receptor binding and low ph coactivate oncogenic ...jvi.asm.org/content/83/22/11447.full.pdf ·...

JOURNAL OF VIROLOGY, Nov. 2009, p. 11447–11455 Vol. 83, No. 220022-538X/09/$12.00 doi:10.1128/JVI.00748-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Receptor Binding and Low pH Coactivate Oncogenic RetrovirusEnvelope-Mediated Fusion�

Marceline Cote, Yi-Min Zheng, and Shan-Lu Liu*Department of Microbiology and Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada

Received 10 April 2009/Accepted 26 August 2009

Fusion of enveloped viruses with host cells is triggered by either receptor binding or low pH but rarelyrequires both except for avian sarcoma leukosis virus (ASLV). We recently reported that membrane fusionmediated by an oncogenic Jaagsiekte sheep retrovirus (JSRV) envelope (Env) requires an acidic pH, yetreceptor overexpression is required for this process to occur. Here we show that a soluble form of the JSRVreceptor, sHyal2, promoted JSRV Env-mediated fusion at a low pH in normally fusion-negative cells and thatthis effect was blocked by a synthetic peptide analogous to the C-terminal heptad repeat of JSRV Env. Incontrast to the receptor of ASLV, sHyal2 induced pronounced shedding of the JSRV surface subunit, as wellas unstable conformational rearrangement of its transmembrane (TM) subunit, yet full activation of JSRV Envfusogenicity, associated with strong TM oligomerization, required both sHyal2 and low pH. Consistently,sHyal2 enabled transduction of nonpermissive cells by JSRV Env pseudovirions, with low efficiency, butsubstantially blocked viral entry into permissive cells at both binding and postbinding steps, indicating thatsHyal2 prematurely activates JSRV Env-mediated fusion. Altogether, our study supports a model that receptorpriming promotes fusion activation of JSRV Env at a low pH, and that the underlying mechanism is likely tobe different from that of ASLV. Thus, JSRV may provide a useful alternate model for the better understandingof virus fusion and cell entry.

Fusion is a fundamental event in the life cycle of envelopedviruses and is essential for viral replication. While viral fusionproteins are highly divergent in primary sequence, their struc-tures and modes of activation share striking similarities, per-mitting their classification into two major groups (41). Class Ifusion proteins, as exemplified by the retrovirus envelope(Env) and influenza virus hemagglutinin (HA), are composedmainly of alpha-helices, and they are present as metastabletrimers on the viral surface (11). Class II fusion proteins, rep-resented by alphavirus E1 and flavivirus E, contain predomi-nantly beta-sheets and exist as dimers in the prefusion state(16). Of note, the vesicular stomatitis virus G (VSV-G) andherpesvirus gB proteins were recently assigned to a newly es-tablished class III, for fusion proteins combining properties ofboth class I and class II (13, 30). Despite these differences, onecommon and intriguing characteristic of all viral fusion pro-teins is their ability to undergo dramatic conformational rear-rangements upon activation, i.e., the formation of trimers ofhairpins, which drive fusion between viral and cellular mem-branes (11, 17).

Retrovirus Env is a typical type I transmembrane proteincomposed of surface (SU) and transmembrane (TM) subunitsand belongs to the class I fusion proteins. SU is responsible forbinding to cognate cellular receptors or cofactors, while TMdirectly mediates membrane fusion (6). Most retroviruses usea pH-independent pathway for entry, during which receptorbinding relieves the ability of SU to restrain TM, resulting inconformational changes in TM and subsequent fusion with the

cell membrane (11). Interestingly, increasing numbers of ret-roviruses have recently been shown to require a low pH (3, 15,24, 28, 31) or pH-dependent protease activities to trigger fu-sion (18); the latter property has also been demonstrated forsome other enveloped viruses (2, 14, 18, 26, 27, 33, 34). Amongthese, avian sarcoma leukosis virus (ASLV) is unique in that ituses a two-step mechanism for fusion, in which receptor bind-ing primes the second trigger of low pH (24).

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovi-rus etiologically responsible for contagious lung tumors insheep (12). The native Env protein of JSRV functions as apotent oncogene that induces cell transformation in vitro andin animals (4, 9, 21, 29, 42). The cell entry receptor for JSRVhas been identified as hyaluronidase 2 (Hyal2), a glycosylphos-phatidylinositol (GPI)-anchored protein belonging to the hy-aluronidase family (29); Hyal2 itself has low hyaluronidaseactivity, and this activity is not associated with JSRV entryand infection (38). Intrigued by the oncogenic nature ofJSRV Env, we recently examined the mechanism of JSRVentry and found that JSRV Env-mediated fusion and cellentry require a low pH (1, 8). These observations led us tohypothesize that the pH-dependent fusion activation ofJSRV Env may be advantageous for its oncogenesis, giventhat extreme cell-cell fusion of the plasma membrane at aneutral pH would result in syncytium formation and oftencell death. Curiously, we noticed that overexpression ofHyal2 is necessary for JSRV Env to induce membrane fu-sion at a low pH in vitro, suggesting that Hyal2 may play anactive role in the pH-dependent fusion process. Here weprovide direct evidence that Hyal2 functions in cooperationwith a low pH to trigger the JSRV Env-mediated fusionactivation yet exhibits some striking differences from themechanism of ASLV fusion. The multistep pathway for

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, McGill University, Montreal, Canada. Phone:(514) 398-4582. Fax: (514) 398-7052. E-mail: [email protected].

