.

34. Roelvink, P. W., I. Kovesdi, and T. J. Wickham. 1996. Comparative analysisof adenovirus fiber-cell interaction: adenovirus type 2 (Ad2) and Ad9 utilizethe same cellular fiber receptor but use different binding strategies forattachment. J. Virol. 70:7614–7621.

35. Roelvink, P. W., A. Lizonova, J. G. Lee, Y. Li, J. M. Bergelson, R. W. Finberg,D. E. Brough, I. Kovesdi, and T. J. Wickham. 1998. The coxsackievirus-adenovirus receptor protein can function as a cellular attachment protein foradenovirus serotypes from subgroups A, C, D, E, and F. J. Virol. 72:7909–7915.

36. Segerman, A., J. P. Atkinson, M. Marttila, V. Dennerquist, G. Wadell, andN. Arnberg. 2003. Adenovirus type 11 uses CD46 as a cellular receptor.J. Virol. 77:9183–9191.

37. Segerman, A., K. Lindman, Y. F. Mei, A. Allard, and G. Wadell. 2006.Adenovirus types 11p and 35 attach to and infect primary lymphocytes andmonocytes, but hexon expression in T-cells requires prior activation. Virol-ogy 349:96–111.

38. Shafren, D. R., R. C. Bates, M. V. Agrez, R. L. Herd, G. F. Burns, and R. D.Barry. 1995. Coxsackieviruses B1, B3, and B5 use decay accelerating factoras a receptor for cell attachment. J. Virol. 69:3873–3877.

39. Shayakhmetov, D. M., A. Gaggar, S. Ni, Z. Y. Li, and A. Lieber. 2005.Adenovirus binding to blood factors results in liver cell infection and hepa-totoxicity. J. Virol. 79:7478–7491.

40. Stone, D., S. Ni, Z. Y. Li, A. Gaggar, N. DiPaolo, Q. Feng, V. Sandig, and A.Lieber. 2005. Development and assessment of human adenovirus type 11 asa gene transfer vector. J. Virol. 79:5090–5104.

41. Swart, P. J., M. E. Kuipers, C. Smit, R. Pauwels, M. P. deBethune, E. deClercq, D. K. Meijer, and J. G. Huisman. 1996. Antiviral effects of milkproteins: acylation results in polyanionic compounds with potent activityagainst human immunodeficiency virus types 1 and 2 in vitro. AIDS Res.Hum. Retrovir. 12:769–775.

42. Thirion, C., H. Lochmuller, Z. Ruzsics, M. Boelhauve, C. Konig, C.Thedieck, S. Kutik, C. Geiger, S. Kochanek, C. Volpers, and H. G. Burgert.2006. Adenovirus vectors based on human adenovirus type 19a have highpotential for human muscle-directed gene therapy. Hum. Gene Ther. 17:193–205.

43. Tomko, R. P., R. Xu, and L. Philipson. 1997. HCAR and MCAR: the humanand mouse cellular receptors for subgroup C adenoviruses and group Bcoxsackieviruses. Proc. Natl. Acad. Sci. USA 94:3352–3356.

44. Trevisan, A., F. Gudat, R. Guggenheim, G. Krey, U. Durmuller, G. Luond,M. Duggelin, J. Landmann, P. Tondelli, and L. Bianchi. 1982. Demonstra-


tion of albumin receptors on isolated human hepatocytes by light and scan-ning electron microscopy. Hepatology 2:832–835.

45. van der Strate, B. W., L. Beljaars, G. Molema, M. C. Harmsen, and D. K.Meijer. 2001. Antiviral activities of lactoferrin. Antivir. Res. 52:225–239.

46. van der Veen, J., and M. Lambriex. 1973. Relationship of adenovirus tolymphocytes in naturally infected human tonsils and adenoids. Infect. Im-mun. 7:604–609.

47. Vogels, R., D. Zuijdgeest, R. van Rijnsoever, E. Hartkoorn, I. Damen, M.P. de Bethune, S. Kostense, G. Penders, N. Helmus, W. Koudstaal, M.Cecchini, A. Wetterwald, M. Sprangers, A. Lemckert, O. Ophorst, B. Koel,M. van Meerendonk, P. Quax, L. Panitti, J. Grimbergen, A. Bout, J.Goudsmit, and M. Havenga. 2003. Replication-deficient human adenovirustype 35 vectors for gene transfer and vaccination: efficient human cell infec-tion and bypass of preexisting adenovirus immunity. J. Virol. 77:8263–8271.

48. Wadell, G., A. Allard, and J. C. Hierholzer. 1999. Adenoviruses, p. 970–982.

In P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover, and R. H. Yolken(ed.), Manual of clinical microbiology, 7th ed. ASM Press, Washington, D.C.

49. Walters, R. W., P. Freimuth, T. O. Moninger, I. Ganske, J. Zabner, and M. J.Welsh. 2002. Adenovirus fiber disrupts CAR-mediated intercellular adhe-sion allowing virus escape. Cell 110:789–799.

50. Walters, R. W., T. Grunst, J. M. Bergelson, R. W. Finberg, M. J. Welsh, andJ. Zabner. 1999. Basolateral localization of fiber receptors limits adenovirusinfection from the apical surface of airway epithelia. J. Biol. Chem. 274:10219–10226.

51. Weinberg, E. D. 2001. Human lactoferrin: a novel therapeutic with broadspectrum potential. J. Pharm. Pharmacol. 53:1303–1310.

52. Wickham, T. J., P. Mathias, D. A. Cheresh, and G. R. Nemerow. 1993.Integrins alpha v beta 3 and alpha v beta 5 promote adenovirus internaliza-tion but not virus attachment. Cell 73:309–319.


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Adenoviruses Use Lactoferrin as a Bridge for CAR-Independent Binding to and Infection of Epithelial Cells