Two Functionally Distinct Forms of a Retroviral Receptor Explain the

JOURNAL OF VIROLOGY,0022-538X/01/$04.0010 DOI: 10.1128/JVI.75.8.3520–3526.2001

Apr. 2001, p. 3520–3526 Vol. 75, No. 8

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Two Functionally Distinct Forms of a Retroviral ReceptorExplain the Nonreciprocal Receptor Interference among

Subgroups B, D, and E Avian Leukosis VirusesHEATHER B. ADKINS,1,2† STEPHEN C. BLACKLOW,2 AND JOHN A. T. YOUNG1,3*

Department of Microbiology and Molecular Genetics1 and Department of Pathology, Brigham and Women’s Hospital,2

Harvard Medical School, Boston, Massachusetts 02115, and Department of Oncology, McArdle Laboratoryfor Cancer Research, University of Wisconsin at Madison, Madison, Wisconsin 537063

Received 7 November 2000/Accepted 16 January 2001

Subgroups B, D, and E avian leukosis viruses (ALV-B, -D, and -E) share the same chicken receptor, TVBS1,a tumor necrosis factor receptor (TNFR)-related protein. These viruses, however, exhibit nonreciprocal re-ceptor interference (NRI): cells preinfected with ALV-B or ALV-D are resistant to superinfection by viruses ofall three subgroups, whereas those pre-infected by ALV-E are resistant only to superinfection by othersubgroup E viruses. In this study, we investigated the basis of this phenomenon by characterizing theinteraction of TVBS1 with ALV-B Env or ALV-E Env. Sequential immunoprecipitation analysis using surfaceenvelope immunoglobulin fusion proteins revealed the existence of two separate types of TVBS1 that areencoded by the same cDNA clone. One form, designated the type 1 receptor, is specific for ALV-B and ALV-E.The other form, the type 2 receptor, is specific for ALV-B. We show that a protein consisting of only the firstand second extracellular cysteine-rich domains of TVBS1 is capable of forming both receptor types. However,the third extracellular cysteine-rich domain is required for efficient formation of the type 1 receptor. We alsodemonstrate that heterogeneous N-linked glycosylation cannot explain the difference in activities of the tworeceptor types. The existence of two types of TVBS1 explains the NRI pattern between ALV-B and -E: subgroupB viruses establish receptor interference with both receptor types, whereas subgroup E viruses interfere onlywith the type 1 receptor, leaving the type 2 receptor available to mediate subsequent rounds of ALV-B entry.The formation of a TVB receptor type that is specific for cytopathic ALV may also have important implicationsfor understanding how some subgroups of ALV cause cell death.

Based on receptor usage in chickens, avian leukosis viruses(ALVs) have been divided into six major subgroups (Athrough E and J). Subgroups B and D viruses (ALV-B and -D)are cytopathic and share with noncytopathic ALV-E the TVBreceptor, a member of the tumor necrosis factor receptor(TNFR) family. TVB is a death receptor that is most structur-ally related to the human TRAIL receptors, TRAIL-R1 (DR4,APO-2) and TRAIL-R2 (DR5) (7, 13, 15, 17, 19, 23), and istherefore likely to play a direct role in cell killing caused byALV-B and ALV-D. By comparing TVB with other TNFR-related proteins, we originally proposed that this ALV recep-tor contains two extracellular cysteine-rich domains (CRDs)that characterize this protein family (6). However, the recentlysolved structure of TRAIL-R2 has revealed the existence of anadditional CRD located at the membrane-distal region of thatreceptor, (9), making it likely that TVB also contains an extraN-terminal CRD (Fig. 1).

Functionally distinct TVB proteins that are encoded by dif-ferent alleles of the chicken tvb locus (tvbs1 and tvbs3) or in-stead by the turkey homolog of this gene (tvbt) have beendescribed: TVBS1, a receptor for ALV-B, -D, and -E (2);TVBS3, a receptor specific for ALV-B and -D (6, 20); andTVBT, a subgroup E-specific viral receptor (1). Although

ALV-E shares the TVBS1 receptor with ALV-B and ALV-D,these viruses display a nonreciprocal receptor interference(NRI) pattern. Cells preinfected with either ALV-B or ALV-Dare resistant to superinfection by ALV-B, -D, and -E presum-ably because the viral receptor is complexed with newly syn-thesized Env glycoproteins (24). This interference pattern isexpected for viruses that share the same receptor. However,cells preinfected with ALV-E are resistant only to furthersubgroup E virus infection, remaining susceptible to ALV-Band -D infection.