� Published ahead of print on 2 September 2009.

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JSRV Env-mediated fusion activation might be importantfor its replication fitness and oncogenesis.

MATERIALS AND METHODS

Cell lines, viruses, and reagents. All mammalian cells used in this study weremaintained in Dulbecco’s modified Eagle medium supplemented with 10% fetalbovine serum using standard procedures. Drosophila Schneider 2 (S2) cells weregrown at 27°C in Express Five SFM medium (Invitrogen, Carlsbad, CA) supple-mented with 2 mM L-glutamine (Invitrogen). Moloney murine leukemia virus(MoMLV) pseudotypes bearing JSRV Env (with a FLAG tag at both the N andC termini [termed F-Jenv-F] [8] or at the N terminus only [called F-Jenv] [20]),human immunodeficiency virus type 1 (HIV-1) Env, or VSV-G were produced aspreviously described (8). Note that the cytoplasmic tail of the HIV-1 Env (strainADA) used here is deleted (a kind gift of Eric Cohen, Institut de RecherchesCliniques de Montreal, Montreal, Canada) so that it can pseudotype theMoMLV retroviral vector. All primary and secondary antibodies were purchasedfrom Sigma (St. Louis, MO), except for the mixture of B3 and C9 monoclonalantibodies against JSRV SU (43), which was kindly provided by Dusty Miller.The peptides corresponding to the JSRV N-terminal heptad repeat (N-HR1)(DKKIEDRLSALYDVVRVLGE) and C-terminal heptad repeat (C-HR2) (FNTNLSLDLLQLHNEILDIENS) were synthesized by Alpha Diagnostic Inter-national (San Antonio, TX), and were solubilized in water and reconstituted in14% dimethyl sulfoxide (C-HR2 is insoluble in water), respectively.

sHyal2 production and purification. Drosophila S2 cells stably expressing sol-uble Hyal2 (sHyal2) (39) (a kind gift of Vladimir Vigdorovich and Dusty Miller)were induced by 1 mM CuSO4 for 5 to 7 days at 27°C; the culture medium washarvested; and sHyal2 protein was purified using nickel-nitrilotriacetic acid col-umns (Qiagen, Valencia, CA). The purity of sHyal2 was determined by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and it wasquantified by Bradford assays.

Cell surface staining. Cell surface staining was performed by flow cytometry aspreviously described (19), except that an anti-FLAG antibody, or 0.5 �g ofsHyal2 followed by an anti-His antibody, was incubated with 293 cells expressingFLAG-tagged JSRV Env.

Syncytium induction assay. Syncytium induction assays were performed asdescribed previously (8), with minor modifications. Briefly, 293 cells were co-transfected with plasmids encoding JSRV Env and pCMV-GFP; 24 h posttrans-fection, cells were incubated with different amounts of sHyal2 for 1 h at 4°C,followed by incubation at 37°C for 30 min. Cells were then treated with pre-warmed pH 7.4 or pH 5.0 buffer for 5 min and were incubated for 1 h at 37°Cbefore being examined for fusion and photographed. For syncytium inductioninvolving heptad repeat peptides, transfected cells were incubated with a peptideor sHyal2 for 1 h at 4°C, followed by incubation at 37°C for 30 min; cells werethen incubated with fresh medium containing either sHyal2 or the peptide at 4°Cfor 1 h, followed by recovery at 37°C for 30 min. The sHyal2- or peptide-treatedcells were incubated with a pH 5.0 buffer for 5 min and were then analyzed forfusion using fluorescence microscopy.

Metabolic labeling. Metabolic labeling was performed as described previously(8), except that different amounts of sHyal2 were incubated with 293 cells duringthe last 3 h of a 6-h chase period. If needed, a pH 5.0 pulse was subsequentlyapplied for 5 min. Cell lysates and culture media containing the [35S]Met-Cys-labeled JSRV Env were harvested, neutralized with 0.1 M Tris (pH 7.5) ifnecessary (pH 5.0 treated), and immunoprecipitated using anti-FLAG beads.Samples were boiled in a buffer containing 1% SDS and 1% �-mercaptoethanolfor 10 min, resolved by 10% SDS-PAGE, and analyzed by autoradiography.

