Single-round selection yields a unique retroviralenvelope utilizing GPR172A as its host receptorPeter M. Mazari1, Daniela Linder-Basso1, Anindita Sarangi, Yehchung Chang, and Monica J. Roth2

University of Medicine and Dentistry of New Jersey–The Robert Wood Johnson Medical School, Department of Biochemistry, Piscataway, NJ 08854

Edited by Stephen P. Goff, Columbia University College of Physicians and Surgeons, New York, NY, and approved February 18, 2009 (received for reviewSeptember 29, 2008)

The recognition by a viral envelope of its cognate host-cell receptoris the initial critical step in defining the viral host-range and tissuespecificity. This study combines a single-round of selection of arandom envelope library with a parallel cDNA screen for receptorfunction to identify a distinct retroviral envelope/receptor pair. The11-aa targeting domain of the modified feline leukemia virusenvelope consists of a constrained peptide. Critical to the bindingof the constrained peptide envelope to its cellular receptor are apair of internal cysteines and an essential Trp required for main-tenance of titers >105 lacZ staining units per milliliter. The receptorused for viral entry is the human GPR172A protein, a G-protein-coupled receptor isolated from osteosarcoma cells. The ability togenerate unique envelopes capable of using tissue- or disease-specific receptors marks an advance in the development of efficientgene-therapy vectors.

library screening � retroviral entry � viral envelope � viral receptor

The binding of the retroviral envelope protein (Env) to itscognate host receptor is the critical first step in infection. The

expression profile of a given receptor therefore regulates theviral host-range and tissue specificity. Viral receptors that arebroadly and abundantly expressed may be a desirable trait innature and for general gene delivery. This distribution, however,represents a significant obstacle in the development of retrovi-ral-based gene-therapy vectors, where a targeted vector, whichutilizes a receptor that is specific to the target tissue or pathologicstate (cancerous vs. healthy tissue), is desired for safety concerns.Recent gene-therapy protocols have successfully circumventedthis problem through ex vivo infection and reimplantation of thetreated cells (1). This method, however, is applicable only to asmall fraction of the disorders that could potentially benefit fromgene delivery. A major research goal is to be able to developalternative Env proteins, which provide the necessary specificitywhile still maintaining highly efficient gene delivery compatiblewith the viral entry pathway.

In gamma-retroviruses, including murine leukemia virus(MuLV) and feline leukemia virus (FeLV), receptor binding ismediated through a variable region (VRA) within the N-terminalhalf of the surface (SU) Env protein. In FeLV, replacing the VRAregion with that of another switches receptor usage accordingly (2).For targeted entry, studies have looked into inserting bindingdomains (3, 4) and single-chain antibody regions (5) into Envbackbones to alter receptor usage to specific proteins. Two commonroadblocks to these methods are that (i) these Env may fail to beincorporated into virions (4, 5), and (ii) adding bulky constituentslike antibodies can interfere with postbinding events (3, 6). Ratherthan direct viral entry, the approach used in our studies randomizesa 10- to 11-aa region within the VRA of FeLV Env (7–9). Thiscreates an Env library of high complexity that can be screened forisolates capable of productive infection. In this method, the uniquebinding region is presented in the context of the Env backbone andin its natural conformation during the initial screening process.Using this method, Env proteins have been identified which useunique host-cell receptors outside the known viral interferencegroups (10).

Key to the development of new retroviral Envs is an under-standing of the potential host receptors, as not all surfaceproteins can provide the necessary structural and functionalrequirements to permit the latter steps in viral entry. Each FeLVsubgroup (11–14), like all other known gamma-retroviruses(15–21), utilizes multipass transmembrane receptors for entry.Furthermore, in some cases, multiple gamma-retroviruses usethe same receptor. MuLV amphotrophic Env, Gibbon-ape leu-kemia virus, and FeLV-B for instance, all use pit-1 as their hostreceptor (14, 15, 19). This suggests that while Envs can betargeted to bind to virtually any protein on the cell surface, notall provide the proper features to mediate fusion and subsequentviral entry.