At least two models have been proposed to explain this NRIpattern. The one-receptor model invokes a single TVBS1 re-ceptor for all three viral subgroups (24). According to thismodel, the subgroup E Env protein has the lowest affinity forthe shared receptor and thus may be displaced by either sub-group B or subgroup D Env proteins contained on incomingvirus particles (24). The alternative two-receptor model pre-dicts one receptor specific for ALV-B, -D, and -E and anotherspecific for ALV-B and -D (24). According to this model, theremay be two closely linked ALV receptor genes located at thechicken tvbs1 locus that encode the distinct receptors (24).

Previously we identified a tvbs1 cDNA clone that encodes acellular receptor for ALV-B, -D, and -E (2). We showed thatthe NRI pattern exhibited by ALV-B and -E could be fullyreconstituted by expressing this receptor in mammalian cellsalong with the respective Env proteins (2). Also, we demon-strated a striking difference in the way that TVBS1 interacts inwith ALV-E Env as opposed to ALV-B Env. Specifically, the

* Corresponding author. Mailing address: McArdle Laboratory forCancer Research, University of Wisconsin—Madison, 1400 UniversityAve., Madison, WI 53706. Phone: (608) 265-5151. Fax: (608) 262-2824.E-mail: [email protected].

† Present address: Biogen, Inc., Cambridge, MA 02142.

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ALV-E Env interaction is influenced by the first four cysteinesof the receptor (Cys-46, Cys-59, Cys-62, and Cys-77), seeminglyrequiring a putative Cys-46–Cys-59 disulfide bond, whereasALV-B can use an altered receptor lacking all four of thesecysteines (2). Taken together, these data suggested that thereis a single type of receptor for ALV-B, -D, and -E and that theNRI pattern among these viruses might be explained by dif-ferences in subgroup-specific Env interactions with that com-mon receptor. However, contrary to these expectations, wehave now found that the tvbsl cDNA clone encodes two func-tionally distinct ALV receptors, one that is specific for ALV-Band -E and another that is specific for ALV-B.

MATERIALS AND METHODS

Cells, viruses, and immunoadhesins. Human 293 cells and transfected 293cells (S1-5 cells) that express TVBS1(DDD), a FLAG epitope-tagged version ofthe receptor without the cytoplasmic death domain, were described previously(2). Pseudotyped murine leukemia virus (MLV) virions with ALV-B Env pro-teins, MLV-LacZ (EnvB), or ALV-E Env proteins, MLV-LacZ (EnvE), wereproduced as described elsewhere (2). The surface envelope (SU)-rabbit immu-noglobulin G (rIgG) fusion proteins, SUB-rIgG and SUE-rIgG, were generatedand purified as described previously (1, 6, 28).

TVB constructs, transfections, and infections. PCR amplification was per-formed using plasmid pHA1 (2) as template DNA to generate (i) the solubleproteins sTVBS1 and sTVBS1(D102-143); (ii) TVBS1(DDD/D102-143), an alteredform of the TVBS1(DDD) receptor that lacks CRD3; and (iii) TVBS1(DDD)-N54A, a mutant TVBs1(DDD) protein bearing the N54A mutation (Fig. 1). Theauthenticity of each construct was validated by DNA sequencing. Human 293cells were transiently transfected with plasmids encoding different TVB proteins

and challenged with pseudotyped MLV virions, and infection was quantitated byb-galactosidase staining as described elsewhere (2).

Flow cytometry. S1-5 cells were prepared for flow cytometry as describedpreviously (28). Briefly, these studies involved incubating the cells with 5 mMpurified SUE-rIgG or SUB-rIgG (in phosphate-buffered saline [PBS] containing1% fetal bovine serum) and then with a fluorescein isothiocyanate-conjugatedsecondary antibody. Samples of 5,000 cells were analyzed on a Coulter Epics XLflow cytometer in the Department of Hematologic Oncology at the Dana-FarberCancer Institute.