Oligomerization assay. JSRV Env pseudovirions were pelleted by centrifuga-tion for 3 h at 185,000 � g on a 1.5-ml 20% sucrose cushion and were resus-pended in phosphate-buffered saline (PBS). Purified pseudovirions were incu-bated with appropriate concentrations of sHyal2 or equal volumes of PBS on icefor 30 min, followed by incubation at 37°C for 30 min. The complex was thentreated with a pH 7.4 or pH 5.0 buffer (acidified with a predetermined volume of0.1 N HCl) for 5 min at 37°C and was neutralized with 0.1 N NaOH if necessary.Unless otherwise stated, the complex was cross-linked by 25 �M dithiobis[suc-cinimidylpropionate] (DSP) (Pierce, Rockford, IL) for 30 min at room temper-ature, and the remaining reactive DSP was quenched by �0.2 M Tris (pH 8.0).For oligomerization assays involving different temperatures, the virion-sHyal2complex was incubated for 5 min at different temperatures before being cross-linked with 25 �M DSP. To assess the stability of the oligomers, the virion-sHyal2 complex, treated with either a pH 7.4 or a pH 5.0 buffer, was incubatedwith different concentrations of SDS or urea for 5 min at 37°C. All samples,unless otherwise specified, were incubated in a nonreducing sample buffer con-taining 0.1% SDS for 5 min at 37°C before being resolved by 7.5% SDS-PAGE,

followed by Western blotting using an anti-FLAG or anti-SU antibody, as pre-viously described (20).

Infection. For infection of nonpermissive cells, green fluorescent protein(GFP)-encoding MoMLV pseudoparticles bearing JSRV Env, HIV-1 Env, orVSV-G were bound to HeLa cells in Dulbecco’s modified Eagle medium con-taining 5 �g/ml Polybrene by spinoculation at 1,680 � g for 2 h at 4°C. Appro-priate amounts of sHyal2 were incubated with the cells for 48 h before the cellswere analyzed by flow cytometry. To assess the effect of sHyal2 on permissivecells, HTX cells were prebound by virions in a medium containing Polybrene for1 h at 4°C; cells were washed to remove unbound virions and were then incubatedwith sHyal2 for 1 h at 4°C. Alternatively, pseudovirions were incubated withappropriate amounts of sHyal2 on ice for 30 min, and the virus-sHyal2 complexwas added to HTX cells for binding at 4°C for 2 h, followed by washes to removeunbound virus. In both cases, cells were incubated for 48 h before analysis usingflow cytometry to measure the percentages of GFP-positive cells. The relativerate of infection was calculated by using cells that had not been treated withsHyal2 as a reference.

RESULTS

sHyal2 mediates syncytium induction by JSRV Env at a lowpH. To address the possible role of Hyal2 in JSRV Env-medi-ated fusion, we produced a soluble form of Hyal2 (sHyal2)using a Drosophila S2 cell line that stably expresses sHyal2(kindly provided by Vladimir Vigdorovich and Dusty Miller).In this system, the GPI anchor of Hyal2 was replaced by ahistidine tag, and the expressed proteins were secreted into thecell culture medium. We used nickel-nitrilotriacetic acid col-umns to isolate sHyal2 to a high purity (�95%) (Fig. 1A), andits ability to interact with JSRV Env was determined by flowcytometry, which demonstrated specific binding of sHyal2 to293 cells expressing FLAG-tagged JSRV Env (F-Jenv-F)(Fig. 1B).

We first examined whether sHyal2 could induce membranefusion of JSRV Env in 293 cells at a low pH. In the absence ofsHyal2, no syncytia were observed (left panel, Fig. 1C), as wepreviously reported (8). However, preincubation of the Env-expressing 293 cells with 0.5 to 15 �g/ml of sHyal2 for 1 h,followed by a pH 5.0 pulse for 5 min, led to syncytium forma-tion, albeit to different extents (central panels, Fig. 1C; alsodata not shown). The concentration of sHyal2 required toinduce syncytia under these conditions (pH 5.0 for 5 min) was�1 �g/ml, and no further increase in fusion was observed whenmore than 5 �g/ml sHyal2 was applied. In contrast, cells pre-incubated with 15 �g/ml of sHyal2, followed by a neutral-pHpulse, showed no apparent fusion (right panel, Fig. 1C). Theseresults indicate that both sHyal2 and a low pH are required forsyncytium induction by JSRV Env.

To show that the effect of sHyal2 on fusion takes placethrough JSRV SU, we performed a similar syncytium forma-tion assay using a JSRV Env chimera that contains the SUfrom a closely related enzootic nasal tumor virus (ENTV)(referred to as EJ in reference 7) and shows reduced binding toHyal2 (7). Indeed, this chimeric Env required a much in-creased concentration of sHyal2 (�15 �g/ml) for syncytiuminduction, and the percentage and size of syncytia were alsomuch lower than those of JSRV Env (Fig. 1D). The reducedfusogenicity of this chimera was not a result of lower Envexpression; it exhibited slightly greater Env expression than didwild-type Env (7). Together, these results demonstrate thatHyal2 is required to induce fusion of JSRV Env at a low pH,most likely by acting through its interaction with the JSRV SU.

