Directed Evolution of a Virus Exclusively Utilizing Human EpidermalGrowth Factor Receptor as the Entry Receptor

Hong-Sheng Dai,a,b Zheng Liu,a Wen Jiang,a Richard J. Kuhna,c

Department of Biological Sciences,a Purdue University Interdisciplinary Life Science Program,b and Bindley Bioscience Center,c Purdue University, West Lafayette, Indiana,USA

Rational design and directed evolution are powerful tools to generate and improve protein function; however, their uses aremostly limited to enzyme and antibody engineering. Here we describe a directed-evolution strategy, named the tandem selectionand enrichment system (TSES), and its use in generating virus with exclusive specificity for a particular cellular receptor. InTSES, evolving viruses are sequentially and iteratively transferred between two different host cells, one for selection of receptorspecificity and the other for enrichment of the fittest virus. By combining rational design and TSES, we generated human epider-mal growth factor receptor (EGFR)-specific virus 1 (ESV1). ESV1 has the backbone of Sindbis virus (SINV) and displays an EGFdomain engrafted onto structural protein E2 after residue Pro192, together with eight amino acid changes stabilizing the E2-EGFchimera. ESV1 uses EGFR to initiate infection and has lost the capacity to interact with all known SINV receptors. A 12.2-Å cryo-electron microscopic (cryoEM) reconstruction of ESV1 reveals that the E2-EGF fusion adopts a fixed conformation, with EGFsitting at the top of the E2 spike; The EGFR binding interface faces outward, and the EGF domain completely masks SINV recep-tor binding. The cryoEM structure of ESV1 explains the desirable properties of ESV1 and provides insights for its further modifi-cation. TSES expands the scope of directed evolution and can be easily extended to other targeting molecules and viral systems.

Rational design and directed evolution are complementarystrategies to create proteins with novel functions (1). The pri-

mary successful application of the two methods has been in theareas of enzymes and antibodies because enzymes and antibodiesare highly evolvable biopolymers and optimized products can beeasily screened for by chemistry- or affinity-based selection meth-ods (2–5). Extending these approaches to create complex molec-ular machines, for example, making a viral vector that uses a tai-lored cellular receptor, is of substantial biological significance butremains a daunting challenge (6, 7).

Oncolytic virotherapy, which uses therapeutic viral vectors toselectively infect and kill cancer cells while sparing normal tissues,has recently emerged as a valuable clinical tool (8, 9). Most virusesdepend on particular cellular receptors to initiate infection (10).Therefore, reconstruction of virus-receptor binding is a widelyused approach to generate cancer-specific viruses by arming scaf-fold viruses with a targeting molecule that binds to the cellularreceptor unique or overexpressed in the cancer cell and abolishingnatural receptor binding (11). Epidermal growth factor receptor(EGFR) signaling, which regulates cell growth, proliferation, anddifferentiation, has been implicated in the development of multi-ple cancers and intensely studied as a therapeutic target (12, 13).Almost 30 years of research has resulted in four anti-EGFR drugsin clinical use for treating epithelial cancers overexpressing EGFR(13). However, these EGFR antagonists frequently have a low re-sponse rate, the development of drug resistance, and lack of spec-ificity (13), necessitating new therapeutic strategies against EGFR.Generation of an oncolytic virus that specifically targets EGFRmight be a promising approach to overcome some problems withcurrent EGFR antagonists.

EGF, a polypeptide of 53 amino acids with three disulfidebonds (14), is the natural ligand for EGFR, and thus, rationaldisplay of the EGF domain on a scaffold virus might target thevirus to EGFR. The advantages of using EGF to target EGFR in-clude the following. (i) The compact structure of EGF minimizes

disturbance to the scaffold virus. (ii) EGF is an independent func-tional entity and large enough to potentially mask the native re-ceptor binding of the scaffold virus. (iii) EGF binding stimulatesEGFR to undergo clathrin-mediated endocytosis (15), the mech-anism by which many viruses enter host cells (10). However, viralvectors generated by this method usually show low specificity andstability and need further optimization (11, 16). Directed evolu-tion is a useful technique to improve protein function, but theapplication of directed evolution to optimize a viral vector is ham-pered by the lack of an effective selection method that takes intoaccount both the stability of the virus and its specificity for thetargeted receptor.

We proposed here a general strategy that combines rationaldesign and directed evolution to make a viral vector with preciseEGFR-targeting ability. Accordingly, this strategy is composed oftwo components, (i) rational design of a virus able to use EGFR forinfection and (ii) a new directed-evolution system, called the tan-dem selection and enrichment system (TSES), to simultaneouslyoptimize EGFR specificity and virus stability. Using this strategy,we developed EGFR-specific virus 1 (ESV1), which shows exclu-sive specificity for human EGFR. Moreover, a 12.2-Å cryoelectronmicroscopic (cryoEM) reconstruction of ESV1 was built, demon-strating the structural basis of the superior EGFR specificity of

Received 26 April 2013 Accepted 1 August 2013

Published ahead of print 7 August 2013

Address correspondence to Hong-Sheng Dai, daihsh@gmail.com, orRichard J. Kuhn, kuhnr@purdue.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JVI.01054-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.01054-13

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ESV1. The structural and functional characteristics of ESV1 vali-dated the value of our strategy in viral vector development.

MATERIALS AND METHODSCells and viruses. ZR7530, SKBR2, MDA231, U87MG, M059J, andBT474 cells were purchased from the American Type Culture Collection,Manassas, VA. MDA435 cells were kindly provided by Shuang Liu (Pur-due University). Raji, DCSIGN Raji, and LSIGN Raji cells were a kind giftof Ted Pierson (NIAID) (17). All cells were cultured in minimum essentialmedium (MEM; Invitrogen catalog no. 61100) supplemented with 10%fetal bovine serum (FBS; Thermo catalog no. SH3039603), except Raji celllines, which were grown in RPMI 1640 medium (Invitrogen catalog no.61870). Toto64, an HRsp derivative having extra restriction sites (18), wasused as the wild-type Sindbis virus (SINV) in all experiments (19). Togenerate cells that stably express human EGFR, BHK cells were transfectedwith a wild-type EGFR plasmid (Addgene catalog no. 11011) and selectedwith 10 �g/ml puromycin (Invitrogen catalog no. A11138). To generateEG435 cells expressing EGFR-green fluorescent protein (GFP), we trans-fected MDA435 cells with the EGFR-GFP construct, which we made byfusing GFP to the C terminus of EGFR.