Immunoprecipitation and deglycosylation studies. Human 293 cells weretransfected with 10 mg of plasmid DNA expressing the different TVB proteins.Cells that expressed transmembrane forms of TVB proteins were metabolicallylabeled at 60 h posttransfection. Briefly, cells were starved in Cys- and Met-freeDulbecco modified Eagle medium for 30 min at 37°C and then incubated for 2to 3 h in medium containing ;1 mCi of [35S]Cys (ICN) per ml. The labeled cellswere then washed in ice-cold PBS and lysed in an NP-40-containing buffer asdescribed elsewhere (1). Cells that expressed soluble TVB proteins were sub-jected to a similar metabolic labeling procedure that was followed by a 3-h chaseperiod in medium containing unlabeled amino acids to allow for secretion ofradioactively labeled proteins into the extracellular supernatant. UnlabeledsTVBS1 was harvested from the extracellular supernatant of transfected human293 cells at 72 h posttransfection.

Sequential immunoprecipitation experiments were performed at 4°C for 1 h,using 1 mg of total cell lysate (transmembrane TVB receptors) or 50 to 150 ml ofextracellular supernatant (soluble TVB receptors) and protein A-Sepharose pre-loaded with SUB-rIgG or SUE-rIgG. The samples were then pelleted, and thesupernatants were subjected to additional rounds of immunoprecipitation. Eachpelleted sample was washed in ice-cold lysis buffer, boiled in sodium dodecylsulfate (SDS) sample buffer under reducing conditions, and electrophoresed ona 12 or 15% polyacrylamide gel containing SDS. Radiolabeled TVB proteinswere then detected by autoradiography, and unlabeled TVB proteins were de-tected by an immunoblotting procedure involving protein transfer to a nitrocel-lulose membrane followed by incubation with an extracellular supernatant con-

FIG. 1. Schematic diagram of the TVBS1 constructs used in these studies. The TVBS1 and TVBS1 (DDD) proteins were described previously(2). The other TVB proteins were generated specifically for these experiments. The amino acid residues are numbered according to a scheme usedpreviously (6). SP, signal peptide; TM, transmembrane region; DD, cytoplasmic death domain.

VOL. 75, 2001 DISTINCT FORMS OF A RETROVIRAL RECEPTOR 3521

taining SUB-rIgG for 1 h at room temperature. The membrane was then washedwith TBSX buffer (10 mM Tris [pH 8.0], 150 mM NaCl, 1% Triton X-100) andincubated for 30 min with TBSX buffer containing a 1:5,000 dilution of a horse-radish peroxidase (HRP)-conjugated antibody specific for rabbit immunoglobu-lins (Amersham). Bound antibodies were detected by enhanced chemilumines-cence.

The deglycosylation experiments were performed at 37°C for 2 h by incubatinga 50-ml aliquot of 35S-labeled extracellular supernatant containing sTVBs1(D102-143) with 1.5 U of N-glycosidase F (Boehringer Mannheim) and 15 U of neur-aminidase (Boehringer Mannheim) in an equal volume of PBS containing 20mM EDTA and 2% NP-40. The deglycosylated proteins were then subjected tosequential immunoprecipitation with the SU-immunoglobulin fusion proteins asdescribed above, and the precipitated proteins were detected by autoradiographyfollowing SDS-polyacrylamide gel electrophoresis.

RESULTS

The TVBS1 receptor is produced as two distinct receptortypes. To biochemically characterize the interaction betweenTVBS1 and ALV Env proteins, a radiolabeled protein lysatewas prepared from transfected human 293 cells that express atruncated TVB receptor, TVBS1(DDD) (2). TVBS1(DDD)lacks a cytoplasmic death domain (Fig. 1) and therefore it doesnot induce any of the nonspecific cell-killing effects that areassociated with overexpression of the full-length receptor intransfected human 293 cells (2). Aliquots of the protein lysatewere subjected to sequential rounds of immunoprecipitationwith SUB-rIgG and with SUE-rIgG (1).