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Membrane fusion mediated by sHyal2 at a low pH is blockedby a C-heptad repeat peptide of JSRV Env. We next sought todetermine the step(s) of the fusion process at which Hyal2 isrequired for syncytium formation by JSRV Env in 293 cells. Tothis end, we incubated Env-expressing 293 cells with sHyal2before, during, or after pH 5.0 treatment and examined the

effects of sHyal2 and low pH on Env-mediated fusion induc-tion. Incubation of sHyal2 after or during low-pH treatmentinduced no fusion or mild fusion (Fig. 1E, central panels),whereas incubation of sHyal2 prior to low-pH treatment, orinclusion of sHyal2 in the entire fusion assay, led to robustsyncytium formation with an increased number and size of

FIG. 1. sHyal2 mediates syncytium formation by JSRV Env in 293 cells at low pH, the effect of which is inhibited by a synthetic C-HR2 peptide.(A) Size exclusion chromatography and SDS-PAGE of sHyal2. sHyal2 (�50 kDa) was purified by fast protein liquid chromatography, and thepurified sHyal2 was analyzed by SDS-PAGE and Coomassie blue staining. (B) sHyal2 specifically binds to 293 cells expressing a FLAG-taggedJSRV Env (top); expression of the latter on the cell surface was confirmed by an anti-FLAG antibody (bottom). (C) Syncytium formation by JSRVEnv in 293 cells (expressing GFP) is induced only by sHyal2 plus pH 5.0 (central panels), but not by either one alone (left and right panels). (D) AJSRV Env chimera, containing the ENTV SU and with reduced binding to Hyal2, requires an increased concentration of sHyal2 for fusion (seethe text for details). (E) sHyal2 acts before but not after the pH 5.0 pulse for fusion induction. These experiments employed 5 �g sHyal2. (F) TheJSRV C-HR2 peptide acts after sHyal2 incubation and before a low-pH pulse to inhibit fusion. The assays employed 5 �g sHyal2 and 30 �g C-HR2peptide, respectively. Arrows are placed to indicate the fused cells only when fusion is not obvious.

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syncytia (Fig. 1E, left and right panels). Importantly, a syn-thetic peptide analogous to JSRV Env C-HR2, but not toN-HR1 (data not shown), substantially blocked syncytium for-mation (Fig. 1F). Of note, the effect of JSRV C-HR2 onEnv-mediated fusion was most pronounced when the peptidewas added between incubation with sHyal2 and the low-pHpulse, not before sHyal2 incubation (Fig. 1F), suggesting thatthe C-HR2 peptide acts after receptor binding but beforelow-pH triggering.

To test if a low pH could modulate the requirement ofsHyal2 for fusion, we preincubated 293 cells with differentamounts of sHyal2, followed by treatment with differentlow-pH conditions for 5 min. We observed that, although 1�g/ml of sHyal2 induced visible syncytia at pH 4.0, 5 �g ofsHyal2 was necessary to trigger discernible fusion at pH 5.0,and even 10 �g or more of sHyal2 barely triggered fusion at pH6.0 (data not shown). These results, together with the effect ofC-HR2 on syncytium formation shown above (Fig. 1F), suggestthat sHyal2 functions in cooperation with a low pH to triggerJSRV Env-mediated fusion, likely by inducing one or morefusion intermediates that are sensitive to binding by the JSRVC-HR2 peptide.

sHyal2 promotes shedding of JSRV SU in cells expressingJSRV Env. SU shedding is regarded as an important, thoughnot necessary, feature of retrovirus fusion and infection (11).To address the possible effects of sHyal2 on JSRV SU shed-ding, Env-expressing 293 cells were pulse-chase labeled with[35S]Met-Cys in the presence of different amounts of sHyal2.While sHyal2 had no effect on JSRV Env levels over the 3-hchase period (Fig. 2A), as would be expected, the shedding of

JSRV SU into the culture medium was clearly detected, in ansHyal2 dose-dependent manner (Fig. 2B). We also treated thelabeled 293 cells with different pH conditions, i.e., pH 7.0, 6.0,5.5, or 5.0, but observed no SU shedding in any of the pH-treated cells, either during the 5-min treatment interval (Fig.2D, lanes 4 to 7), or in the 1-h recovery period (Fig. 2D, lanes12 and 13). In the presence of sHyal2, while SU shedding wasdetected at pH 5.0 during the 1-h recovery period, its level wassimilar to that at pH 7.0 (Fig. 2D, compare lane 15 with lane14). Again, as expected, different pH treatments had no effecton JSRV Env expression and processing (Fig. 2C). Collec-tively, these data demonstrate that sHyal2, but not low pH,promotes JSRV SU shedding in Env-expressing 293 cells, sug-gesting that sHyal2 may cause conformational changes in SUon the cell surface, resulting in a weakened association be-tween SU and TM (see Discussion).