Molecular cloning and virus production. The EGF module was in-serted into the SINV E2 sequence by a standard cloning technique (Fig.1A). The sequences of Toto64 and all of the primers used are availableupon request. To facilitate library construction and restrict mutations toE2 domains A and B and EGF, silent mutations of 192PEGF were made tointroduce a unique PvuI site at the N terminus of E2 and an SphI site at thejunction of E2 domains B and C (see Fig. 4A). The coding sequence be-

tween the PvuI and SphI sites was subjected to error-prone PCR (Strat-agene catalog no. 200550) and cloned back into the 192PEGF(PS) vector.Ligation products were used to transform MC106 competent cells. Thelibrary size was calculated on the basis of transformation efficacy. Theconstruction of red fluorescent protein (RFP)-SINV has been describedpreviously (20). To construct RFP-ESV1, RFP coding sequences were am-plified with PvuI-flanked primers and cloned into PvuI-digested ESV1plasmids. Virus production has been described previously (19, 20).

Immunostaining and immunoblotting. HeLa cells were infected withvirus at a multiplicity of infection (MOI) of 1 for 16 h. Raji cells wereinfected with virus at an MOI of 5 for 48 h. Cells were fixed with 2%paraformaldehyde, permeabilized, and blocked in phosphate-buffered sa-line (PBS) containing 0.3% Triton and 2% bovine serum albumin for 20min prior to incubation with SINV capsid antibodies (1:500) for 45 mineach at room temperature. Subsequently, cells were stained with fluores-cently labeled secondary antibodies (Invitrogen catalog no. T2767, 1:500)and 4=,6-diamidino-2-phenylindole (DAPI; Invitrogen catalog no.D21490). Stained samples were analyzed on an Olympus ZX-1 epifluores-cence microscope with an X-Cite 120Q light source. Membranes wereincubated overnight at 4°C with antibodies against actin (MilliporeMAB1501, 1:10,000), EGFR (Cell Signaling Technology D38B1, 1:2,500),SINV E2 (Milton Schlesinger, Washington University, St. Louis, MO;1:5,000), or SINV capsid (1:5,000). Membranes were washed and thenincubated for 45 min with the appropriate IRDye-labeled secondary an-tibody (LI-COR, 1:10,000) at room temperature and protected from light.Membranes were scanned with an Odyssey scanner at wavelengths of 700and 800 nm. Quantification was performed with Odyssey V3.0 software.

FIG 1 Rational design of viruses displaying an EGF domain. (A) Schematic representation of the SINV genome and the organization of EGFR-targetingconstructs. The EGF domain (light green) flanked by the linker sequence (red) was inserted right after the designated E2 residues. Residue numbers correspondto the sequence with PDB code 3J0F. (B) Sequence of the EGF cassette. The linker is shown in red with the NotI and XhoI restriction sites underlined, and the EGFcoding sequence is in light green. (C) BHK cells were electroporated with in vitro-transcribed viral RNA. Expression of E2-EGF fusion protein was determinedby immunoblotting against actin, EGF, and SINV E2 and capsid 16 h later. The values to the left are molecular sizes in kilodaltons. (D) Plaque sizes and titers ofrecombinant viruses on BHK cells. The titers at the bottom are averages of duplicate samples. (E) Six clones of each virus were picked after seven passages, andtheir E2-EGF fragments were amplified by RT-PCR and digested with NotI and XhoI. Black arrowheads indicate the EGF cassette.

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Plaque assays. Serial dilutions or �100 PFU of viruses were reconsti-tuted in 400 �l PBS. To competitively inhibit virus binding, the virusdilutions were added to confluent BHK or EGFR� BHK cells grown onsix-well plates in the presence or absence of 100 �g/ml heparin (Sigma-Aldrich catalog no. H3149), 160 �M ammonium ferric citrate (Sigma-Aldrich catalog no. F5879), or 0.4 �g EGF antibody (R&D Systems catalogno. AF236), respectively. After a 30-min incubation at room temperature,virus-reagent mixtures were removed and cells were layered with MEMsupplemented with 5% FBS and 1% agarose and incubated at 37°C in 5%CO2. Forty-eight hours later, cells were stained with neutral red for 6 h at37°C to visualize the plaques.

EGFR knockdown. Twenty percent confluent HeLa cells grown onsix-well plates were transfected with 3 �l small interfering RNA1(siRNA1) or siRNA2 (Cell Signaling Technology catalog no. 6481) mixedwith 7.5 �l RNAiMax (Invitrogen catalog no. 13778-075) in accordancewith the manufacturer’s instructions. Forty-eight hours later, cells wereinfected with viruses at an MOI of 1 or lysed with 2� loading buffer(Thermo catalog no. 39000) for immunoblotting. To increase the knock-down efficacy, 100 ng EGF (R&D Systems catalog no. 236-EG) was addedto each well during the first 24 h of knockdown.

Cell viability assay. Cell viability was evaluated with the Quick CellProliferation Assay kit (BioVision catalog no. K301) according to themanufacturer’s protocol. Briefly, cells (1 � 104/well) were seeded into thewells of a 96-well plate in culture medium at 100 �l/well. After 24 h ofincubation at 37°C, cells were infected with viruses at an MOI of 5. Atspecified time points, 10 �l 2-(4-iodophenyl)-3-(2,4-dinitrophenyl)-5-phenyl-3H-tetrazolium-2=,4=-disulfonate sodium salt solution was addedto each well. Absorbance at 450 nm was determined by spectrometer after2 h of incubation at 37°C.