This study demonstrates the development and screening of aunique Env library generated through the randomization of 11aa within the VRA of a FeLV-A and -C chimera. The 11-aareceptor targeting constrained peptide (CP) encodes uniquestructural features required for the presentation of an essentialTrp residue. The receptor for this Env isolate was identified asthe G-protein-coupled receptor, GPR172A, which serves as thehuman gamma-hydroxybutyrate (GHB) receptor (22), as well asthe human receptor for porcine endogenous retrovirus A(PERV-A) (16). Furthermore, GPR172A has been shown to becommonly up-regulated in human malignancies (23). Thus, asingle-round of selection and infection has yielded high titervirus (1.4 � 105 LSU/ml on the human osteosarcoma cell line143B), which has independently selected, as a cell-surface re-ceptor, a protein that has coevolved as the receptor for anendogenous virus from a distant species. The implications of thisfinding with respect to receptor usage and gene therapy will bediscussed.

ResultsDeveloping an 11-aa Random Env Library Within a FeLV A/C Backbone.Retroviral entry involves a complex series of interactions be-tween the viral Env protein and the host-cell receptor. Receptorbinding is the initial step of entry, triggering a series of confor-mational changes that ultimately yield membrane fusion. Formultiple retroviral Env proteins, various domains of SU havebeen implicated in receptor binding (24–28). Within the FeLV-Abackbone, however, the primary epitope lies within the VRAregion alone (2). Through randomizing a limited primary se-quence within this VRA region, a library of Env proteins havebeen generated and screened for productive infection into targetcells. Through this process, it has been established that retroviralentry can be targeted to novel host-cell receptors (7, 8, 10). To

Author contributions: M.J.R. designed research; P.M.M., D.L.-B., A.S., and Y.C. performedresearch; P.M.M., D.L.-B., A.S., and M.J.R. analyzed data; and P.M.M., D.L.-B., and M.J.R.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

1P.M.M. and D.L.-B. contributed equally to the manuscript.

2To whom correspondence should be addressed. E-mail: roth@umdnj.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0809741106/DCSupplemental.

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understand the scope of the potential receptor usage and thecorrelation with the evolution of known endogenous and exog-enous retroviruses, a new FeLV library was generated random-izing 11 amino acids within the VRA region.

Fig. 1 outlines the modified vector system. Two highlights ofthe modified vector (RIP) are that (i) the drug selection cassettehas been modified to encode an IRES-puromycin cassette ratherthan an internal SV40 promoter expressing neo, and (ii) the biasfor an arginine was removed from the library at position 6 (7).A constitutive library expressing the random Env cassettes wasgenerated through a 2-step process. The Env library was tran-siently expressed in 293TCeB cells (expressing Gag/Pol) in thepresence of vesicular stomatitis virus G glycoprotein (VSV-G)and the viral particles released were used to infect TECeBF2cells (29) at a low multiplicity of infection to ensure no more than1 viral particle per infected cell. The complexity of the librarywas 2 � 105. Viral supernatants from the constitutive librarywere then used to infect the Caki-1 renal carcinoma cell line,resulting in �100 puror Caki-1 colonies, which were maintainedas a population. PCR analysis of the bulk population yielded 1predominant Env isolate with the 11-aa insert shown (see Fig.1B). The general features of the insert are outlined in Fig. 1B.The randomized insert (indicated in bold) contains 3 arginineresidues as well as the potential for an alternative cysteine bond.

Host Range and Tissue Specificity. To determine the host and tissuespecificity of the CP Env, cell lines derived from a range ofspecies and tissue types were challenged with viral particlesbearing CP and packaging lacZ as a marker of gene transfer(Tables 1 and 2). Titers were measured as lacZ-staining units permilliliter of viral supernatant (LSU/ml).

The infectivity of virus bearing the CP Env was tested onmurine, rat, feline, canine, and human cell lines. Significantly, noinfection was detected on the feline fibroblast cell line AH927,the permissive cell line for both FeLV-A and -C viruses. Thisindicates that the CP Env is using a receptor outside of the knowninterference groups of FeLV. Similarly, no infection was ob-served on murine tail fibroblasts (NIH/3T3), eliminating recep-tor usage of the known ecotropic, polytropic, and amphotropic

MuLV viruses (18, 20, 21). High titer was observed on the canineD17 osteosarcoma cells (1.2 � 105), but not on the canine kidneycell line MDCK (2.2 � 101), indicating tissue specificity withinthe canine family. The rat osteosarcoma UMR-106 cells werenonpermissive to infection with CP-pseudotyped virus (�10LSU/ml), although infection with VSV-G pseudotyped virus wasefficient, eliminating the possibility of a postentry block toinfection (data not shown).

