Retroviral vector targeting through insertion of epidermal growth factor into receptor binding deficient influenza A hemagglutinin results in fusion defective particles

Journal of Virological Methods 120 (2004) 189–199

Retroviral vector targeting through insertion of epidermal growth factorinto receptor binding deficient influenza A hemagglutinin results in fusion

defective particles

Catherine Haynes, Barbara S. Schnierle∗

Institute for Biomedical Research, Georg-Speyer Haus, Paul-Ehrlich-Str. 42-44, D-60596 Frankfurt/Main, Germany

Received 20 October 2003; accepted 4 May 2004

Available online 2 July 2004

Abstract

Targeting retroviral entry is a central theme in the development of vectors for gene therapy. The host range of a retrovirus is dependentupon the interaction of its envelope glycoprotein (Env) with a specific cell surface receptor protein, which allows viral entry. In contrast, thepH-dependent viruses enter cells through receptor-mediated endocytosis and the subsequent acidification produces conformational changesin the viral envelope protein(s) which lead to membrane fusion. We attempted to redirect retroviral vectors to epidermal growth factor (EGF)receptor expressing cells by using the pH-dependent influenza A virus hemagglutinin (HA). Wild type receptor binding was avoided either bypoint mutations or by deletion of the globular head structure of HA and also inserted EGF into HA. Replacement of the whole head domainwas not tolerated. Two of the EGF-HA proteins bearing point mutations could be incorporated into retroviral particles, but unfortunately theirfusion activity was lost. The data indicate that care must be taken when mutating multiple sites in HA, and that targeting HA requires furtheranalysis of appropriate sites for the insertion of foreign sequences.© 2004 Elsevier B.V. All rights reserved.

Keywords:Targeting; Retroviral vector; Hemagglutinin; Epidermal growth factor

1. Introduction

A significant improvement of retroviral gene transfer vec-tors would be the ability to target gene transfer to specificcell types. This is particularly important when using suchvectors to introduce cytotoxic genes into cancer cells. Spe-cific targeting would avoid the need for cell isolation and exvivo transduction, and, in the case of cancer therapy, shouldalso enable vectors to be used in the treatment of metastasesand minimal residual disease.

The host range of a retroviral vector is largely dependentupon its envelope glycoprotein (Env), which recognizes andbinds to a specific cell surface receptor protein. The Envprotein consists of two subunits, SU which contains the re-ceptor binding domain, and TM which contains the fusionpeptide. Binding of SU to the receptor is thought to inducea conformational change in TM, resulting in the exposure

∗ Corresponding author. Tel.:+49 69 63395 218;fax: +49 69 63395 297.

E-mail address:[email protected] (B.S. Schnierle).

of the fusion peptide and activation of the process of fusionof the viral and cellular membranes, and eventual deliveryof the viral core into the cell. Several modified Env proteinshave been constructed that allow specific binding to targetedreceptors. However, the titers of vectors bearing these pro-teins tend to be very low or non-existent. This is believedto be a result of an inability of ligand binding to inducethe conformational changes required for fusion, and/or se-questration of vectors into endosomes and their subsequentdegradation (Russell and Cosset, 1999; Haynes et al., 2003).

An alternative method that can be used to alter the hostrange of a retroviral vector involves the replacement of theEnv protein with an envelope protein from another virus, aprocess known as pseudotyping. The influenza envelope pro-tein, hemagglutinin, appears to be a good candidate for thisapproach. It has been well characterized, both structurallyand functionally (reviewed by (Skehel and Wiley, 2000)),and can be incorporated into retroviral particles (Dong et al.,1992; Hatziioannou et al., 1998). In contrast to retroviruses,the cell entry of influenza viruses appears to occur indepen-dently of an interaction with a specific receptor. The HA pro-

0166-0934/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.jviromet.2004.05.009

190 C. Haynes, B.S. Schnierle / Journal of Virological Methods 120 (2004) 189–199

tein binds to sialic acid residues present on a wide range ofcell surface proteins, and the virus is internalized by endocy-tosis. The low pH within the endosome induces the confor-mational changes required to initiate fusion. These low pHinduced changes can occur in the absence of receptor bind-ing, suggesting that the binding serves only as a means oftargeting the virus to endosomes. This property of influenzaHA implies that this protein should retain its fusion activityfollowing retargeted binding, as long as this binding resultsin endocytosis and subsequent acidification.