TSES. Sixty percent confluent MDA231 cells were infected with theevolutionary library. Following a 40-min incubation at 37°C, virus-con-taining supernatants were aspirated and cells were flushed twice with freshmedium to remove unbound virus. Subsequently, MDA231 cells weregrown in fresh medium for 2 days until supernatants were collected for theinfection of EGFR� BHK cells. Supernatants from EGFR� BHK cells wereharvested at 24 h postinfection, diluted, and added to MDA231 cells forsecond-round selection. Heparin (100 �g/ml) was included in all of theselection steps. After four cycles of TSES, supernatants were collected andsubjected to a plaque assay. Individual plaques were picked from theplaque assay and transferred to infect EGFR� BHK cells. Twenty-fourhours later, supernatants were individually collected, subjected to clonepurification, or stored at �80°C for future use; infected cells were lysedwith TRIzol reagent for RNA extraction. The E2-EGF region was ampli-fied by reverse transcription (RT)-PCR and sequenced or, in some cases,cloned back into 192PEGF(PS) to make cDNA clones.

Virus purification and deglycosylation. Viruses were purified ac-cording to previously published protocols (21), except that ESV1 wasgrown on EGFR� BHK cells. Four micrograms of purified SINV or ESV1was digested at 37°C for 4 h with 1,250 U of endoglycosidase H (EndoH;NEB catalog no. P0702) or peptide N-glycosidase F (PNGase F; NEB cat-alog no. P0704) under native conditions.

Single virus particle imaging and analysis. EG435 cells were plated inglass bottom dishes (MatTek catalog no. P35G010) and grown in MEMcontaining 1% FBS overnight prior to imaging. Cells were transferred to aprewarmed (37°C) live-imaging chamber (Tokai Hit TIZW) and equili-brated for 30 min before the commencement of imaging. Purified RFP-SINV or RFP-ESV1 was added to cells and visualized with a Nikon A1Rconfocal microscope with a Piezo stage. Samples were simultaneouslyexcited at 488 and 561 nm, and the emitted light was collected by aPlanApo 60�/1.4 oil immersion objective, split, and passed through 500-to 550-nm and 570- to 620-nm band-pass filters. Image series were re-corded at 4 frames/min with a galvano scanner or at 120 frames/min witha resonant scanner, and five z stacks spaced 0.5 �m apart were acquired.Galvano scanner images taken at 4 frames/min were subjected to mini-mum processing with NIS-Elements software, including convolution

with a Gaussian spatial filter, intensity equalization, and maximum-inten-sity projection.

CryoEM reconstruction. CryoEM images were collected with aPhilips CM200 at 200 kV and recorded on Kodak SO163 film under low-dose (�20 e/Å2) conditions at a nominal magnification of �38,000 withunderfocus values of 1.5 to 5.0 �m. Particles were manually selected frommicrograph images with the graphic program boxer in the EMAN soft-ware package (22). The contrast transfer function parameters were auto-matically fitted and verified with the EMAN ctfit program (23, 45). Thewhole data set (21,291 particles) was divided into two half subsets (even

FIG 2 192PEGF displays a functional EGF domain. (A) EGF neutralizationexperiment conducted with BHK cells. Fifty to 100 PFU of each virus wereincubated with PBS, IgG control, or EGF antibody (Ab) (0.4 �g) for 30 min atroom temperature prior to plaque assays. (B) Examination of EGFR expres-sion in EGFR� BHK cells by immunoblotting. The values to the left are mo-lecular sizes in kilodaltons (k). (C) Fifty to 100 PFU of each virus were equallyloaded onto BHK and EGFR� BHK cells for plaque assays. Enhancement ofinfectivity was calculated by dividing the number of plaques each virus formedon EGFR� BHK cells by the number of plaques it formed on BHK cells (n � 3).(D) BHK and EGFR� BHK cells were preincubated with 1 �g IgG control orincreasing amounts of EGFR blocking antibody for 30 min, and then viruseswere equally loaded onto cells for plaque assay. BHK cells, which do not ex-press EGFR, were included to show the EGFR-specific inhibition by the block-ing antibody. Plaque numbers were normalized to the number of plaquesformed on PBS-pretreated cells (n � 5). All data are means � SEM.

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and odd subsets), and all subsequent image processing was performedindependently for the two half data sets. The initial icosahedral model wasbuilt de novo by using 300 randomly selected particles with sequentialassignment of random orientations, iterative refinements, and impositionof icosahedral symmetry. All image processing, including the initialmodel, two-dimensional (2D) alignments, and 3D reconstruction, weredone by the in-house-developed jspr.py program using the EMAN/EMAN2 programs and library function. By pooling particles from the twohalf data sets, the final density map was reconstructed from 2,290 of21,291 particles, with the majority of the particles rejected on the basis ofalignment scores and stabilities of orientation assignments at differentstages of the refinements. The final resolution (12.2 Å) of the reconstruc-tion was determined from the two independently refined density mapsbased on Henderson’s 0.143 Fourier shell cross-correlation coefficients(FSC) criterion (24). VEDA was used to fit the atomic models of SINV andEGF with symmetry constraints (25). The SINV structure (Protein DataBank [PDB] code 3j0f) was first manually placed in the correspondingposition in the density map. The final positions were obtained by optimiz-ing the correlation between the electron microscopic density map and theatomic model with the URO fitting algorithm. After fitting of the SINVasymmetric unit, the EGF (PDB code 1jl9) was subsequently fitted into theextra density at the tip of surface trimer. The EM reconstruction has been

deposited in the Electron Microscopy Data Bank (EMDB) under acces-sion code EMD-5699.

Statistical analysis. The quantitative values in this work are means �the standard errors of the means (SEM) unless otherwise noted. Data setswere statistically evaluated with a t test.

RESULTSSelection of a scaffold virus. To rationally design a virus capableof binding EGFR, we first searched for a scaffold virus that shouldbe able to present EGF in a functional way while still maintainingthe original virus structure. The criteria for choosing the scaffoldvirus were (i) the availability of a high-resolution virus structure,(ii) receptor binding separated from other functions of viral pro-teins, (iii) no linkage to severe human disease, and (iv) reportedoncolytic activity. SINV, a positive single-stranded RNA virus,stands out because of its structural features (21), oncolytic efficacy(26), and minimal biosafety issues (27).