Productive infection into a wide-range of human cell lines wasobserved. Interestingly, although VSV-G was used to establishthe constitutive Env producer cells, titers on the 2 cell lines thatwere used to create the Env library, 293T and TE671, were high(4 � 105 and 2.5 � 105 LSU/ml, respectively). Similar titers,ranging from 8.9 � 104 to 2.2 � 105 LSU/ml, were found on all3 of the human osteosarcoma cell lines that were tested: 143B,MG63, and HOS. Furthermore, titers of 1.2 � 108 LSU/ml on143B cells were readily obtained with CP viral preparationsconcentrated by ultracentrifugation. High titers (�104 LSU/ml)were also seen on other cancer cell lines, including the cervicalcarcinoma line HeLa, and the liver carcinoma cell lines Huh-7and HepG2. Despite poor titers (�102 LSU/ml) on the prostatecancer cell line DU-145, much higher titers (3 � 104 LSU/ml)were seen on the PC-3 cell line, which is a prostate cancer lineisolated as a bone metastasis. Interestingly, titers in the range of103 or lower were found on all 4 of the renal carcinoma cell linesthat were tested, Caki-1, Caki-2, ACHN, and A498, despite thefact that the library was initially screened on the Caki-1 cell line.

Mutation Analysis of the CP Env. The 11-aa insert in the CP Envpresented several interesting characteristics (see Fig. 1), includ-

A

B

Fig. 1. Schematic of the Env library vector and CP-targeting peptide. (A)Schematic of the randomized Env-IRES-puro (RIP) library vector. The SV40promoter and the neomycin selection marker from pRVL (7) were replaced byan expression cassette in which an internal ribosomal entry site (IRES) drivesthe expression of puro. Position of the LTRs and viral genes are marked; �,packaging signal. (B) Sequence of the Env isolated from Caki-1. The 11-aaencoding the CP new binding insert are numbered in bold and flanked by theconserved regions of the FeLV envelope. The putative cystine bonds arerepresented and alternative configurations of bonds between cysteines arepossible. The cysteine loop in the parental FeLV Env consists of the outerresidues.

Table 1. Animal cell-line host range and tissue specificity

Cell lLine Host Tissue (pathology) Titer (LSU/ml)

D17 Dog Bone (osteosarcoma) 1.2 � 1.6 � 105

MDCK Dog Kidney (normal) 2.2 � 3.4 � 101

AH927 Cat Fibroblast (normal) �10UMR-106 Rat Bone (osteosarcoma) �10B16F1 Mouse Skin (melanoma) �10MC3T3 Mouse Bone (pre-osteoblast) �10NIH3T3 Mouse Fibroblast (normal) �10Primary

Calvaria OBMouse Bone (osteoblast) �10

Table 2. Human cell-line and tissue specificity

Cell lLine Human tissue (pathology) Titer (LSU/ml)

Caki-1 Kidney (carcinoma) 2.1 � 1.6 � 103

Caki-2 Kidney (carcinoma) �10A498 Kidney (carcinoma) 7.7 � 1.5 � 103

ACHN Kidney (carcinoma) 3.9 � 3.8 � 102

293T Kidney (normal) 4.0 � 2.0 � 105

TE671 Muscle (rhabdomyosarcoma) 2.5 � 1.3 � 105

143B Bone (osteosarcoma) 1.4 � 0.5 � 105

1.3 � 0.3 � 108a

HOS (CRL1543) Bone (osteosarcoma) 8.9 � 0.9 � 104

MG63 (CRL1427) Bone (osteosarcoma) 2.2 � 0.2 � 105

HeLa Cervix (carcinoma) 1.6 � 1.3 � 105

HepG2 Liver (carcinoma) 3.5 � 2.9 � 105

Huh-7 Liver (carcinoma) 2.0 � 0.9 � 104

DLD-1 Colon (carcinoma) 8.3 � 6.9 � 103

PC3 Prostate (metastatic carcinoma) 2.7 � 1.5 � 104

DU-145 Prostate (carcinoma) 7.0 � 4.7 � 101

HFF Foreskin fibroblasts (normal) 5.1 � 1.8 � 103

aVirus stock concentrated 56-fold by centrifugation.

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ing a putative cysteine bond and a clustering of basic residues. Toassess the role that each of the 11 residues plays as it relates toviral entry, alanine scanning was performed followed by site-directed mutagenesis of the residues that were found to benecessary for infection. The relative activity of each Env mutantwas measured by its ability to mediate gene transfer of the lacZmarker as compared to wild-type CP (Fig. 2). All of the Envs thathad lost infectivity were still incorporated into viral particles atlevels comparable to that of wild-type CP, as demonstrated byWestern blot (data not shown).