In order to show fusion activity, the HA protein must firstbe cleaved by host proteases into the two fragments HA1and HA2. The major part of HA1 forms the globular headregion, which contains the sialic acid binding domain. Thestem region is formed from HA2 and the N- and C-termini ofHA1. HA2 contains the fusion peptide and transmembranedomain. The sequence of the cleavage site is a major de-terminant of viral pathogenicity (Garten and Klenk, 1999).HA proteins of pathogenic avian influenza strains, such asfowl plague virus (FPV), possess a cleavage site containingmultiple basic residues (consensus R-X-K/R-R). This site iscleaved by furin and related proteases present in the Golginetwork of a wide range of host cells. As a result theseviruses leave the host cell with a fully processed, fusion ac-tive HA protein.

In this study attempts were made to remove the naturalbinding site of FPV HA and target its binding specifically tothe epidermal growth factor (EGF) receptor. This receptoris over expressed in a number of different tumors makingit an attractive target for cancer therapy. In addition, it hasbeen shown recently that influenza virus entry and EGF re-ceptor trafficking and degradation can both be blocked byinhibition of protein kinase C�II (Sieczkarski et al., 2003),implying that similar routes of endocytosis are employed.It has been shown previously that insertion of peptides atthe N-terminus of the FPV HA protein can target binding tospecific cell surface molecules (Hatziioannou et al., 1999).However, vectors bearing these proteins still display the nor-mal host range of FPV since the natural receptor bindingpocket is retained. In order to overcome this problem weintroduced point mutations within conserved regions of thesialic acid binding pocket in an attempt to remove or greatlyreduce the natural binding activity, without disturbing fu-sion. Unfortunately, not all of these mutations were toleratedin the presence of EGF, and the proteins showed reduced lev-els of cleavage and cell surface expression. Although someof the proteins were incorporated into vector particles nofusion activity was observed.

By comparing the crystal structures of HA and thehemagglutinin-esterase-fusion protein of influenza C virus,Rosenthal et al. (1998)have proposed that during evolutionof the HA protein, the globular head region containing thereceptor binding pocket became inserted into a loop of aprecursor fusion protein. This suggested to us that the re-placement of the entire head domain with a new targetingdomain may be tolerated. This would remove the sialic acid

binding domain while leaving the protein regions involvedin fusion intact.Sagawa et al. (1996)reported an influenzaHA construct from which they had deleted the head region.This mutant was recognized by a neutralizing antibodythat binds to a conformational epitope within the stem re-gion, indicating that the protein was properly folded. Wetherefore tried replacing this domain with EGF as a secondstrategy. This resulted in a protein that was well expressed,but was retained within the cell making it unavailable forincorporation into budding vector particles.

2. Materials and methods

2.1. Cell lines

All cells were maintained at 37◦C and 10% CO2 andpropagated in Dulbecco’s modified Eagles medium supple-mented with 10% heat-inactivated fetal calf serum, 2 mMglutamine, 100 U/ml penicillin and 10�g/ml streptomycin.

The TELCeB6 cell line (Cosset et al., 1995) expressesMoloney murine leukemia virus Gag/Pol and an nlsLacZretroviral vector. Other cell lines used were human kidney293T, murine NIH 3T3 and African green monkey COS 7.

Transfections were carried out using a standard calciumphosphate precipitation protocol.

2.2. Plasmid construction

The hemagglutinin protein of influenza A/FPV/Rostock/34 (H7N1) (a generous gift from W. Garten, Universityof Marburg, Germany) was expressed from the mam-malian expression vector pSG5 (Green et al., 1988), usingthe SV40 early promoter and rabbit�-globin intron. TheHA cDNA was inserted into the blunt endedBamHI siteand the construct was named pSG5.HA. Point mutationswere introduced into the receptor binding pocket using theQuickChangeTM site-directed mutagenesis kit (Stratagene,Heidelberg). To minimize the risk of introduced errors theMscI fragments containing the mutations were insertedback into the original construct. Mutants are named afterthe altered residues: Y for Y88F, H for H174F and L forL185A. Numbers are based on the mature FPV HA protein.