SINV has an 11-kb genomic RNA encoding four nonstructuralproteins (nsP1 to nsP4) and three major structural proteins, cap-sid, E2, and E1 (Fig. 1A) (27). E1 and E2 are transmembrane

FIG 3 192PEGF uses EGFR to infect human cancer cell lines. (A) Cell lines were infected with SINV expressing an mCherry reporter. Images were captured at24 h postinfection. All scale bars are 100 �m, except that of ZR7530, which is 50 �m. (B) Expression of EGFR in 10 human cancer cell lines was examined byimmunoblotting. (C) Cell lines were infected with SINV or 192PEGF virus, and cell viability was determined at specified time points by measuring optical density(OD) at 450 nm (n � 10 replicates from three independent experiments; see Materials and Methods for details). All data are means � SEM.

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glycoproteins, and their ectodomains form heterodimers with E1sequestered beneath E2; 240 E1-E2 heterodimers are assembledinto 80 trimeric spikes to form the icosahedral SINV particle (21).E2 ectodomains are composed of three immunoglobulin do-mains, A (residues 1 to 135), B (residues 172 to 232), and C (res-idues 264 to 343), linked by connector peptides. Domain A con-stitutes the central region of the spike; domain B forms the distalhead; and domain C forms the stem and glues three E1-E2 het-erodimers together (21, 28, 29). Other than maintaining virusstructure, E2 (specifically, domains A and B) binds to cellular

receptors and E1 mediates membrane fusion under low-pH con-ditions within endosomes (21). Functional separation of E2 andE1 suggests that substitution of SINV receptor binding with othertarget molecules may not damage the membrane fusion activity ofE1. Structurally, domains A and B protrude away from the spher-ical virus particle and form a perfect platform for displayingEGFR-targeting molecules without interfering with other func-tions of the virus.

Rational design of viruses displaying the EGF domain. Onthe basis of the structures of SINV E2 and EGF, we rationallydesigned 11 constructs in which an EGF domain was individuallyinserted into 11 surface-exposed loops in E2 domains A and B(Fig. 1A and B). The EGF cassette consists of a NotI restriction site,an extra T following the NotI site to maintain the reading frame,the EGF coding sequence, and a short linker at the 3= end thatcontains an XhoI site (Fig. 1B). Because E2 domains A and B areresponsible for receptor binding, this insertional design is in-tended to simultaneously obtain EGFR binding and disrupt nat-ural SINV receptor binding.

We first tested the expression of E2-EGF fusion proteins byWestern blotting (Fig. 1C). SINV structural proteins are firstlytranslated as a polypeptide and then cleaved by capsid and cellularsignalase to yield individual proteins; therefore, the E2 glycopro-tein in cell lysates exists mostly in the form of E3-E2 or E3-E2-E1(27). SINV E2 blotting showed two bands, representing E3-E2/E3-E2-EGF (lower band) and E3-E2-E1/E3-E2-EGF-E1 (upperband), respectively. EGF blotting specifically showed E3-E2-EGFand E3-E2-EGF-E1 bands. The extra EGF domain caused a mo-

TABLE 1 EGFR expression and SINV susceptibility of human cancercell lines

Cell line SINV susceptibilitya EGFRb

BT474 � �MDA231 � �SKBR3 � �ZR7530 � �MDA435 � �Huh7 � �HepG2 � �HeLa � �U87MG � �M059J � �a Susceptibility to SINV infection was determined by infecting cells with SINVexpressing RFP (Fig. 3A). �, susceptible; �, resistant.b Expression of EGFR was confirmed by Western blotting (Fig. 3B). �, expression; �,no expression.

FIG 4 Construction of a 192PEGF evolution library and TSES. (A). Construct modifications for the generation of a 192PEGF evolutionary library. The PvuI andSphI sites were removed from the original 192PEGF plasmid by mutagenesis. A new PvuI site was introduced at the junction of E3 and E1, and an SphI site wasintroduced after EGF-E2 residue Ala332 by silent mutagenesis. The fragment between the unique PvuI and SphI sites, containing the EGF domain and E2domains A and B, was subjected to error-prone PCR and cloned back into the 192PEGF(PS) vector to generate the library. (B) TSES flow chart. The 192PEGFlibrary contains viruses that vary in EGFR specificity and stability. MDA231 selection eliminates viruses with little or no EGFR specificity, and EGFR� BHKenrichment increases the sensitivity of the system by amplifying viruses with higher stability. Four cycles of TSES were conducted before single-clone purification.Products of TSES can be evolved further (dashed line).

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bility shift, and thus, all E3-E2-EGF and E3-E2-EGF-E1 proteinslagged behind the E3-E2 and E3-E2-E1 proteins, respectively. Theresult demonstrated that all 11 constructs had normal expressionand cleavage of viral proteins. However, only three viable viruseswere recovered, 71QEGF, 162TEGF, and 192PEGF, with the EGFsequence being inserted after E2 Gln71, Thr162, and Pro192, re-spectively (Fig. 1D). The resulting viruses were attenuated, form-ing pinpoint-size plaques and very low titers, indicating that EGFinsertion affects virus stability, production, or receptor binding.Because of the low fidelity of viral RNA-dependent RNA polymer-ase (30) and the large number of virus particles produced in eachSINV infection cycle, SINV revertants appear very fast and recom-binant viruses tend to suffer from genome instability (31). Todetermine whether the three EGF-tagged viruses are geneticallystable, viruses were continuously passaged seven times in BHKcells. Following passages, six clones of each virus were picked andtheir E2-EGF fragments were amplified by RT-PCR and digestedwith NotI and XhoI (Fig. 1E). Restriction enzyme digestionshowed that the EGF sequence was still presented in the virusgenome after continuous passages, indicating that the short EGFinsertion does not affect viral genome stability. This was also con-firmed by sequencing of these E2-EGF fragments (not shown),which showed no additional mutations after seven passages.