Replacement of the cysteines in position 4 or 9 with alanine(C4A or C9A) resulted in a complete loss of titer (see Fig. 2).Furthermore, the more conservative substitution of both cys-teines with serines in these positions (C4S;C9S) was unable torescue infectivity (see Fig. 2). To assess whether a disulfide bondexisted between these 2 positions, the residues in positions 4 and9 were replaced with glutamate and lysine, respectively(C4E;C9K) to determine whether a salt bridge between thesepositions could rescue Env function. This mutation resulted in nomeasurable titer (see Fig. 2), suggesting that these residues areforming disulfide bonds, but with residues outside of the ran-domized region. The glycine in position 8 also proved to benecessary for proper infectivity, as its replacement with alanine(G8A) resulted in a greater than 4 log decrease in titer (see Fig.2). The positioning of this f lexible residue immediately next to

a cysteine (position 9) may provide the necessary rotation topermit cystine bond formation, which could be sterically con-strained by alanine in the G8A mutant.

Beyond the amino acids implicated in the structural backbone,the tryptophan in position 11 proved to be essential for Envfunction. Replacement with alanine (W11A) resulted in a com-plete loss of function (see Fig. 2). Significantly, conservativereplacement with other aromatic residues (W11F, W11H, andW11Y) yielded similar results. Substitution of the second aro-matic residue, tyrosine in position 2 with alanine (Y2A) resultedin a 1 log decrease in titer (see Fig. 2), which could be restoredby replacement with other aromatic residues (Y2F, Y2H, Y2W)(see Fig. 2).

Although sequence scanning highlighted the presence of acluster of basic residues in the amino terminus of the insert,replacement of any of the individual basic residues with alanine(R1A, R5A, R6A) did not result in any appreciable decrease intiter (see Fig. 2), suggesting that these individual charges are notnecessary for binding. Additional mutations having no signifi-cant effect on binding or entry include L3A, R5K, T7A, andS10A (see Fig. 2.).

Binding Assay. To confirm that loss of infectivity associated withmutants C4A, C9A, C4E;C9K, G8A, and W11A were the resultof a loss of receptor binding and not a result of a defect in anylater step in viral entry, the ability of each of these viral Envs tobind to the cell surface was measured (Fig. 3). The virus boundto the cell surface was detected with FACS analysis usingantibodies recognizing a conserved epitope of the FeLV Env,outside of the receptor-binding domain conjugated to an FITCsecondary Ab (30). Using this assay, viral-binding correlatedwith viral titer. No significant viral-binding was observed forC4A, C9A, C4E;C9K, W11H, and W11A. The G8A mutant,which had a measurable but significantly decreased level ofinfectivity (see Fig. 2), similarly had a detectable but significantlylower binding than that observed by wild-type CP. These datademonstrate that the loss of infectivity noted with these Envmutants was directly related to a decrease in binding to the hostreceptor.

Isolation of the CP Receptor. The host-range of virus bearing theCP Env indicated that it utilizes a receptor outside the knownreceptors of the FeLV and MuLV. To isolate the host receptorfor the CP Env, a cDNA library was derived from the permissivecell line 143B. The library was inserted into the retroviralbackbone pMX1 and transduced into the nonpermissive cell lineUMR-106 (see Table 1). Two independent screens for the CPreceptor were performed, differing in the quantity of 143BcDNA introduced into the UMR-106 cells. UMR-106 cellsexpressing the 143B cDNA library were challenged with virus

Fig. 2. Titer of the CP Env mutants. The assay is quantifying the transfer ofthe lacZ marker via wild-type CP or mutant CP Envs. The figure represents theaverage of the 3 values, with error bars representing 1 SD. Mutations arenumbered according to their position in the randomized region of the wild-type CP insert.

Fig. 3. Binding studies of virus bearing mutant CP on human 143B osteosarcoma cells. Virus-binding to 143B cells was detected by flow-cytometric analysis usingthe C11D8 mAb recognizing the FeLV Env backbone and goat anti-mouse IgG conjugated with FITC. For each panel, the black line represents the control assayperformed in the absence of virus and the red line represents the binding of the wild-type CP virus to the 143B cells. The green line represents the virus bearingthe mutated CP Env as indicated on each panel.