In order to insert the EGF sequence,HindIII and XhoIrestriction sites were introduced into the HA sequencebetween the signal peptide and HA1 coding regions. Thedetailed cloning strategy can be obtained from the authorsupon request. The resulting construct was confirmed bysequence analysis and named pSG5.HA-HX. The EGF se-quence was obtained from pSG5.EGFenv (Erlwein et al.,2002) and inserted via theHindIII andXhoI sites to producepSG5.EGF-HA. To construct pSG5.EGF-G4S-HA, the oli-gos G4S-1 5′-GAT CCT CGA GGG AGG TGG CGG ATCCGG TGG AGG GGG TTC AGG AGG CGG TGG TTCAGG GGG CGG AGG TTC TCT CAG GGA TC-3′ andG4S2 5′-GAT CCT CGA GAG AAC CTC CGC CCC CTG

C. Haynes, B.S. Schnierle / Journal of Virological Methods 120 (2004) 189–199 191

AAC CAC CGC CTC CTG AAC CCC CTC CAC CGGATC CGC CAC CTC CCT CGA GGA TC-3′ were an-nealed, digested withXhoI and inserted into pSG5.EGF-HA.The receptor binding pocket mutations were introducedinto pSG5.EGF-HA and pSG5.EGF-G4S-HA by standardcloning procedures and details can be obtained from theauthors.

In order to remove the globular head region, aHindIII/XhoIcloning site flanked by two (G4S)3 linkers was insertedbetween theMscI and BamHI sites at positions 207 and824 respectively of HA. This resulted in the constructpSG5.HArdel. The EGF sequence was again inserted be-tween theHindIII and XhoI sites to produce the constructpSG5.HArdelEGF. The detailed cloning strategy can beobtained from the authors upon request.

All constructs were confirmed by restriction digest andsequence analysis.

2.3. Fusion assay

One day prior to transfection, 2× 105 TELCeB6 cellswere plated in wells of six well plates. Cells were trans-fected with 5�g of each plasmid encoding HA constructs.Two days after transfection, cells were washed with phos-phate buffered saline (PBS) and then incubated for 5 minat 37◦C with fusion buffer (10 mM HEPES, 10 mM MESin PBS, pH 5.2). Cells were then washed in warm serumfree medium and then incubated with complete mediumat 37◦C for 4 h to allow the formation of syncytia. Thecells were fixed in 0.02% glutamine and stained with5-bromo-4-chloro-3-indoyl-�-d-galactopyranoside (X-gal).This resulted in blue staining of the nuclei as a consequenceof the nuclear localization signal of the LacZ expressed bythese cells.

2.4. Western blot analysis

2–3×106 293T cells were plated onto 10 cm dishes 1 dayprior to transfection. The cells were transfected with 5�gpSFG.EGFP, a retroviral vector encoding the enhanced greenfluorescent protein (Lindemann et al., 1997), 10�g pHIT60,encoding MLV Gag and Pol (Soneoka et al., 1995) and 10�gHA plasmid. For HArdelEGF, TELCeB6 cells were platedat 2× 106 cells, and transfected with 30�g plasmid. Twodays after transfection, cells were lysed and analyzed bystandard Western blotting procedures except that cells werelysed in 20 mM Tris–HCl pH 7.5, 1% Triton X-100, 0.05%sodium dodecyl sulfate and 5 mg/ml sodium deoxycholate.Blots were analyzed with a rabbit anti-EGF antibody (SantaCruz Biotechnology, Heidelberg) and horseradish peroxi-dase (HRP)-conjugated protein A (Amersham Life Science,Freiburg) and developed with an enhanced chemilumines-cence kit (Amersham Life Science, Freiburg).

Supernatants of transfected cells were examined as de-scribed previously before (Erlwein et al., 2002) and the West-ern blot was carried out as described above.

2.5. Cell surface staining

Transfected cells were detached from plates using0.02% EDTA in PBS. Cells were washed in PBS contain-ing 2% fetal calf serum, and stained with polyclonal ratanti-HA (Genovac, Freiburg) or rabbit anti-EGF (SantaCruz Biotechnology, Heidelberg) antibodies, followed byFITC-conjugated anti-rat (PharMingen, Heidelberg) orPE-conjugated anti-rabbit (Dianova, Hamburg) antibod-ies. Fluorescence of the stained cells was analyzed with afluorescence-activated cell sorter (FACScan, Becton Dick-inson, Heidelberg).

2.6. Binding assay

2.5 ml of filtered vector supernatant was incubated with5 × 105 COS 7 cells for 1 h at 4◦C. The cells were thenwashed and then stained with rat anti-HA followed by FITC-conjugated anti-rat antibodies. Fluorescence was measuredusing a fluorescence-activated cell sorter.