sHyal2 and a low pH induce conformational refolding of TMin JSRV Env-pseudotyped retroviral particles. To acquire bio-chemical evidence for the role of Hyal2 in JSRV Env-mediatedfusion activation, we developed an oligomerization assay andassessed possible conformational changes of JSRV TM in-duced by sHyal2, a low pH, or both. In the absence of thecross-linker, DSP, JSRV TM exists predominantly as a mono-mer of �37 kDa, although a minor species of �90 kDa, pos-sibly a TM dimer, was also detected in all samples (Fig. 3A,lanes 1 to 4). In the presence of DSP, in addition to theenhanced 90-kDa form, a �120-kDa species was apparentlyinduced by a low pH (Fig. 3A, lane 6), and its intensity wassubstantially enhanced by preincubation with sHyal2 (Fig. 3A,lane 8). Interestingly, sHyal2 alone also appeared to induce a

FIG. 2. sHyal2, but not low pH, promotes shedding of JSRV SU. (A and B) 293 cells expressing JSRV Env were metabolically labeled for 1 hand then chased for 3 h before the addition of the indicated amounts of sHyal2. Cells were chased for an additional 3 h before being lysed. Celllysates and culture media were harvested and immunoprecipitated using anti-FLAG beads. Samples were resolved by SDS-PAGE and analyzedby autoradiography. (A) Env expression and processing in cell lysates. (B) JSRV SU shedding into the culture medium. (C and D) Metaboliclabeling was performed as described for panels A and B, except that cells were incubated in the absence or presence of 5 �g sHyal2. The culturemedium was harvested (pre-pH), and the cells were treated with the indicated pH buffers for 5 min at 37°C. The pH buffers were harvested andneutralized (during pH), and the cells were cultured for an additional 1 h. The culture medium was harvested (post-pH), and the cells were lysed.The cell lysates (C) and harvested supernatants or neutralized pH buffers (D) were immunoprecipitated using anti-FLAG beads and were analyzedby autoradiography. Env, JSRV Env precursor; NC, parental 293 cells not expressing JSRV Env. The strong band appearing in the parental 293cells (panels A and C, lanes 1), and sometimes in the Env-expressing 293 cells, is likely a cellular protein that was nonspecifically pulled down byanti-FLAG beads.



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slight formation of the �120-kDa species (Fig. 3A, lane 7; notethe very faint band underneath the smeared bands), as well asto enhance the 37-kDa monomers as opposed to the larger,high-molecular-weight (HMW) species (�130 kDa) (Fig. 3A,compare lane 7 with lane 5). The latter observations werefurther confirmed by an independent experiment showing thatsHyal2 induces the formation of 120-kDa species, increases theproportions of TM monomers, and reduces those of HMWspecies (Fig. 3C, lanes 1 to 4).

Given that the JSRV pseudovirions used for the oligomer-ization assay harbor an Env that is FLAG tagged on both SUand TM (called F-Jenv-F), it is possible that the 120-kDa forminduced by sHyal2 and a low pH also contains JSRV SU and/orsHyal2, in addition to TM. This is despite the fact that, forunknown reasons, the anti-FLAG antibody does not normallydetect the FLAG-tagged JSRV SU by Western blotting (7, 20)yet functions efficiently for flow cytometry (Fig. 1B) and im-munoprecipitation (Fig. 2). Reblotting of the polyvinylidene

difluoride membrane that was used for Fig. 3A with an anti-Hisantibody (sHyal2 is tagged with His) revealed two major bands,i.e., �50- and �100-kDa species (likely corresponding to themonomer and dimer of sHyal2, respectively) (Fig. 3B), but noband corresponding to �120 kDa was observed, suggestingthat the 120-kDa species observed in Fig. 3A does not containsHyal2. To exclude the possibility that the 120-kDa speciesmay contain JSRV SU, we performed an oligomerization assayusing highly concentrated JSRV pseudovirions bearing an Envthat is FLAG tagged only on the SU (F-Jenv) and compared itsoligomerization patterns with those of F-Jenv-F. Again, no120-kDa species was detected for the F-Jenv construct (Fig.3D, lane 7), despite the loading of �5-fold more of this con-struct than of F-Jenv-F (Fig. 3D). Notably, a 55-kDa band wasfound in the samples treated with sHyal2 and in those treatedwith sHyal2 plus a low pH (Fig. 3D, lanes 6 and 7, respec-tively); this size is similar to that of boiled viral particles (Fig.4D, lane 8), confirming that sHyal2 does promote JSRV SU