EGFR mediates 192PEGF infection. To test whether EGF dis-played on 71QEGF, 162TEGF, and 192PEGF is surface accessible,we treated viruses with PBS, IgG control, or EGF neutralizing

antibody (0.4 �g) for 30 min at room temperature prior to plaqueassays. The EGF neutralization experiment showed that EGF an-tibody was able to specifically block the infection of all three EGF-tagged viruses but not that of SINV (Fig. 2A), demonstrating thatEGF is presented on the surface of these recombinant viruses. Toinvestigate whether the surfaced-exposed EGF domain can medi-ate virus infection, we generated the EGFR� BHK cell line, whichstably expressed human EGFR (Fig. 2B). We conducted plaqueassays with both BHK and EGFR� BHK cells by loading each cellline with the same amount of diluted virus. While SINV, 71QEGF,and 162TEGF formed almost the same number of plaques on bothBHK and EGFR� BHK cells, 192PEGF formed 10 times more plaqueson EGFR� BHK cells than it did on BHK cells (Fig. 2C). Therefore,ectopic expression of human EGFR enhanced the infectivity of192PEGF virus. Furthermore, the enhanced infectivity of 192PEGFwas specifically inhibited by an EGFR antibody that blocked EGF-EGFR interaction (Fig. 2D). These data suggest that 192PEGF has afunctional EGF domain that can bind human EGFR.

However, SINV evolved to use multiple receptors to infect dif-ferent host cells (32–35). It is thus important to validate the dataobtained with BHK by using other cell lines. We screened 10 hu-man cancer cell lines for EGFR expression and susceptibility toSINV infection (Fig. 3A and B; Table 1) and identified two breastcancer cell lines, MDA231 and SKBR3, that express high levels ofEGFR but are resistant to SINV. We thus determined whether192PEGF can infect MDA231 and SKBR3 cells by measuring cell

FIG 5 Phenotypes of viruses with improved EGFR specificity and stability. (A) Plaque sizes and titers of each ESV. Below the chart are listed the mutations arisingin each ESV. E2-EGF fragments were amplified, sequenced, and aligned with the original 192PEGF sequence. Plaque sizes (left y axis) were measured on BHK(black box) and EGFR� BHK (red box) cells. The box-and-whisker plot shows the median, minimum, and maximum sizes of plaques (n � �10). Virus titers(right y axis and blue bar, n � 3) were determined by plaque assay with EGFR� BHK cells. (B) EGFR specificity indexes of ESVs. Virus plaque formation assayswere conducted with both BHK and EGFR� BHK cells. EGFR specificity indexes were calculated as described in the text. Data are means � SEM.

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viability following infection. We also included Huh7 (liver cancercell line) and MDA435 (melanoma cell line [36]) cells for com-parison. While they are both susceptible to SINV infection, Huh7cells express EGFR but MDA435 cells do not (Fig. 3A and B). Theinfection dynamics revealed that SINV was unable to infectMDA231 cells and infected SKBR3 cells less efficiently than Huh7and MDA435 cells, while the ability of 192PEGF to actively infectMDA231 and SKBR3 cells was enhanced (Fig. 3C). The differen-tial effects of SINV and 192PEGF virus infections of MDA231 andSKBR3 cells further validated the suggestion that 192PEGF dis-played a functional EGF, acquired the ability to bind EGFR, andused this receptor to infect cells. The failure of SINV to infectMDA231 and the acquired ability of 192PEGF to infect MDA231(Fig. 3A and C) suggest that (i) MDA231 does not have the entrymachinery required for SINV infection and (ii) infection ofMDA231 cells by 192PEGF is mediated purely by EGF-EGFR en-gagement. However, 192PEGF was still able to infect MDA435cells (Fig. 3C), implying that 192PEGF still retained SINV receptortropism.

TSES for directed evolution of viruses. 192PEGF is far fromperfect, showing an attenuated phenotype, mediocre EGFR spec-ificity, and residual SINV tropism. We speculated that the E2-EGF

fusion was not in a fully functional state and therefore disturbedvirus stability and EGFR interaction. Directed evolution has beenwidely used to improve protein function and usually uses eitherenzyme activity or binding affinity as selection pressure for thesingle purpose of enhancing the performance of enzymes or anti-bodies (2–4). To optimize a virus with a tailored receptor by di-rected evolution, however, we have to focus on both receptorbinding and virus stability, which cannot be done by existingchemical or affinity-based screening methods.

We thus developed a new directed-evolution strategy calledTSES to achieve full integration of the two functional componentsof the E2-EGF chimera, as well as to remove the residual SINVtropism. A 192PEGF evolution library was first generated by in-troducing random mutations into 192PEGF E2-EGF residues 1 to331, covering domains A and B and the EGF domain (Fig. 4A). Toevaluate the quality of the evolution library, we picked 96 individ-ual clones from it and sequenced them. Fifty-three of the 96 se-quences yielded high-quality reads, and thus, they were trimmedto 700 high-quality bases and aligned with the 192PEGF sequence(not shown). Sequence analysis revealed that mutations are evenlydistributed throughout the E2-EGF coding sequence, error-pronePCR introduces predominately point mutations, and the average

FIG 6 EGFR is the entry receptor for ESV1 infection. (A) Immunoblot assays showing the expression of EGFR in EG435 cells. The values to the left are molecularsizes in kilodaltons (k). (B) EG435 cells were stimulated with EGF (200 nM), and images were taken every 4 min. Scale bar, 10 �m. (C) Viability of MDA435 andEG435 cells following virus infection. Cells were infected with SINV or ESV1 at an MOI of 5. Cell viability was determined at 0, 24, 48, and 72 h postinfection (n �7 replicates from two experiments). Data are means � SEM. OD 450, optical density at 450 nm. (D and E) A 1:1 mixed culture of MDA435 (gray) and EG435(green) was infected with RFP-SINV (D) or RFP-ESV1 (E). Cells were observed by microscopy or fixed at different time points for flow cytometry. Scale bar, 100�m. Lower panels show the percentages of EG435 cells determined by flow cytometry. d, day(s).