2.7. Tyrosine phophorylation assay

Transfected 293T cells were incubated overnight inmedium containing 2% fetal calf serum. 4× 105 COS7 cells were plated in wells of six well plates and incu-bated overnight in 5% serum and then for approximately1 h in serum free medium. Supernatants from transfectedcells was added to a well of COS 7 cells and incubated for10 min at 37◦C. The COS 7 cells were then lysed as abovebut with buffer supplemented with phosphatase inhibitorcocktails I and II (Sigma, Taufkirchen). Thirty microgramsof each lysate was analyzed as described inErlwein et al.,2002. A mouse monoclonal anti-phospho-tyrosine antibody(Cell Signaling Technology, Frankfurt/Main) and a rabbitanti-EGF receptor antibody (Santa Cruz Biotechnology,Heidelberg) were used to analyze the Western blot.

2.8. Immunofluorescent staining

2 × 105 TELCeB6 cells were plated on glass coverslips.The next day, the cells were transfected with 2�g HA con-struct. Two days after transfection, the cells were fixed in4% paraformaldehyde/PBS and either stained immediatelyor following permeabilization with 0.3% Triton X-100/PBSfor 5 min. HA proteins were detected with anti-EGF anti-body (Santa Cruz Biotechnology, Heidelberg) and a Cy3TM

labeled secondary antibody (Zymed, Berlin).

3. Results

3.1. Construction of EGF-HA and EGF-G4S-HA mutants

The sequence encoding the 53 amino acids of EGF wasobtained from pSG5.EGFenv (Erlwein et al., 2002), and


was inserted between the signal peptide and the N-terminusof HA1, resulting in the construct EGF-HA. In agreementwith the results of others (Hatziioannou et al., 1999), theEGF-HA protein is properly processed, is incorporated intoMLV vector particles and shows specific increased bindingto EGF receptor positive cells (data not shown). As vectorsbearing this protein still display the wide host range of thewild type HA protein, we attempted to remove the naturalsialic acid binding activity through the introduction of pointmutations within the receptor binding pocket. Although thebinding domain of FPV HA has not yet been fully charac-terized, the equivalent site in the HA protein of H3 strainshas been extensively studied through crystallographic andmutational analyses (Skehel and Wiley, 2000; Martin et al.,1998). Mart́ın et al. (1998) showed that the substitutionsY98F, H183F and L194A in HA1 almost entirely abolishthe receptor binding activity of H3 strain HA. Althoughthese residues are conserved in FPV HA, their role in re-ceptor binding may differ since HAs from human strainspreferentially bind sialic acids in�2,6 linkages, while thosefrom avian strains prefer�2,3 linkages. However, the crystalstructure of the H5 avian strain HA receptor binding pocketbound to a receptor analog indicates that these residues arealso involved in the binding of�2,3-linked sialic acids (Haet al., 2001). Using site-directed mutagenesis, we thereforemade mutants containing double or triple combinations ofthese substitutions. Each of these mutants was found to be

Fig. 1. Characterization of HA receptor binding domain mutants. (A) FACS analysis of TELCeB6 cells transiently transfected with wild type HA and HAproteins containing point mutations within the receptor binding domain. Cells were stained with rat anti-HA antibody followed by FITC-conjugated anti-ratantibody, and HA surface expression was detected by a shift to green fluorescence. Thick line, HA protein; thin line, negative control of non-transfectedcells. (B) Syncytia formation of transiently transfected TELCeB6 cells. Following a 5 min exposure to pH 5.2, the multinucleated cells were stained withX-gal. This resulted in blue staining of the nuclei as a consequence of the nuclear localization signal of the LacZ expressed by these cells.

well expressed on the cell surface (Fig. 1A) and capable ofinducing cell–cell syncytia following exposure to low pH(Fig. 1B). Vectors bearing these mutants show reduced titerson target cells compared to those bearing wild type HA,indicating a reduction in receptor binding. Similar resultshave recently been published by (Lin and Cannon, 2002).We therefore introduced these mutations into the sialic acidbinding pocket of EGF-HA. We also made a second seriesof mutants in which an oligo encoding a (G4S)4 linker wasinserted into theXhoI site between EGF and HA1. This wasdone for two reasons: to increase exposure of EGF on thevector surface, and because the presence of a linker is ex-pected to increase the proper folding of the separated pro-tein domains. The construction of the mutant HA genes isdepicted inFig. 2.