Virtually all of the TVBS1(DDD) protein was removed fromthe lysate after two rounds of precipitation with SUB-rIgG(Fig. 2A, lanes 1 and 2). In fact, only a trace amount ofreceptor protein was precipitated by a third round of SUB-rIgG precipitation (Fig. 2A, lane 3), and no additional receptorprotein bound to SUE-rIgG (Fig. 2A, lane 4). Therefore, asexpected, all forms of TVBS1(DDD) that were expressedbound to the subgroup B-specific SU-immunoglobulin protein.By contrast, only a fraction of the total receptor protein wasprecipitated after two rounds of incubation with SUE-rIgG(Fig. 2A, lanes 5 and 6). Although a third round of incubationwith SUE-rIgG failed to precipitate more of the TVB protein(Fig. 2A, lane 7), a substantial fraction of the receptor was stillpresent in the lysate, and this form was precipitated specificallyby SUB-rIgG (Fig. 2A, lane 8).

Taken together, the results of this experiment led to thesurprising finding that there are two types of TVBS1 receptor,designated here type 1 and type 2. The type 1 protein is areceptor for ALV-B and ALV-E (Fig. 2A, lanes 1, 2, 5, and 6);the type 2 receptor is specific for ALV-B (Fig. 2A, lane 8).

Flow cytometric analysis performed with the SU-immuno-globulin fusion proteins confirmed that there are more sub-group B than subgroup E viral receptors on the surfaces oftransfected 293 cells that express TVBS1(DDD) (Fig. 2B).These experiments involved binding the SU-immunoglobulinproteins to cells and then detecting the bound protein by in-cubation with a fluoresceinated secondary antibody. In theseexperiments, the mean fluorescence of each cell population isdirectly related to the amount of each SU-immunoglobuinfusion protein bound, which in turn is a measure of the relativenumber of ALV-B versus ALV-E receptors. When these ex-periments were performed under conditions that led to satu-rable binding of SUE-rIgG (data not shown), a much greateramount of SUB-rIgG was bound to the cells (Fig. 2B). There-fore, we conclude from these studies that there are more sub-

group B viral receptors than subgroup E viral receptors presentat the surface of these cells, a result that is fully consistent withthe existence of the two types of TVBS1 receptor.

To test whether the ability to form both receptor types is aproperty of the extracellular domain of TVBS1, the sTVBS1

protein (Fig. 1) was produced. This soluble protein was alsosubjected to the same type of sequential immunoprecipitationanalysis that was used to characterize the transmembrane re-ceptor, with the exception that in this case the precipitatedproteins were detected by immunoblotting following SDS-polyacrylamide gel electrophoresis with SUB-rIgG and anHRP-coupled secondary antibody. Like the transmembranereceptor, sTVBS1 was produced both as type 1 (Fig. 2C, lanes1, 2, and 5) and type 2 (Fig. 2C, lanes 1, 2, and 8) forms.Therefore, we conclude that the ectodomain of the TVBS1

receptor is by itself capable of forming both receptor types.The first two CRDs of TVBS1 are sufficient for forming both

receptor types. To test whether the formation of both receptortypes is a property associated with the first two extracellularCRDs of this protein, the TVBS1(DDD/D102-143) and

FIG. 2. TVBS1 is produced as two distinct receptor types. (A) Ali-quots of [35S]cysteine-labeled protein lysate prepared from transfectedhuman 293 cells that express TVBS1(DDD) were subjected to threerounds of sequential immunoprecipitation with protein A-Sepharosebeads loaded with either SUB-rIgG (lanes 1 to 3) or SUE-rIgG (lanes5 to 7) and then with SUE-rIgG (lane 4) or SUB-rIgG (lane 8). Theprecipitated TVB proteins were visualized by autoradiography. (B)Flow cytometric analysis of 293 cells expressing TVBS1 (DDD) wasperformed with SUB-rIgG or SUE-rIgG, followed by a fluoresceinisothiocyanate-conjugated secondary antibody. The results of a repre-sentative experiment performed in triplicate are shown as the meanfluorescence values of each cell population with the standard devia-tions of the data indicated with error bars. (C) An experiment simi-lar to that described for panel A was performed with extracellularsupernatant containing sTVBS1. In this case, the immunoprecipitatedproteins were detected by immunoblotting with SUB-rIgG and anHRP-conjugated secondary antibody followed by enhanced chemilu-minescence.