FIG. 3. JSRV TM oligomerization induced by sHyal2 and low pH. (A) Purified JSRV Env pseudovirions were incubated with or without 1.5�g of sHyal2, followed by treatment with a pH 7.4 or pH 5.0 buffer. Samples were either cross-linked by 25 �M DSP or left untreated, and TMoligomerization was analyzed by Western blotting using an anti-FLAG (�-FLAG) antibody. (B) The membrane was then stripped and reblottedusing an anti-His (�-His) tag antibody to detect sHyal2. (C) Effect of sHyal2 on TM oligomerization. Purified virions were incubated with theindicated amounts of sHyal2 or were treated with a neutral or a low pH, which served as negative and positive controls, respectively. Samples werecross-linked and analyzed by Western blotting. (D) Purified JSRV pseudovirions bearing F-Jenv-F or F-Jenv were either treated with 1.5 �gsHyal2, alone or in combination with pH 5.0, or left untreated. Samples were either cross-linked by DSP (lanes 1 to 3 and 5 to 7) or boiled withoutcross-linking (lanes 4 and 8). Note that fivefold more virus was used for F-Jenv than for F-Jenv-F, in order to enhance the detection of SU. (E andF) Purified pseudovirions bearing F-Jenv-F were either left untreated or treated with 1.5 �g sHyal2 plus pH 5.0; then they were cross-linked withDSP. The cross-linked virus samples (indicated as “37°C”) and boiled virions and cell lysates (“Boiled”) were resolved by SDS-PAGE and analyzedby Western blotting using an anti-FLAG antibody (E) or an anti-JSRV SU (�-SU) antibody (F).



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shedding from the pseudovirions, as revealed in Fig. 3. Theeffect of sHyal2 on SU shedding, as well as the lack of SU in the120-kDa species, was further confirmed by using an anti-SU an-tibody on the F-Jenv-F construct (Fig. 3F), although the signalfor SU was very weak, even with prolonged exposure (Fig. 3F,lane 2). Again, it is evident that the anti-FLAG antibody wasunable to detect JSRV SU by Western blotting, even fromsamples of boiled viral particles and cell lysates (compare Fig.3E, lanes 3 and 4, with Fig. 3F, lanes 3 and 4). Taken together,these results strongly support the notion that the 120-kDaspecies is predominantly derived from JSRV TM and does notappear to contain JSRV SU or sHyal2. However, we cannotcompletely rule out the possibility that the anti-FLAG and/oranti-SU antibody used here may not recognize the JSRV SUpossibly present in the 120-kDa form.

sHyal2 modulates the threshold of low pH or heat requiredfor TM oligomerization. We next tested if sHyal2 could mod-ulate the thresholds of low pH and/or high temperature re-quired for TM oligomerization. In the absence of sHyal2, thepH threshold for inducing the �120-kDa species was �pH 5.5(Fig. 4A, lane 5), but it shifted to �pH 6.5 or 7.0 in thepresence of 1.5 �g of sHyal2 (Fig. 4A, lanes 9 and 10). ThesepH thresholds appeared to be relatively higher than thoseobserved in the fusion assays (7), possibly because the bio-chemical oligomerization assay is more sensitive than the phe-

notype-based fusion assay. Similarly, the temperature thresh-old for detecting the �120-kDa species was reduced from 65°Cto 50°C in the presence of sHyal2 (Fig. 4B, compare lane 9 withlane 4). Taken together, these data demonstrate that sHyal2effectively reduces the energy barriers that must be overcomeby low pH or heat in order to form the �120-kDa species. Ourresults also demonstrate that the JSRV Env protein in themature MoMLV pseudoparticles is metastable, like those ofinfluenza virus HA and ASLV Env (5, 22, 32, 37).

Stability of JSRV TM oligomers induced by sHyal2 and lowpH. The stability of JSRV TM oligomers, in particular the�120-kDa species, which was specifically induced by a low pHand/or sHyal2, was also assessed. TM oligomers induced by acombination of low pH and sHyal2 were stable in urea atconcentrations up to 2 M (Fig. 5A, lanes 6 to 9), whereassHyal2-induced oligomers were relatively unstable and disas-sociated even with 0.5 M urea (Fig. 5A, lanes 2 to 5). Inter-estingly, the �120-kDa species, induced either by a low pHplus sHyal2 or by sHyal2 alone (Fig. 5B, lanes 1 to 5), weresensitive to SDS treatment, even at very low concentrations(0.01% or higher). These results are in sharp contrast to thosereported for ASLV, whose six-helix bundles (6-HBs) and otherHMW species are virtually resistant to 1 to 2% SDS, evenwithout cross-linking (22, 24). However, our data are similar to

FIG. 4. sHyal2 modulates the thresholds of low pH and temperature required for JSRV TM oligomerization. JSRV pseudovirions, preincu-bated with 1.5 �g sHyal2 or an equal volume of PBS, were treated with the indicated pH conditions (A) or temperatures (B) for 5 min, followedby cross-linking with DSP, and were analyzed by Western blotting using an anti-FLAG antibody.

FIG. 5. Stability of TM oligomers induced by sHyal2 and low pH. JSRV Env pseudovirions, preincubated either with 1.5 �g sHyal2 alone orwith 1.5 �g sHyal2 plus pH 5.0, were subjected to the indicated concentrations of urea (A) or SDS (B) for 5 min at 37°C before being cross-linkedby DSP and analyzed by Western blotting.