3.2. Expression of EGF-HA and EGF-G4S-HA mutants

Each of the constructs was transiently transfected into293T cells along with a MLV Gag/Pol expression plasmidand a GFP encoding retroviral vector, and 2 days later,the cells were lysed in order to analyze protein expression.Fig. 3A and Bshow the results of Western blot analysis ofthe cell lysates. All of the proteins show good expression,although the receptor binding pocket mutants show reducedor absent HA1, indicating poor processing. This is especiallytrue for the HL and YHL mutants. In order to check for in-


Fig. 2. Schematic representation of constructs. The restriction sites used for cloning are indicated. SP, signal peptide; rbd, receptor binding domain; fp,fusion peptide; tm, transmembrane domain.

corporation into vectors, supernatants of transfected 293Tcells were ultracentrifuged through a 20% sucrose cushion,and the pellets were subjected to Western blot analysis. Theresults are shown inFig. 3C and D. Incorporated proteincould only be detected for constructs containing the wildtype receptor binding pocket or the YH and YL substitu-tions. The presence of the (G4S)4 linker appears to slightlyincrease incorporation (Fig. 3D). The proteins that can bedetected in association with vector particles are those that

Fig. 3. Expression of HA mutants and their incorporation into vector particles. (A) Western blot analysis of lysates of 293T cells transiently transfectedwith EGF-HA proteins. (B) Western blot analysis of lysates of 293T cells transiently transfected with EGF-G4S-HA proteins. (C) Western blot analysisof vector supernatants released from 293T cells transiently transfected with EGF-HA proteins. Supernatants were purified by ultracentrifugation through20% sucrose. (D) Western blot analysis of vector supernatants released from 293T cells transiently transfected with EGF-G4S-HA proteins. Supernatantswere purified by ultracentrifugation through 20% sucrose. All blots were analyzed using rabbit anti-EGF antibody followed by HRP-conjugated protein A.

show a higher level of processing in the cell lysate blots,and, as for wild type HA, it is the cleaved HA1 form thatis preferentially incorporated. As furin cleavage takes placeduring passage through thetrans-Golgi, it seems likely thatonly the processed proteins are reaching the cell surfaceand are therefore available for incorporation into vectors. Inorder to directly test cell surface expression we performeda fluorescence activated cell sorter analysis of transfected293T cells. As shown inFig. 4, those constructs previously


Fig. 4. FACS analysis of 293T cells transiently transfected with EGF-HA and EGF-G4S-HA proteins. Cells were stained with rabbit anti-EGF antibodyfollowed by PE-conjugated anti-rabbit antibody, and surface expression of proteins was detected by a shift to red fluorescence. Thick line, HA protein;thin line, negative control of cells transfected with only Gag/Pol and vector plasmids.

observed to be better processed and incorporated, also showhigher cell surface expression levels. The presence of the(G4S)4 linker also appears to slightly increase the level ofsurface expression, particularly for the HL mutant. Unfor-tunately, unlike both the EGF-HA and EGF-G4S-HA pro-teins containing wild type receptor binding pockets, none ofthe mutants were able to induce cell–cell syncytia forma-tion following exposure to low pH (pH 5.2) buffer (data notshown), indicating that precursor cleavage and cell surfaceexpression are not sufficient for proper function.

3.3. Binding to the EGF receptor

In order to test the ability of our proteins to bind tothe targeted EGF receptor we incubated vector supernatantsof transfected 293T cells with EGF receptor positive COS7 cells. Binding was then analyzed by FACS using an an-tibody directed against HA. Only the YH and YL mutantswere tested since these were shown to be incorporated intovector particles. The results are shown inFig. 5A. Wild typeHA shows only a low level of binding, since the bindingto sialic acid is barely detectable at the temperature of 4◦Cused in the experiment (Hatziioannou et al., 1999). In con-trast, both EGF-HA and EGF-G4S-HA show a distinct in-crease in binding, presumably due to their interaction withthe EGF receptor. However, none of the receptor bindingpocket mutants show any increase in binding compared towild type. None of the constructs showed specific bindingto 3T3 cells that do not express the EGF receptor (data notshown).