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bearing the CP Env and packaging the GFP-IRES-puro vector.UMR-106 cells expressing the CP receptor will be permissive toinfection and be identified as puror, GFP� colonies. Afterselection with puromycin, 8 GFP� colonies from the first screenand 84 from the second were isolated and subjected to asecondary challenge with virus bearing the CP Env and pack-aging lacZ to confirm that these cells were now permissive toinfection. From the first screen, 2 of the 8 colonies resulted intiters of 1.5 � 103 and 3.3 � 103 LSU/ml, respectively, uponsecondary challenge with CP/lacZ virus. Using pMX-specificprimers, a common 2-kb band was amplified from genomic DNAfrom both colonies (clones 4 and 5). Sequence analysis of thePCR products revealed identical cDNA inserts encoding theG-protein coupled receptor 172A (GPR172A) (Accession # NM024531.3). This protein has been reported to function as theGHB receptor (22) as well as one of the receptors for PERV-A(16). An additional 4 clones, which were puror, GFP�, and lacZ�,were identified from the second cDNA library screening whenchallenged with virus bearing CP Env. PCR analysis of these 4colonies amplified a similar 2-kb product using the pMX prim-ers. Sequence analysis indicated that 2 of these clones encodedthe identical 1941 base GPR172A cDNA (Fig. 4, isolate 1). Inaddition, 2 clones encoded a GPR172A cDNA of 1961 bases(Fig. 4, isolate 2). The 2 cDNAs differ by 20 bases in their 5�UTR.

Confirmation that the GPR172A cDNA functions as thereceptor for virus bearing the CP Env was obtained throughreconstruction and reintroduction of the 1941 base cDNA intononpermissive cells. The cDNA was reinserted into the pMX1backbone, and viral particles packaging the GPR172A cDNAwere introduced into the nonpermissive cell lines UMR-106 andAH927 [CP titer of �10 and 1.5 � 101 LSU/ml, respectively (seeTable 1)]. For UMR-106 cells, a GPR172A� population wasselected, whereas total GPR172A-transduced AH927 cells wereused for CP titer analysis. In the presence of GPR172A cDNA,the CP titers were 1.3 � 104 and 2.2 � 104 LSU/ml for AH927and UMR-106 cells, respectively (Fig. 5A).

Further confirmation that GPR172A served as the CP recep-tor was obtained by shRNA knockdown in 143B cells. Knock-down of GPR172A expression by 2 specific shRNAs (11421 and8272) resulted in a 98.4% and 99.5% reduction in viral titer in143B cells, respectively. RT-PCR analysis showed a markeddecrease in GPR172A expression although detectable levels stillremained after shRNA treatment (data not shown). The empty

vector control (pLKO.1) had minimal effect on CP viral titer(Fig. 5B).

DiscussionThe results of these studies highlight the remarkable flexibilityas well as the limitations of retroviral receptor usage. Byproviding the virus with an appropriate backbone in which ashort primary sequence can determine receptor binding, asingle-round of infection can identify unique functional Envisolates of high titer. Through this Env library system, viralreceptor usage has been expanded to use cell-surface proteinspreviously not reported as viral receptors (10). In the currentstudy, the receptor identified is one used by a porcine endoge-nous retrovirus.

In this Env library screen as well as in the screening of apreviously derived library (10), the receptors isolated weremultipass transmembrane receptors, consistent with that re-ported for all known gamma-retroviruses. This demonstrates anadvantage for viral entry to proceed through this class ofproteins. However, the independent selection of an identicalreceptor from a single-round of infection implies an additionalbenefit for this particular protein. This could include readilyfacilitating membrane fusion or perhaps a preference for theintracellular pathway used for recycling and trafficking of thereceptor. The use of identical receptors by different viral speciesis not unusual; the Pit receptors are used by murine, feline, andgibbon ape leukemia viruses (14, 15, 19).

Of particular interest is the fact that the VRA regions betweenthe PERV-A and CP Envs are unrelated, although alternativedomains within PERV SU could contribute to receptor recog-nition (25, 26). The GPR172A protein, beyond its function as a

Fig. 4. Schematic diagram of the GPR172A mRNA from 143B cells. TheGPR172A gene is encoded on chromosome 8 at position q24.3 (top line).Shown is the 5� terminus of the gene including the 5� UTR, with the Met startencoded at position 731 within the gene. Multiple mRNA start sites and splicejunctions have been reported within the 5� UTR for the GPR172A mRNA andare individually listed. Key positions with respect to the transcription start sitein the chromosome, as well as the sequence of the 5� terminus of the mRNAs,are indicated. The 2 cDNAs isolated from 143B cells are shown in comparisonwith the reported GPR172A mRNAs.