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those for MoMLV, whose TM oligomers are also less stable inSDS (35).

sHyal2 enables JSRV Env pseudovirions to transduce non-permissive cells but blocks their entry into permissive cells.The apparent role of sHyal2 in JSRV Env-mediated fusionprompted us to further test whether sHyal2 can induce JSRVvector transduction of nonpermissive cells. To this end, JSRVEnv pseudovirions were allowed to bind to nonpermissiveHeLa cells at 4°C for 2 h, and the virion-cell complexes wereincubated with sHyal2 for 48 h before the cells were analyzedfor infectivity by flow cytometry. As shown in Fig. 6A, sHyal2

enabled the transduction of HeLa cells by JSRV pseudovirions(albeit with low efficiency, �2 to 3%) but had no effect on thatby HIV-1 Env (nonpermissive in HeLa cells; efficiency, �0.16to 0.3%) or VSV-G (permissive in HeLa cells; efficiency,�35%). In contrast, sHyal2 markedly reduced the transductionefficiency of JSRV-permissive HTX cells, which were pre-bound with JSRV pseudovirion particles, in a dose-dependentmanner (Fig. 6B). As would be expected, preincubation ofJSRV pseudovirions with sHyal2 (1.5 �g/ml) before binding toHTX cells virtually blocked vector transduction (Fig. 6B).These results strongly argue that, in addition to blocking virus

FIG. 6. sHyal2 enables transduction of nonpermissive cells by JSRV Env pseudovirions (A) but blocks their entry into permissive cells (B).(A) HeLa cells were bound by JSRV Env, HIV-1 Env, or VSV-G pseudovirions encoding GFP by spinoculation, followed by incubation with theindicated amounts of sHyal2. The transduction efficiency was analyzed by flow cytometry 48 h postinfection. The titers of JSRV pseudovirions withthe presence of 0, 1.5, and 10 �g of sHyal2 in HeLa cells are 34, 360, and 550 GFP� cells/ml, respectively. The titers of HIV-1 Env and VSV-Gpseudotypes in HeLa cells are �35 and 3.6 � 106 GFP� cells/ml, respectively. Note that the low titer of HIV-1 Env pseudovirions in HeLa cellswas not due to the viral stock preparation or infection, because the titer in HeLa-TZM-bl cells expressing CD4 and CCR5 was 5 � 104 GFP�

cells/ml. Representative dot plots are shown, and the percentages of GFP� cells are given at the lower right. The changes (n-fold) in transductionefficiency in four independent experiments were averaged and plotted. (B) HTX cells either were infected with JSRV Env or VSV-G pseudovirionsthat had been preincubated (before binding) with the indicated amounts of sHyal2 or were first bound with pseudovirions and then incubated withthe indicated amounts of sHyal2 (after binding); see details in Materials and Methods. In both cases, unbound sHyal2 and virions were removedfrom cells by washing, and viral infectivity was assessed by flow cytometry 48 h postinfection.



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binding to target cells, sHyal2 also inactivates JSRV Envpseudovirion infectivity, possibly by triggering premature con-formational changes of TM, as shown in Fig. 3. The potenteffect of sHyal2 on the infectivity of JSRV Env pseudovirionswas in sharp contrast to that of low pH, which has no signifi-cant effect (8). These results are also consistent with the ob-servation that syncytium induction mediated by sHyal2 was notstrictly dose dependent (Fig. 1C), and they may explain, atleast in part, why the sHyal2-mediated transduction of a JSRVvector into nonpermissive HeLa cells was inefficient and didnot occur in a linearly dose dependent manner (Fig. 6A). It istherefore possible that the sHyal2-mediated activation ofJSRV Env fusion is cooperative, where multiple interactionsbetween and within the Env trimers of JSRV may occur upona single binding by sHyal2. A similar mechanism has beenreported previously for ASLV (10).

DISCUSSION

JSRV is an interesting retrovirus in that its native Env func-tions as an active oncogene in addition to mediating entry intohost cells. However, unlike most retroviruses, which are pHindependent, JSRV entry requires dynamin-dependent endo-cytosis and a low pH (1, 8). Moreover, we observed that mem-brane fusion mediated by JSRV Env in vitro could be inducedonly by a low pH in cells that overexpress functional Hyal2 (8),suggesting that Hyal2 may play an active role in the fusionactivation process. Using a soluble form of the JSRV receptor,sHyal2, we demonstrate here that a low pH is indeed notsufficient to induce fusion by JSRV Env but that receptorbinding is also required for this process to occur (Fig. 1). Weconclude that, like ASLV, oncogenic JSRV has borrowed fea-tures of both pH-independent and pH-dependent viruses forits fusion activation and entry, which involve stepwise andcooperative triggers from both the receptor and low pH. Thismultistep fusion pathway used by JSRV and ASLV is alsoanalogous to HIV entry, where CD4 binding to HIV Envtriggers initial conformational changes of gp120 that allowsubsequent coreceptor binding and fusion, leading to produc-tive infection (11).