As an alternative approach to test binding to the EGF re-ceptor, we made use of the ability of EGF to induce tyrosine

phosphorylation of its receptor. Following a 10 min exposureto vector supernatants, COS 7 cells were lysed and subjectedto Western blot analysis using an antibody directed againstphosphorylated tyrosine residues. As a positive control wemade use of vectors bearing EGF-Env, an MLV Env pro-tein containing EGF that has been previously shown by ourgroup to induce tyrosine phosphorylation of the EGF recep-tor (Erlwein et al., 2002). As shown inFig. 5B, an increase inthe density of the band at approximately 180 kD can be seenin the sample exposed to EGF-Env in comparison to thoseexposed to wild type HA or negative control vectors bearingno envelope glycoprotein. The binding of an anti-EGF recep-tor antibody to a protein running at the same level in a sec-ond blot confirms the identification of this band as tyrosinephosphorylated EGF receptor. All six of the EGF contain-ing HA proteins appear to induce tyrosine phosphorylation,indicating an interaction with the receptor. No significantvariation can be seen in a second protein detected by the an-tibody, suggesting that the interaction is specific for this re-ceptor. A stronger induction is seen with the constructs con-taining the wild type receptor binding pocket, which corre-lates with the results from the FACS analysis. Although vec-tor particles containing the EGF-HA or EGF-G4S-HA YHand YL mutants bound to EGF-receptor expressing cells, asexpected from the fusion assays transduction of EGF recep-tor positive or negative cells could not be detected (data notshown).

3.4. Replacement of the globular head of HA with EGF

In order to test whether direct replacement of the globularhead region of HA could be tolerated we removed this do-


Fig. 5. Vector particle binding to EGF receptor. (A) Binding of EGF-HA and EGF-G4S-HA proteins to COS 7 cells. Cells were incubated withvector-containing supernatants and analyzed by FACS. Staining was carried out with rat anti-HA antibody followed by FITC-conjugated anti-rat antibody,and bound HA protein was detected by a shift to green fluorescence. Thick line, bound HA protein; thin line, negative control of cells incubated withvector supernatant containing no HA protein. (B) Tyrosine phosphorylation assay. COS 7 cells were exposed to vector-containing supernatants for 10minand cell lysates were then analyzed by Western blot. Blots were analyzed using mouse anti-phospho-tyrosine antibody followed by HRP-conjugated sheepanti-mouse antibody (upper blot), or rabbit anti-EGF receptor followed by HRP-conjugated protein A (lower blot).

main using natural restriction sites and inserted EGF, flank-ing it with two (G4S)3 linkers. This resulted in the constructHArdelEGF. Transfected TELCeB6 packaging cells (Cossetet al., 1995) were lysed and analyzed by Western blot. Asshown inFig. 6A, a single band was detected in transfectedcell lysates, again indicating a lack of precursor cleavage.No protein could be detected in vector pellets. In order totest cell surface expression of the construct we again carriedout FACS analysis. As shown inFig. 6B, in contrast to wildtype HA and EGF-HA, no cell surface HArdelEGF could bedetected. This result was confirmed by immunofluorescentstaining of transfected cells. Staining of EGF-HA was seenin both permeabilized and non-permeabilized cells (Fig. 6C).In contrast, although strong staining of HArdelEGF couldbe seen in permeabilized cells, little or no protein was de-tected in non-permeabilized cells, indicating no cell surfaceexpression.

4. Discussion

We attempted to redirect retroviral vectors to EGF recep-tor expressing cells. MLV vectors were used pseudotypedwith FPV HA which had the wild type receptor binding ab-rogated either by point mutations or by deletion of the glob-ular head structure of HA and into which the EGF sequencewas also inserted. Replacement of the whole head domainwith EGF was not tolerated. Moreover, most receptor bind-ing mutants did not permit the additional insertion of EGF,which lead to a reduced or abrogated surface expression.Only two binding deficient EGF-fusion proteins were ex-pressed on the cell surface and could be incorporated intoretroviral particles. Although these proteins appeared to bindto the EGF receptor their fusion activity was lost and trans-duction of EGF receptor positive or negative cells could notbe detected.