Fig. 5. GPR172A functions as CP Env viral receptor. (A) Introduction ofGPR172A cDNA renders cells permissive to CP infection. Human GPR172AcDNA was introduced into the nonpermissive AH927 cells and UMR-106 cellsby retroviral infection (see Materials and Methods). Cells were challengedwith virus bearing the CP Env and packaging lacZ. Titers of CP/lacZ virus(LSU/ml) are indicated: nonpermissive cells (light gray); GPR172A expressed innonpermissive cells (Gray); control 143B permissive cells (black). (B) Introduc-tion of shRNA against GPR172A in permissive 143B cells. VSV-G pseudotypedlentiviral vectors packaging shRNA were used to infected 143B cells, selectedfor puror and challenged with virus bearing the CP Env and packaging lacZ.Viral titer on 143B was 3.0 � 105 LSU/ml. Relative rates of infectivity are shownfor shRNAs 11421 and 8272, as well as for the empty vector control (pLKO.1)compared to that of wild-type 143B cells. The figures represent the average ofthe 3 values with error bars representing 1 SD.

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PERV-A receptor, has also been discovered to serve as thehuman GHB receptor. From an evolutionary point of view, theendogenous retroviruses might have evolved to bind this recep-tor without altering the function of the host protein, whereas theCP Env does not have this constraint and could function similarlyto known substrates or inhibitors. It is of interest to examine ifthe CP acts as an antagonist or agonist to the known physio-logical responses characteristic of GHB receptors.

All 6 of the cDNAs isolated from receptor-positive cellsencoded the GPR172A gene, although they differed in their 5�termini. It is not known whether these reflect previously un-identified start sites or incomplete cDNAs. The splicing patterncorresponds with a cDNA that was previously isolated fromRamos cells, a human Burkitt’s lymphoma cell line (CR606512).As studies have indicated that GPR172A is commonly up-regulated in human cancers (23), it would be of interest todetermine whether this 5�UTR results in an altered level ofexpression or mRNA stability in the osteosarcoma cell line orother transformed cells. The absence of the cDNA for theGPR172B gene, which also functions as the PERV-A receptor(16), is intriguing. It is possible that the remaining titers seen inthe shRNA-treated cells are the result of additional receptorsincluding GPR172B. However, the isolation of multiple cDNAsencoding GPR172A from 2 independent library screenings inconcert with the �98% decrease in titer observed uponGPR172A-targeted shRNA treatment, confirms that in the 143Bcell line GPR172A is the predominant receptor responsible forentry of virus bearing CP Env.

Published data using PERV-A Env pseudotyped onto MuLV-based particles have generally yielded titers in the range of 104

on HeLa and TE671 cells (31), while the CP Env produced titersgreater than 105 LSU/ml. This single round of molecular evo-lution and selection has yielded an Env that is capable of usinga known retroviral receptor with higher efficiency than itsnaturally evolved counterpart, and which can be easily concen-trated to produce extremely high titers on human cancer celllines. This method of retargeting has now been shown to be ableto create unique and functionally efficient retroviral Envs withtherapeutic potential.

Materials and MethodsLibrary Plasmids. The vector pRVL-SV40-neo (7) used for the initial libraryconstruction was modified by replacing the SV40-neo expression cassette withIRES-puro (see SI Text and Table S1 for a list of primers and sequences). Thenew vector, named RIP, was used for the creation of the randomized envelopelibrary. The plasmid pHIT-G (32) expresses VSV-G.

Cell Lines. All of the cell lines used for the assays were maintained in mediacontaining 10% FBS and 10 �l/ml of antibiotic-antimycotic mixture (Gibco).The producer cell lines 293TCeB (7), TECeBF2, and TELCeB (29) were main-tained in DMEM containing 9 �g/ml of Blasticidin S (Invivogen). The cell line293TCeB/GIP was created as previously described (10) and maintained inDMEM containing 2.5 �g/ml puromycin, 400 �g/ml G418, and 9 �g/ml Blasti-cidin S. The cell lines used for screening and host- and tissue-specificity assayswere maintained as follows: Caki-1 and Caki-2 in McCoy’s 5A medium;CRL1543, CRL1427, Huh7, UMR106, NIH 3T3, D17, and MDCK in DMEM; 143Band AH927 in MEM with 2 mM L-glutamine; PC3 and DU145 in RPMI withnonessential amino acids; ACHN, A498, Hep-G2, HeLa, and DLD-1 in MEM with2 mM L-glutamine, nonessential amino acids, 1 mM sodium pyruvate, and 1.5g/L sodium bicarbonate; MC3T3 and primary calvaria preosteoblasts in �MEMmedium and human foreskin fibroblasts (HFF) in DMEM/F12 medium. For allinfections, the media was filtered (0.45 �m filters) and performed in thepresence of 8 �g/ml polybrene. Viral concentration was performed by over-night utracentrifugation as previously described (8).