However, there are some striking differences between JSRVand ASLV in terms of fusion activation by receptor binding orpriming. First, we show that sHyal2 induces pronounced JSRVSU shedding in 293 cells at a neutral pH (Fig. 2), and this is insharp contrast to findings for ASLV, where soluble Tva doesnot cause apparent disassociation between SU and TM (36)(see detailed discussions below). Second, we find that the�120-kDa species, likely the 6-HB, or a pre-6-HB intermedi-ate, of JSRV Env, is much less stable in SDS (Fig. 5A) than itsASLV Env counterpart, which is resistant to SDS at concen-trations up to 2% (22, 24). This could be due to the relativelyshort lengths of the N- and C-HRs of JSRV Env compared tothose of ASLV (25) and/or to their intrinsic differences inspecific amino acid interactions, or technical limitations. Therelative low stability of JSRV TM oligomers may explain, atleast in part, why cross-linking is required for the detection ofJSRV TM oligomerization (Fig. 3). Third, sHyal2 potentlyinactivates the infectivity of JSRV Env pseudovirions in astep(s) after virus binding (Fig. 6B), which is consistent withthe observation that sHyal2 induces the TM conformational

refolding of JSRV Env at a neutral pH (Fig. 3C). This featureappears also to be different from that of ASLV, in which bothsoluble Tva and a low pH are required to inactivate viralinfectivity (37).

We propose the following model for JSRV Env-mediatedfusion activation. Upon receptor binding, JSRV SU undergoesconformational changes that cause it to disassociate from TM.This would relieve its intrinsic restriction on TM, resulting inexposure of the fusion peptide and its subsequent insertioninto the plasma membrane. Although our current study did notdirectly address the conformational changes of SU and theassociation of TM with the cell membrane, it was evident thatsHyal2 promotes JSRV SU shedding in both Env-expressingcells (Fig. 2) and Env pseudovirions (Fig. 3). It is notable thatJSRV SU contains a CXXC motif, a conserved amino acidsequence that has previously been shown to be important forfusion activation in other retroviruses by mediating the switchfrom an intersubunit (SU-TM) to an intra-SU subunit disulfidebond (40). Interestingly, pretreatment of JSRV pseudovirionswith an alkylating agent, N-ethylmaleimide, failed to block theability of sHyal2 and low pH to induce the formation of the120-kDa species (data not shown). While we cannot rule outthe possibility that this might be due to the experimental con-ditions we have used, or the nature of the pseudovirions thatwere produced from the Hyal2-positive 293 cells and couldhave been prematurely activated, it is noteworthy that betaret-roviruses, including JSRV and ENTV, were recently shown tocontain no disulfide bond between SU and TM (John M. Cof-fin, personal communication). Whether or not the CXXC mo-tif of JSRV SU, and/or the potential formation of receptor-induced thiolates, mediates JSRV SU shedding and fusionactivation remains to be investigated.

Once the fusion peptide inserts into the plasma membrane,the TM of JSRV Env may become elongated and form one ormore prehairpin intermediates that are sensitive to JSRV C-HR2 peptide binding. At this point, a low pH is necessary totrigger dramatic TM conformational refolding in order to forma 6-HB and to enable subsequent fusion. We found, however,that although the JSRV C-HR2 peptide effectively inhibitedJSRV Env-mediated membrane fusion (Fig. 1F; also data notshown), it is unable to block the formation of the �120-kDaspecies, even at extremely high concentrations (up to 2 mg/ml)(data not shown). It is possible that the �120-kDa form is aheterogeneous mixture, containing both pre-6-HB intermedi-ates and stable 6-HBs; alternatively, this form may be a pre-cursor of JSRV 6-HB, with a true 6-HB form remaining to beidentified. It is noteworthy that the ASLV C-HR2 peptide,R99, does not always block the formation of 6-HB species yetpotently inhibits ASLV Env-mediated fusion and infection (22,23, 25). We should mention that all the JSRV Env pseudoviri-ons used in this study were produced from 293T or 293 cells(because of their high transfection efficiency), and it is there-fore possible that the endogenous Hyal2 expressed in thesecells may have prematurely activated JSRV Env in the viralparticles and contributed to the formation of the �120-kDaspecies by low pH alone, even without sHyal2 preincubation(Fig. 3). Work is under way to address this issue and to furthercharacterize the JSRV fusion intermediate(s) and the forma-tion of its 6-HBs.



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ACKNOWLEDGMENTS

We thank Vladimir Vigdorovich and Dusty Miller for the generousgift of the S2 cell line stably expressing sHyal2. We also thank theanonymous reviewers for comments and suggestions that improved thearticle.

This work was supported by the Canadian Institutes of Health Re-search (CIHR) (to S.-L.L.). M.C. was supported by scholarships fromthe Natural Sciences and Engineering Research Council of Canada(NSERC). S.-L. Liu holds a Canada Research Chair in Virology andGene Therapy.

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