Fig. 6. Characterization of the HArdelEGF mutant. (A) Western blot analysis of cell lysates and vector supernatant from TELCeB6 cells transientlytransfected with HArdelEGF. Vector supernatant was purified by ultracentrifugation through 20% sucrose. The arrow indicates the size of the banddetected in the cell lysate. Both blots were analyzed using rabbit anti-EGF antibody followed by HRP-conjugated protein A. (B) Surface expression ofHArdelEGF, analyzed by FACS. Transiently transfected TELCeB6 cells were stained either with rabbit anti-EGF antibody followed by PE-conjugatedanti-rabbit antibody, or with rat anti-HA antibody followed by FITC-conjugated anti-rat antibody. Cell surface HA proteins were detected by a shiftto redfluorescence or to green fluorescence, respectively. Thin line, HA protein; Thick line, negative control of non-transfected cells. (C) Immunofluorescentstaining of transiently transfected TELCeB6 cells. Cells were fixed in 4% paraformaldehyde and either stained immediately or following permeabilizationwith 0.3% Triton X-100/PBS. HA proteins were detected with rabbit anti-EGF antibody followed by Cy3TM-conjugated anti-rabbit antibody.

The most likely explanation for the lack of or reduction incell surface expression of our mutants is that they are mis-folded and trapped within the endoplasmic reticulum (ER).During normal folding, wild type HA interacts with twolectin-like ER chaperones, calnexin and calreticulin (Hebertet al., 1996). The function of these proteins is to bind to im-mature and misfolded proteins, retaining them in the ER (re-viewed inBraakman and van Anken, 2000). Misfolded HAhas also been shown to form complexes with the HSP70 ho-mologue BiP, which are retained in the ER for long periodsbefore degradation (Hurtley et al., 1989). The proposal thatthe mutants are retained within the ER is supported by thefact that the level of surface expression and incorporationinto vector particles appears to correlate with the degree ofprecursor cleavage. Lack of cleavage could indicate that theprotein does not reach thetrans-Golgi complex, the region

of furin protease activity. In addition, the insertion of the(G4S)4 linker into the EGF-HA constructs increases cleav-age, surface expression and incorporation of some mutants.This linker is expected to increase proper folding by separat-ing EGF and HA, thereby preventing interference betweenthe two domains.

It is perhaps not surprising that the globular head re-placement mutant HArdelEGF was retained within thecell, since we replaced a large section of the protein withunrelated sequences.Zhang et al. (1997)generated a se-ries of C-terminally truncated fragments of HA by addingpuromycin to influenza virus infected cells. They found thatthe majority of these fragments were retained within the ERin association with calnexin and calreticulin. Less than 2%of the fragments were secreted, indicating a very stringentquality control. The fragments that were secreted consisted


of entire folding domains, suggesting that complete do-mains are required to pass the quality control mechanism.Since we removed the whole globular head region, thoughtto fold as a complete domain (Chen et al., 1995; Braakmanet al., 1992), replacing it with EGF and spacers, it mightbe expected that our construct consisted only of complete,independently folding domains. However, although EGF isa small molecule, its N- and C-termini are not adjacent toone another when it is folded properly (Cooke et al., 1987).This could prevent the bringing together of the separateHA1 polypeptide regions of the stem domain. A secondconstruct in which the EGF sequence was replaced withthe Myc tag was also retained in the cell (data not shown),although this tag is much shorter. However, the presence ofthe spacer regions could also hinder this process.

Despite reaching the cell surface, the EGF-HA andEGF-G4S-HA mutants containing YH and YL were unableto induce cell–cell fusion (data not shown) under conditionssufficient to induce large syncytia in the absence of EGF(Fig. 1B). This suggests that although the proteins havepassed through the quality control mechanisms of the cellthey are nevertheless non-functional. Since EGF-HA andEGF-G4S-HA are able to induce syncytia formation (datanot shown) the lack of fusion cannot be due to the presenceof EGF. It is possible that the level of fully cleaved proteinon the cell surface is too low to induce fusion. However, thefact that the cleaved protein is preferentially incorporatedinto vectors (Fig. 3C and D) indicates that the majority ofprotein on the cell surface is processed. It seems unlikelythat cleavage is a pre-requisite for incorporation since theHA proteins of non-pathogenic strains of influenza virus canbe incorporated into budding virions in an uncleaved state.However, it remains feasible that the total level of cell sur-face protein of these mutants is too low. The modificationsmay also prevent proper trimer formation, although this hasbeen reported to prevent cell surface expression (Copelandet al., 1986). Another possibility is that the introduced mod-ifications have altered the pH sensitivity of these mutants.Several reports have described HA mutants that show an in-creased sensitivity to higher pH values (e.g. (Daniels et al.,1985; Lin et al., 1997)). We have not looked at this questionin detail since we never saw any transduction with thesemutants, a vital property for use in the targeting of vectorsfor gene therapy. A closer study of the biochemical and bio-physical properties of these mutants may however provideinteresting information on the fusion process of HA.