RIP Library Construction. The 11 random amino acid library with the RIP vectorwas generated through insertion within 2 back-to-back BbsI sites using 3oligonucleotides, as previously described (7). The oligonucleotides encodingthe random library contained the sequence 5�-GTGGGAGACACCTGG-GAACCT(NNB)11TCCTCCTCAAAATAT-GGA-3�, where B stands for T, C, or G.

The elimination of A in the third position of the codon biases the libraryagainst stop codons. The library plasmid DNA was introduced into ElectromaxDH10B competent cells (Invitrogen) by electroporation. Ampr colonies wereharvested and their plasmids were purified by CsCl gradient. DNA analysis of10 individual colonies indicated 6 contained random inserts, with the remain-ing 4 representing the parental pRIP vector encoding a stop codon betweenthe BbsI sites, and thus would not yield functional Env protein.

A TECeB-based cell line constitutively expressing the randomized Env li-brary was obtained as previously described (8), with the modification thattransfections were performed with lipofectamine 2000 and cells were selectedwith 2.5 �g/ml puromycin. After drug selection, the number of colonies wascounted and the complexity of the library was determined to be 2 � 105.

Screening of the RIP Library and Identification of a Unique Env Peptide. The RIPlibrary was used to screen for envelopes capable of productively infecting thehuman renal cell carcinoma cell line Caki-1. Viral supernatants from TECeBF2cells expressing the RIP library were collected in the absence of puromycin andapplied to the target cells at 15 ml per 15-cm plate. A total of 100 puror

colonies grew and were maintained as a population.Genomic DNA was extracted from puromycin-resistant Caki-1 cells using

the DNeasy kit (Qiagen). The Env was PCR amplified with the FeLV-specificprimers FeLV1 and FeLV2, and sequenced using FeLV1 primer. The Env isolate(named CP) was subsequently reconstructed through PCR amplification, withthe primers RANBBS5 and RANBBS3 flanking the randomized 11 aa, as previ-ously described (7), and reinserted into the pRVL-SV40-neo plasmid.

Mutations of CP Env Isolate. Mutagenesis of the 11 randomized amino acids inthe CP Env isolate was performed by overlapping PCR for mutations T7A andG8A and PCR for the remaining mutations (see SI Text and Table S2). Reinser-tion into the Env backbone was facilitated by the presence of an AgeI sitewithin the 33-bp portion encoding the randomized region and by HpaI andApaI sites flanking this region. The mutations were verified by sequencingwith the primer FeLV1.

Viral Titers. A stable producer cell line capable of producing virus particlespseudotyped with CP-Env and transferring the lacZ marker has been obtainedvia transfection of the TELCeB cell-line with pRVL-SV40-neo-CP and subse-quent G418 selection. Viral titers were determined as previously described (7).

Titration with the mutant CP envelopes was performed by transfecting asubconfluent 10-cm plate of TELCeB cells with the 10 �g of pRVL-SV40-neo-CP(mut) or pRVL-SV40-neo-CP with lipofectamine 2000 (Invitrogen). The nextday, the cells were induced with 10-mM sodium butyrate. Viral supernatantwas collected 24 h after induction and used to infect 143B cells at 0 and 1/10dilutions.

Virus Binding Assay. The binding assay was performed as previously describedon 143B cells (30), with the modification that the cells were not fixed withparaformaldehyde. The binding analysis was performed on a BeckmanCoulter Cytomics FC500 at the Cytometry Core Facility at the Environmentaland Occupational Health Sciences Institute of the University of Medicine andDentistry of New Jersey.

Construction of 143B cDNA Library. Poly(A)� mRNA was isolated and purifiedfrom 5 � 107 143B cells using an mRNA isolation kit (Stratagene) and 5 �g wasused as template for cDNA synthesis using a cDNA synthesis kit (Stratagene) inthe absence of radioactive dNTPs.