3522 ADKINS ET AL. J. VIROL.

sTVBS1(D102–143) proteins were generated (Fig. 1). TheTVBS1(DDD/D102-143) receptor was expressed in transfectedhuman 293 cells that were challenged with pseudotyped MLVvectors encoding b-galactosidase (MLV-LacZ) and containingeither ALV-B Env (EnvB) or ALV-E Env (EnvE). Comparedto wild-type TVBS1(DDD), the receptor lacking CRD3 wasfully functional for subgroup B viral entry but mediated ap-proximately 30-fold-reduced levels of subgroup E viral entry(Fig. 3A). Nevertheless, the level of subgroup E viral entrymediated by TVBS1(DDD/D102-143) was significantly (1,000-fold) greater than that seen with nontransfected human 293cells (data not shown), demonstrating that the protein is in-deed a competent subgroup E viral receptor. These data indi-cated that TVBS1(DDD/D102-143) might be capable of form-ing both receptor types but the amount of protein with type 1receptor activity may be lower than that with type 2 receptoractivity.

Sequential immunoprecipitation experiments performedwith the SU-immunoglobulin fusion proteins confirmed thatthe type 1 receptor is reduced in level relative to the type 2receptor in the protein with only two extracellular CRDs. Theamount of TVBS1(DDD/D102-143) that was precipitated spe-

cifically by SUE-rIgG (Fig. 3B, lane 7) was much less than thatamount precipitated by SUB-rIgG (Fig. 3B, lane 12). Similarresults were obtained with sTVBS1(D102-143), a soluble formof the receptor lacking CRD3 (Fig. 3C, compare lanes 7 and 8with lane 12). Taken together, these studies demonstrate thatthe CRD1 and CRD2 regions of the TVBS1 receptor are suf-ficient for the formation of both receptor types. However, theCRD3 region is also important either because it is needed forefficient formation of the type 1 receptor or instead because itis required for that receptor to bind strongly to subgroup EEnv.

The distinct functional activities of both types of TVBS1

receptor cannot be explained by heterogeneous N-linked car-bohydrate additions. Since N-linked glycosylation gives rise toseveral distinct forms of TVBS1 (2) (Fig. 2A), we reasoned thatsubtle differences in the types of carbohydrate added mightaccount for both receptor forms. To test this possibility, thesTVBS1(D102-143) protein, which contains a single putativeN-linked glycosylation site at residue Asn-54 (Fig. 1), was sub-jected to enzymatic deglycosylation. Extracellular supernatantcontaining sTVBS1(D102-143) was treated with a combinationof N-glycosidase F and neuraminidase prior to sequential im-munoprecipitation with the SU-immunoglobulin fusion pro-teins. This combination of enzymes was more effective thanN-glycosidase F alone in deglycosylating the TVB protein(data not shown), presumably because cleavage by neuramin-idase allowed N-glycosidase F better access to the sugar moi-eties. The deglycosylated sTVBS1(D102-143) protein, whichmigrated on SDS-polyacrylamide gels with an apparent molec-ular mass of 13 kDa was still capable of forming both receptortypes (Fig. 4A, lanes 5 and 10). Therefore, differences in N-linked glycosylation do not appear to explain the distinct ac-tivities of the two receptor types.

To formally rule out any possible role for carbohydratesadded at residue Asn-54, this residue was changed to an ala-nine to generate the TVBS1(DDD)-N54A receptor (Fig. 1).This altered protein supported subgroup B and subgroup Eviral entry at levels comparable to those mediated by wild-typeTVBS1(DDD) (Fig. 4B). Consistent with this result, the exis-tence of both type 1 and type 2 forms of TVBS1(DDD)-N54Areceptor was confirmed by sequential immunoprecipitationstudies using the SU-immunoglobulin fusion proteins (Fig. 3C,lanes 7, 8, and 12). Together, these data argue that the distinctfunctional activities of the two classes of TVBS1 receptor can-not be explained by heterogeneous N-linked carbohydratemodifications.