The incorporated mutants do appear to be able to bindto the targeted EGF receptor, as detected by the inductionof tyrosine phosphorylation (Fig. 5B). The lower level ofphosphorylation seen with the YH and YL mutants is likelyto be a consequence of the lower protein levels. This mayalso be the reason for the lack of binding detected in theFACS experiment (Fig. 5A), since this is expected to be a lesssensitive method. It is also possible that these mutants bindto the receptor with lower affinity. This may result in barelydetectable binding at 4◦C, as observed with wild type HA

(Fig. 5A; (Hatziioannou et al., 1999)). The possibility cannotbe excluded that the polyclonal anti-HA antibody used forthe FACS analysis is unable to recognize these mutants,although it does recognize all of the binding mutants in theabsence of the EGF domain.

Taken together, the results indicate that the addition ofboth EGF and point mutations within the receptor bindingpocket destabilizes the HA protein, leading to poor process-ing and poor function. It may be expected that the HA pro-tein is already inherently unstable due to its priming for ac-tivation through a reduction in pH. In fact, one function ofthe M2 protein of influenza virus is to raise the pH within in-tracellular vesicles, thereby preventing premature activationof cleaved HA protein (Ciampor et al., 1992). Such activa-tion of HA within the cell leads to permanent inactivation(Takeuchi and Lamb, 1994; Ohuchi et al., 1994). Althoughwe had no problems expressing wild type HA in our system,it is possible that the mutants undergo premature activationwithin the cell, leading to degradation or to the cell surfaceexpression of inactivated proteins. Since a reverse geneticssystem for influenza virus is now available (Neumann et al.,2000), it would be interesting to test the function of thesemutants in an influenza virus background, since M2 andother viral proteins may have a stabilizing influence.

In conclusion, it appears that the inherent instability ofHA coupled with the stringent quality control mechanismswithin the ER make the retargeting of this protein to spe-cific cell types more complex than previously thought.However, with careful choice of targeting ligand and in-sertion/mutation site this may still be possible. In our ex-perience EGF is the ligand best tolerated by HA. We havealso tried inserting the closely related transforming growthfactor � (TGF�), the Myc tag and single chain antibodiesat the N-terminus of HA, all of which lead to poor pro-cessing and incorporation into MLV vectors. Interestingly,other work in our group has provided similar results forMLV Env, i.e. that EGF is well tolerated, but not TGF� orthe Myc tag (Erlwein et al., 2002). It therefore appears thatcareful choice of ligand is required for successful targeting.The site of insertion is also likely to be very important.Several regions within retroviral Env proteins, including theN-terminus, proline rich region and the variable region A,have been found to tolerate insertions (Russell and Cosset,1999; Lavillette et al., 2001; Haynes et al., 2003). With fur-ther analysis, suitable sites may also be found in HA. Caremust be taken, however, when mutating multiple sites. Aswe have shown here, insertion of EGF at the N-terminus orpoint mutation within the receptor binding domain is toler-ated, but insertion into the same construct results in loss offunction. Replacement of the whole head domain was alsonot tolerated. While this may still be possible with care-ful choice of ligand and spacers, it seems likely that moresubtle insertions within this region will be better tolerated.It was found recently that the insertion of short peptideswithin an external loop close to the receptor binding pocketcan sometimes allow retention of fusion activity. The bind-


ing and transducing activity of such constructs is, however,yet to be tested.

It has been shown previously that it is possible to tar-get retroviral vectors by separating the binding and fusionevents on two separate proteins using binding-defective FPVHA and a chimeric MLV Env that facilitates binding (Linet al., 2001). Although these data made HA appear a veryattractive protein to be used to directly target gene transferto cancer cells, we found that introduction of EGF in com-bination with the abrogation of the natural receptor bindingproperties is not tolerated. Until better insertion sites in HAare found coexpression of a targeted MLV Env protein anda binding-defective influenza HA protein will be the methodof choice to target HA containing vectors.

Acknowledgements

The authors wish to thank Wolfgang Garten for the FPVHA cDNA and Alexandra Bittner for expert technical assis-tance. This work was funded by the Sander Stiftung.

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Retroviral vector targeting through insertion of epidermal growth factor into receptor binding deficient influenza A hemagglutinin results in fusion defective particles