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mutation rate of the library is 9.6/kb. The library displayed greatdiversity, with 1 � 106 independent clones.

To specifically and sensitively screen the library for the idealclones with the highest stability and specificity (Fig. 4B), libraryclones were sequentially and iteratively selected with MDA231cells and enriched with EGFR� BHK cells. The flat curve of192PEGF infecting MDA231, compared with that of 192PEGFinfecting Huh7 and MDA435 cells (Fig. 3C), indicates that thereplication of 192PEGF in MDA231 cells is restrained at a lowlevel. These features of MDA231 could impose strong selectionpressure on the library, eliminating mutants having little or noEGFR binding ability and retaining only viruses with the highestadaptability and EGFR specificity. Those EGFR-specific clones aresubsequently enriched and amplified in EGFR� BHK cells.

EGFR� BHK cells are supersusceptible to SINV and producehigh-titer virus; therefore, EGFR� BHK enrichment also increasesthe sensitivity of the system by amplifying viruses that are under-represented in the starting pool. Heparin has been reported tononspecifically facilitate many viral infections through electro-static interactions (10); thus, it is added at both steps to competi-tively reduce nonspecific binding.

Phenotypes of EGFR-specific viruses. In TSES, high-titer vi-rus could outgrow low-titer viruses and only EGFR-specific vi-ruses could survive MDA231 selection. Therefore, viruses havingevolved the optimal structure and specific EGFR binding willeventually become predominant in the pool. After four cycles ofiterative selection and enrichment, we purified 47 individual vi-ruses, amplified their E2-EGF fragments by RT-PCR, and se-

FIG 7 Single virus particle tracking of the virus entry process. Purified RFP-SINV (A) or RFP-ESV1 (B) particles were added to EG435 cells (see Materials andMethods for details). Insets are enlargements of boxed areas. Virus particles are red, EGFR is green, and the overlap is yellow. Scale bar, 10 �m.

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quenced them. We found that they belonged to six independentESVs and contained a very diverse mutation spectrum (Fig. 5A).We conducted plaque assays with BHK and EGFR� BHK cells todetermine their titers, plaque sizes, and EGFR specificities (Fig. 5Aand B). To quantitatively evaluate virus specificity for EGFR, wecreated an EGFR specificity index, which is the number of plaquesformed on EGFR� BHK cells divided by the number of plaquesformed on BHK cells. All six ESVs had larger plaque sizes and atleast a 2-log increase in titer compared with the parental 192PEGFvirus. ESV1, containing eight amino acid changes, reached titersnear the SINV titer and formed 0.8-mm plaques on BHK cells but3.5-mm plaques on EGFR� BHK cells. Accordingly, its EGFRspecificity was approximately 500 (Fig. 5B), which means that99.8% of infectious ESV1 cannot initiate an infection in the ab-sence of EGFR. ESV2 was about 10-fold more specific for EGFRthan the 192PEGF virus was, while the other four clones showedlittle or no improvement in EGFR specificity. To exclude the pos-sibility that the improved phenotype of ESV1 and ESV2 was aconsequence of unrelated mutations elsewhere in the viral ge-nome, we recloned the E2-EGF fragments of ESV1 and ESV2 intothe original SINV genome, thereby ensuring that no other muta-tions were present. We found that they maintained the same phe-notype; confirming that this improved phenotype was the directresult of directed evolution. We then focused our study on ESV1because of its high specificity for EGFR.

EGFR is the entry receptor for ESV1 infection. To determinethe role of EGFR in mediating ESV1 infection and monitor thetraffic of EGFR upon virus infection, we fused GFP to the C ter-minus of EGFR. We transfected MDA435 cells and generated theEG435 cell line, which stably expresses EGFR-GFP (Fig. 6A). Aspreviously reported (37), EGFR-GFP was localized at the plasmamembrane and EGFR endocytosis occurred very quickly upon

EGF treatment (Fig. 6B). The parental 192PEGF virus retainedSINV tropism and was able to infect EGFR null MDA435 cells(Fig. 3C); however, ESV1 lost its ability to infect MDA435 cells,while it aggressively killed EG435 cells (Fig. 6C). This result sug-gests that the eight additional mutations on ESV1 might abolishthe residual SINV tropism and thus contribute to the higher EGFRspecificity seen.

To visualize the virus entry process, we also generated RFP-SINV and RFP-ESV1, which have mCherry fused to the N termi-nus of E2 and E2-EGF, respectively. We infected a 1:1 mixed cul-ture of MDA435 and EG435 cells with RFP-SINV or RFP-ESV1(Fig. 6D and E). SINV killed both cell types within 3 days (Fig.6D). However, RFP-ESV1 selectively infected and depleted EG435cells while leaving MDA435 cells essentially uninfected and dom-inant in the mixture (Fig. 6E). This experiment further confirmedESV1’s high specificity for EGFR.

To get a real-time view of virus entry and virus-receptor inter-action, we infected EG435 cells with RFP-labeled viruses andtracked the entry process by confocal microscope. SINV particlesloosely attached to and randomly moved on the cellular mem-brane (Fig. 7A; see Movie S1 in the supplemental material). Wedid not observe any consistent colocalization of SINV and EGFR.SINV particles did not enter EG435 cells during the 20-min ob-servation period (Fig. 7A; see Movie S1). In comparison, ESV1bound EGFR and triggered EGFR accumulation and endocytosiswithin 2 min (Fig. 7B; see Movie S2), which paralleled the rate ofEGF uptake (Fig. 6B) (15). At every single point of entry, ESV1particles were encapsulated in and moved together with EGFRendocytic vesicles. Single virus particle tracking not only corrob-orated that ESV1 used EGFR as the entry receptor but also re-vealed that EGF-mediated endocytosis was far more efficient thanSINV entry. Because ESV1 and SINV have the same replication

FIG 8 EGFR knockdown especially and proportionally inhibits ESV1 infection of HeLa cells. (A) Western blot assay showing the knockdown of EGFR by twodifferent siRNAs in the presence or absence of EGF (100 ng/ml). The relative EGFR levels shown above the gel were normalized to �-actin. The values to the rightare molecular sizes in kilodaltons (k). (B) Following siRNA silencing, HeLa cells were infected with viruses and stained for SINV capsid. Scale bar, 100 �m; cont,control. (C) Percentages of capsid-stained cells (capsid %) were generated from three randomly picked microscopic fields of each sample. Cont, control.