DISCUSSION

In this report, we have provided compelling evidence thatthere are two distinct types of TVBS1 receptor. The type 1receptor is specific for ALV-B and ALV-E, whereas the type 2receptor is specific for ALV-B. Previously we have shown thatTVBS1 differs from TVBS3 (a protein which exhibits only type2 receptor activity) by a single amino acid difference, namely,that residue 62 is a cysteine in the former and a serine in thelatter (2). This fact, coupled with our finding that a putativedisulfide bond located between residues Cys-46 and Cys-62seems to be important for subgroup E receptor function (2),raises the possibility that the two types of TVBS1 may differ in

FIG. 3. The first two CRDs of TVBS1 are sufficient for formation ofthe type 1 receptor. (A) Human 293 cells were transiently transfectedwith a plasmid expressing TVBS1(DDD) or TVBS1(DDD/D102-143)and then challenged with MLV-LacZ (Env B) or MLV-LacZ (Env E).Infected cells were identified by staining for b-galactosidase activity.The data from three independent experiments with standard devia-tions are shown. Aliquots of [35S]cysteine-labeled protein lysate con-taining TVBS1(DDD/D102-143) (B) or supernatant containing sTVBS1

(D102-143) (C) were subjected to the sequential immunoprecipitationprocedure outlined for Fig. 2A, and the precipitated proteins werevisualized by autoradiography following SDS-polyacrylamide gel elec-trophoresis.


their intrachain disulfide bonding patterns, although this idearemains to be tested.

The existence of two types of TVBS1 receptor leads to anattractive model to explain why these viruses exhibit NRI (Fig.5A). According to this model, following subgroup B viral in-fection the newly synthesized ALV-B Env proteins interferewith the function of both receptor types leading to a block tosuperinfection by ALV-B, -D, and -E. Given that subgroup Dviruses have the same receptor-interference properties as sub-group B viruses, we fully expect that ALV-D Env will alsointeract with both types of receptor, although this remains tobe formally shown. By contrast to these two viral subgroups,ALV-E infection leads to the production of Env proteins thatinterfere only with the function of the type 1 receptor, leavingthe type 2 receptor still available to mediate subsequent roundsof subgroup B (and presumably also subgroup D) viral entry(Fig. 5A). Thus, the NRI pattern between these viruses isexplained by a two-receptor model. The interesting twist to thisstory is that the two receptor types are derived from preciselythe same mRNA species and not from separate genes whichare closely linked at tvbs1.

NRI has also been observed with other retroviruses such asxenotropic and polytropic MLVs (X-MLVs, and P-MLVs, re-spectively). X-MLVs establish receptor interference to bothviral types in Mus dunni cells, whereas P-MLVs only partiallyinterfere with X-MLVs. The Modunni receptor shared by theseviruses has been isolated and characterized (3, 21, 27). Al-though receptor determinants that are specifically involved inX-MLV entry have been defined (14), it is not known yet if,like TVBS1, this cellular protein, is produced as two distinctreceptor types (i.e., one type that is specific for X-MLV andP-MLV and a second type specific for X-MLV).

The existence of two types of TVBS1 protein may also haveimportant implications for understanding the mechanism ofcell death that is induced by ALV-B and ALV-D (25, 26). Thecell-killing events caused by these viruses are associated withmassive rounds of viral superinfection which give rise to theaccumulation of many copies of unintegrated viral DNA withincells that are destined to die (25, 26). Several lines of evidencesupport a direct role for the TVB receptor in these viral cyto-pathic effects. First, the determinants on Env that are requiredfor cell killing are the same as those needed for TVB interac-tion (8). Second, the TVB receptor is a death receptor of theTNFR family, and this protein can activate avian cell deathafter binding to either subgroup B or subgroup E SU-immu-

FIG. 4. The existence of both receptor types cannot be explainedby heterogeneous N-linked glycosylation. (A) An aliquot of [35S]cys-teine labeled extracellular supernatant containing sTVBS1(D102-143)was incubated with N-glycosidase F and neuraminidase and then sub-jected to serial rounds of immunoprecipitation with SUB-rIgG andSUE-rIgG. The precipitated proteins were subjected to SDS-polyacryl-amide gel electrophoresis and visualized by autoradiography. (B) Hu-man 293 cells expressing TVBS1 (DDD) and TVBS1(DDD)-N54A werechallenged with MLV-LacZ (Env B) or MLV-LacZ (Env E) and in-fected cells were identified and counted after staining for b-galactosi-dase activity. The data shown represents the average values obtained inthree independent experiments with the standard deviations given byerror bars. (C) An aliquot of [35S]cysteine-labeled protein lysate con-taining TVBS1(DDD)-N54A was subjected to the sequential immuno-precipitation assay, and proteins were visualized by autoradiographyfollowing SDS-polyacrylamide gel electrophoresis.