The pMX vector was modified as follows to allow for insertion of the library.The vector was digested with BstXI and a linker region encoding a cloning sitecontaining EcoRI and XhoI restriction sites was inserted. Next, pMX�EcoRI/XhoI was digested with EcoRI and XhoI and a 2-kB stuffer region was insertedto reduce background of singly cut vector to produce pMX-EcoRIstufferXhoI.The cDNA library was inserted into this vector after digestion with EcoRI andXhoI, electroporated into DH10B electromax ultracompetent cells accordingto the manufacturers protocol, and plated on four 245 � 245-mm platescontaining LB-carbenecillin (100 ng/ml). After overnight incubation, theplates yielded �2 � 106 colonies. The resulting colonies were harvested andplasmids were purified using the Endofree Maxiprep kit.

To determine that the cDNA library contained full-length cDNAs, regionscorresponding to the 5� and 3� ends of the clathrin mRNA-coding region aswell as to the 5� end of the GAPDH mRNA-coding region were amplified afterfirst-strand cDNA synthesis, second-strand synthesis, as well as from the com-pleted library after endotoxin-free plasmid preparation, as previously de-scribed (33). Amplification of the cDNA regions that correspond to the 5� and3� portions of the clathrin mRNA-coding region assures that at each step thelibrary contained full length cDNAs up to at least 6 kb.

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cDNA Library Screening for the CP Receptor. Viral particles were assembled asdescribed above by transfecting 30 to 60 �g of the cDNA library and 30 �g ofpHIT-G in each of 3 15-cm plates of 293TCeB. This viral supernatant was addedto each of 3 15-cm plates of UMR-106 cells overnight.

Two days after infection, the cells were challenged with virus bearing theCP Env and packaging GIP (25 ml viral supernatant/15 cm plate) overnight. Thisvirus was generated by transfecting 293TCeB/GIP cells with 30 �g of pRVL-SV40-neo-CP, as described above. The viral supernatant was harvested andused to infect the library containing UMR-106 cells. Beginning 2 days afterinfection, the UMR-106 cells were submitted to selection with puromycin (2.5�g/ml). Surviving colonies were then isolated and challenged with viruspackaging the lacZ gene and bearing the CP Env (see Viral Titers, above).

PCR and Sequence Analysis of cDNA Inserts. Genomic DNA was harvested from�5 � 106 cells using the DNeasy kit (Qiagen). The viral insert was then PCRamplified with virus-specific primers MX11 and MXcDNA R. The PCR productswere isolated and sequenced using the primers MXcDNA F, GPR172A603 F,and GPR172A1066 F.

Receptor cDNA Cloning. The receptor cDNA was PCR amplified from genomicDNA isolated from the 2 clones (numbers 4 and 5) derived from our first libraryscreening with primers MX11 and MXcDNA R using Fidelitaq Polymerase (USB)and subcloned into the Topo2.1 vector (Invitrogen). The insert was removedusing EcoRI and XhoI yielding a 0.8 kB 5� EcoRI/XhoI cDNA fragment and a 1.1kB 3� XhoI/XhoI cDNA fragment. The 2 fragments were inserted sequentially

into pMX1�EcoRIstufferXhoI. The resulting pMX-GPR172A was sequenced asdescribed above. The pMX�GPR172A vector was packaged into viral particlesby transfecting 293TCeB cells with 10 �g each of pMX-GPR172A and pHIT-G,as described above. The viral supernatant was used to infect UMR-106 andAH927 cells. Two days later, lacZ titers were measured on the AH927 cellswhile the UMR-106 cells were infected with CP-GIP and placed under puro-mycin selection. LacZ titers were then measured on the puror GFP� UMR-106population.

shRNA Knockdown of GPR172A. Receptor expression was knocked down usingthe lentiviral based plasmid pLKO.1 expressing the GPR172A specific shRNA’sTRCN0000008272 (8272) and TRCN0000011421 (11421) (Open Biosystems).Lentiviral particles were produced by transfecting 293T cells with 5 �g each ofpHIT-G, pCMVR8.2vpr (34), and pLKO.1 either containing the shRNA or asan empty vector control. The viral supernatant was used to infect 143B cells.After puromycin selection, titers based on lacZ transfer were measured on theshRNA� population as described above.

Sequence Analysis and Alignment. DNA and protein sequences were analyzedusing the Basic Local Alignment and Search Tool provided by the NationalInstitutes of Health (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). Protein se-quence alignment was performed using the MultAlin software (35).

ACKNOWLEDGMENTS. This work was supported by Grant 1RO1CA49932 (toM.J.R.) and Fellowship 707060 from the New Jersey Commission on CancerResearch (to P.M.M.).

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