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machinery and thus the same rate of replication, the acceleratedentry step of ESV1 infection may explain why the plaques of ESV1on EGFR� BHK cells were larger than those of SINV althoughESV1’s titer was slightly lower (Fig. 5A).

No involvement of canonical SINV entry mechanisms inESV1 infection. ESV1’s high specificity for EGFR drove us to askwhether EGFR is the only receptor for ESV1 to initiate infection.HeLa cells are susceptible to SINV infection and express high lev-els of EGFR (Fig. 3A and B); hence, we used HeLa cells to deter-mine the effect of decreasing EGFR expression on ESV1 infection.Knockdown of EGFR expression with siRNA (Fig. 8A) did notaffect SINV infection but protected HeLa cells from ESV1 infec-tion (Fig. 8B). EGF treatment has been reported to shorten thehalf-life of EGFR (15). We combined siRNA transfection and EGFtreatment to further reduce the EGFR level (Fig. 8A) and observeda lower level of ESV1 infection (Fig. 8B). A statistical summary ofthe results revealed that ESV1 infectivity was proportional to thelevel of EGFR expressed on HeLa cells (Fig. 8A and C). Theseresults strongly indicate that EGFR is the only cellular receptorthat mediates ESV1 infection of HeLa cells.

SINV has a very broad tropism and infects very diverse hostcells (27). Postulated SINV receptors/attachment factors includeglycan binging protein DCSIGN and LSIGN (35), natural resis-tance-associated macrophage protein 2 (NRAMP2) (33), andheparin (38). We next individually explored the roles of theseSINV receptors in ESV1 infection. SINV E2 Asn196 is an N-gly-cosylation site and has been reported to augment SINV infectivityby interacting with glycan-binding proteins DCSIGN and LSIGN(35). Ectopic expression of either DCSIGN or LSIGN on Raji cellsenhanced SINV infectivity, but ESV1 infection was restrictedamong the three Raji cell lines tested (Fig. 9A). The inability ofDCSIGN and LSIGN to enhance ESV1 infection may be caused byloss of glycosylation because of EGF insertion or by blocking ofglycan-SIGN interaction by the EGF domain. On ESV1 virions,the N-glycosylation residue Asn257 (corresponding to SINVAsn196) and consensus sequence are intact, suggesting that loss ofglycosylation is unlikely to happen. To experimentally excludethis possibility, we purified SINV and ESV1 and digested viruseswith EndoH and PNGase F, which cleave N-linked glycan differ-ently (39). We conducted a mobility shift analysis to examine the

FIG 9 Canonical SINV receptors are not involved in ESV1 infection. (A) Native Raji cells, DCSIGN�Raji, and LSIGN�Raji cells were infected with viruses andstained for SINV capsid. Scale bar, 100 �m. (B) Immunoblotting against SINV E2 and capsid was conducted to evaluate the mobility shift of E2 (black arrow) andEGF-E2 (red arrow). Cont, control. (C) EGFR expression in Raji cell lines was examined by Western blotting. The values to the left are molecular sizes inkilodaltons (k). (D) BHK and EGFR� BHK cells were treated with PBS or 160 �M Fe3� prior to and during a plaque assay. Plaque reduction efficacy of Fe3� wasnormalized to the plaque numbers of a PBS control (n � 3). All data are means � SEM. (E) Viruses were incubated with heparin (100 �g/ml) or PBS during thebinding step of the plaque assay.

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molecular weight change following deglycosylation (Fig. 9B).SINV capsid is not a glycoprotein, and thus, its molecular weightwas not changed after glycosidase digestion. The SINV E2 andESV1 E2-EGF proteins shifted faster upon glycosidase treatmentand had the same N-glycosylation pattern. Because the EGF inser-tion site, Pro192, is so close to the Asn196 glycosylation site, wespeculated that the EGF domain covered glycan and blockedDCSIGN and LSIGN binding. This was later confirmed by thecryoEM structure of ESV1. Furthermore, all Raji cell lines showedno EGFR expression (Fig. 9C), explaining why ESV1 cannot infectRaji cell lines.

NRAMP2 is a ferric transporter and has been recently definedas a SINV receptor (33). Rose et al. reported that Fe3� treatmentdepleted NRAMP2, reduced SINV binding, and protected U2OScells from infection (33). We confirmed here that Fe3� treatmentinhibited SINV infection of BHK cells. However, we did not seeFe3� treatment inhibiting ESV1 infection of either BHK orEGFR� BHK cells (Fig. 9D), implying that NRAMP is not in-

volved in ESV1 infection. In addition, ESV1 was selected underheparin competition and independent experiments also con-firmed that heparin competitively inhibited SINV infection andhad no effect on ESV1 (Fig. 9E). Taken together, the results ob-tained with different infection systems demonstrate that ESV1infectivity is dependent solely on EGFR.

cryoEM structure of ESV1. To relate the functional propertiesof ESV1 to its structure, we used cryoEM (Fig. 10A) and 3D re-construction to generate a 12.2-Å resolution structure of ESV1(Fig. 10B) (40). ESV1 preserves all of the structural features ofSINV, including (i) 80 trimeric spikes arranged on the T�4 ico-sahedral lattice, (ii) a lipid bilayer penetrated by paired transmem-brane helices of E1 and E2, and (iii) a nucleocapsid core anchoredto the inner leaflet of the lipid bilayer (Fig. 10C and D) (21). TheE2-EGF fusion protein forms a heterodimer with E1 the same wayas the wild-type E1-E2 heterodimer (Fig. 10E and F) (21). How-ever, ESV1 has a diameter of 730 Å (Fig. 10C and D), which islarger than the 700-Å diameter of SINV. The extra diameter of