FIG. 5. Implications of the two receptor types for understandingNRI between ALV-B and ALV-E and for ALV-induced cytopathiceffects. (A) NRI between ALV-B and ALV-E; (B) possible role ofTVBS1 in cell death induced by ALV-B. These models are discussed indetail in the text.


noglobulin fusion proteins, at least in the presence of cyclo-heximide which presumably acts to extinguish the expression ofcellular survival factors (5, 6).

With the identification of two types of TVBS1, at least twodifferent models can now be envisaged to explain why ALV-Band ALV-D may kill cells whereas ALV-E does not. The firstmodel proposes that subgroup B Env can induce death follow-ing infection by interacting with either the type 1 or type 2receptor (Fig. 5B). If this were the case, then ALV-E might beunable to induce cell death following infection because it in-teracts with the type 1 receptor in a fundamentally differentway that may not activate cell killing unless the action ofcellular survival factors is also blocked (Fig. 6B)(2, 5). Muta-tional studies have in fact shown that subgroup B and subgroupE Env proteins interact with the shared receptor by distinctmeans (2). In a second model, subgroup B Env can elicit celldeath only by interacting with the type 2 receptor (Fig. 5B). Ifthis were the case, the type 1 receptor would be unable toparticipate in cell killing even when complexed with ALV-BEnv (Fig. 5B).

Given that subgroup E viruses are endogenous in thechicken germ line (4), it is tempting to speculate that the twotypes of TVBS1 evolved in response to selective pressures im-posed by these endogenous viruses. For example, there may bea selective pressure to maintain the expression of some form ofTVBS1 protein (the type 2 form) in chicken cells that containendogenous subgroup E viruses and thus exhibit type 1 recep-tor interference. Indeed, the TVB receptor may play an im-portant role in the host response to viral infection. Support forthis idea has come from studies of the highly related TRAILreceptors, which have been shown to mediate ligand-inducedapoptosis of cells infected by a variety of viruses includingcytomegalovirus (18), measles virus (22), and human immuno-deficiency virus type 1 (10, 11).

Additional evidence that TVB receptors may have evolvedunder the influence of selective pressures imposed by endog-enous subgroup E viruses comes from comparing TVBS1 withTVBS3 and with TVBT. TVBS3 is an ALV-B- and ALV-D-specific receptor that is encoded by a different allele of thechicken tvb gene (6, 20). TVBT is an ALV-E-specific receptorencoded by the turkey homolog of tvb (1). The only differencebetween TVBS3 and TVBS1 is that the former protein containsa serine residue at position 62 whereas the latter protein con-tains a cysteine at the same position (2). Since the highlyrelated TRAIL receptors also contain a cysteine residue at thatcorresponding position, it seems intuitive that TVBS3 arosefrom TVBS1 by accquiring a mutation that selectively abro-gates subgroup E Env binding. Further support for this idea isfound with the turkey TVBT receptor, which has evolved in aspecies lacking endogenous subgroup E viruses. TVBT hasretained a cysteine residue at position 62 and supports sub-group E viral entry (1). Work is ongoing to characterize thefunctional consequences of different subgroup-specific ALVEnv-TVB interactions with the goals of understanding themechanisms of entry used by these viruses and establishinghow some of these viruses elicit cell death, information that inturn will provide new insights into the coevolution of theseavian retroviruses with their hosts.

ACKNOWLEDGMENTS

We thank members of the Young and Blacklow laboratories forstimulating discussions and for constructive criticisms. We especiallythank John Naughton for assistance in preparing the figures and SaraKlucking and Dan Knauss for critical reading of the manuscript.

This work was supported by NIH grants CA 70810 (to J.A.T.Y) andRO1-HL61001 (to S.C.B.). S.C.B. is a Pew Scholar in the BiomedicalSciences.

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