FIG 10 cryoEM reconstruction of ESV1. (A) Typical micrograph of ESV1 following cryogenic freezing. Scale bar, 50 nm. (B) The FSC (0.143) of the recon-struction indicates the effective resolution to be 12.2 Å. (C) Radially colored, surface-shaded representation of the 3D reconstruction of ESV1. (D) Central slabof the reconstructed density map revealing internal features of the ESV1 structure. (E) Top view of the trimeric spike. The EGF, E2, and E1 structures were fittedinto the density map. E1 is red, E2 is dark blue, and EGF is yellow. Green dots show the positions of Asn257 glycosylation sites, which are hidden under EGFdensity. (F) Side view of the spike showing the alpha-carbon trace of EGF, E1, E2 and capsid molecules fitted into the reconstruction. (G) Closeup view of the spikestructure. Domain A is green, domain B is cyan, and EGF is orange. Mutated residues are highlighted as magenta balls. Residue numbers correspond to theE2-EGF sequence of 192PEGF. The green dot shows the position of the Asn257 glycosylation site.

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ESV1 is contributed by the density of EGF, which is about 40 Å(length) by 30 Å (width) by 20 Å (height) and sits at the top of theglycoprotein spike (Fig. 10F and G). Fitting of the crystal struc-tures of EGF (PDB code 1jl9), SINV E2, and E1 (PDB code 3j0f) asa rigid body into the density reveals that E2-EGF adopts a fixedconformation and the EGFR binding interface faces outward (Fig.10G). Although the EGF domain is inserted after E2 Pro192,which is in the middle of domain B, domain B maintains its orig-inal structure, as well as its position relative to domain A and theE1 protein.

The EGF domain completely covers E2 domain B and partiallycovers domain A (Fig. 10F and G). The Asn257 glycosylation site ishidden under EGF (Fig. 10E), which explains why DCSIGN andLSIGN do not facilitate ESV1 infection. Furthermore, three EGFdomains form a 20-Å-high tripod-like cushion that deepens thecavity of the trimeric spike (Fig. 10F and G). Because of sterichindrance, such a cushion very likely minimizes potential interac-tions between SINV cellular receptors and the remaining accessi-ble surface of domain A. Thus, the structure clearly explains whyESV1 has lost the ability to interact with DCSIGN, NRAMP, andheparin. Mapping of the eight mutations of ESV1 onto the recon-struction reveals that five mutations (H73N, A193E, D206N,V230E, and S254P) are located at the interface between E2 domainB and the EGF domain (Fig. 10G). Although we are unable toassign each of the point mutations an exact role at the currentresolution, these mutations likely improve the interface and thusstabilize the E2-EGF fusion protein and contribute to higher-titerviruses.

DISCUSSION

Viruses are the product of millions of years of evolution with verysophisticated inter- and intramolecular interplay among viralproteins, nucleotides, and lipids. The generation of a virus with atailored receptor (EGFR in our case) is not a trivial task. In thisstudy, we divided this task into two sequential subtasks, i.e., ac-quisition of EGFR binding ability by rational design and optimi-zation of EGFR specificity via directed evolution. First we built the192PEGF virus by rational design. The 192PEGF virus displaysfunctional EGF domains, as exemplified by its ability to infectEGFR-expressing MDA231 cells, which SINV is unable to infect.However, the 192PEGF virus is attenuated and has residual SINVtropism. To optimize 192PEGF, we constructed a 192PEGF evo-lutionary library. Current library selection methods, including af-finity maturation and genetic/chemical complementation, are in-tended for the adaptation of antibodies and enzymes and are notapplicable to virus evolution (1). On the basis of the supersuscep-tibility of BHK to SINV infection and special features of MDA231,we developed the TSES to screen the 192PEGF evolutionary li-brary for virus with exclusive specificity for EGFR. TSES usesMDA231 cells to maintain EGFR specificity and EGFR� BHK cellsto increase the sensitivity of selection and virus stability.

TSES yielded ESV1, which shows superior specificity, losinginteractions with all known SINV receptors and infecting onlyEGFR-expressing cells. Five of the eight amino acid changes inESV1 are located at the interface between EGF and E2 domain B;this fact highlights the ability of TSES-backed directed evolutionto optimize viral vectors. The successful generation of ESV1 vali-dates the idea that TSES can simultaneously achieve three goals, (i)increased virus stability, (ii) enhanced receptor specificity, and(iii) removal of the residual tropism of the scaffold virus. The

extensive knowledge of the SINV structure and finding or gener-ating permissive cell lines (MDA231 in the case of SINV) thatexpress the targeting receptor but were not susceptible to the scaf-fold SINV contributed to the successful retargeting of SINV toEGFR. Given that cell lines permissive for a particular virus existnaturally or can be easily generated by transfection or knockout ofspecific cellular factors, TSES could be a general approach formaking viral vectors with precise targeting ability.

Cancer specificity is the crucial element for therapeutic tools tobe effective against malignancy and has hardly been achieved suf-ficiently to date. Random mutagenesis has been used to modifythe host range of influenza virus, retrovirus, and adeno-associatedvirus (41–44). However, without coupling with rational design,random mutagenesis cannot effectively direct viral vectors towardany particular cellular receptor. To our knowledge, this is the firsttime that rational design and directed evolution have been com-bined to generate a virus exclusively specific to a cellular receptor.Because of its high specificity for EGFR and the demonstratedoncolytic efficacy of SINV (26), Esc1 can now be exploited foroncolytic uses in vivo.

ACKNOWLEDGMENTS

We thank S. Liu (Purdue University) for MDA435 cells, T. Pierson(NIAID) for Raji cell lines, D. Griffin (John Hopkins University) for SINVantibody, and A. Tyler at the Bindley Bioscience Imaging core facility fortechnical support.

This study is supported by NIH grant GM056279 to R.J.K.

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