Effect of Epitope Position on Neutralization by An
http://www.100md.com
病菌学杂志 2006年第5期
Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 4, Room 336, Bethesda, Maryland 20892
ABSTRACT
The membrane-proximal region of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein (TM) is critical for envelope (Env)-mediated membrane fusion and contains the target for broadly reactive neutralizing antibody 2F5. It has been proposed that 2F5 neutralization might involve interaction of its long, hydrophobic, complementarity-determining region (CDR) H3, with adjacent viral membrane. Using Moloney murine leukemia virus (MLV) as a tool, we examined the effect of epitope position on 2F5 neutralization. When the 2F5 epitope was inserted in the proline-rich region of MLV Env surface protein (SU), 2F5 blocked cell fusion and virus infection, whereas MLV with a hemagglutinin (HA) epitope at the same position was not neutralized by anti-HA, even though the antibodies bound their respective Envs on the surface of infected cells and viruses equally well. When the 2F5 epitope was inserted in the MLV Env TM at a position comparable to its natural position in HIV-1 TM, 2F5 antibody blocked Env-mediated cell fusion. Epitope position had subtle effects on neutralization by 2F5: the antibody concentration for 50% inhibition of cell fusion was more than 10-fold lower when the 2F5 epitope was in SU than in TM, and inhibition was less complete at high concentrations of antibody; we discuss possible explanations for these effects of epitope position. Since membrane proximity was not required for neutralization by 2F5 antibody, we speculate that the CDR H3 of 2F5 contributes to neutralization by destabilizing an adjacent protein rather than by inserting into an adjacent membrane.
INTRODUCTION
Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS, is highly immunogenic but notoriously poor at generating broadly reactive, neutralizing antibodies. This is a crucial problem for vaccine development. The major target for neutralizing antibodies is the envelope glycoprotein (Env). Although the Env sequence is highly variable between viruses and over time in infected individuals, some regions of Env are highly conserved. Nevertheless, only a few broadly reactive, anti-Env neutralizing antibodies have been identified (5, 29, 36, 37, 41, 46, 49, 58, 59, 63).
HIV-1 Env is translated as a precursor (gp160) that undergoes posttranslational modification including trimerization, glycosylation, and proteolytic processing to form surface protein (SU; gp120) and transmembrane protein (TM; gp41) as it travels from the endoplasmic reticulum (ER) to the cell surface. SU and TM remain associated through noncovalent interactions, forming a trimer of heterodimers. The process of Env-mediated membrane fusion has been extensively analyzed. SU binding to the HIV-1 receptor CD4 and a coreceptor, usually CCR5 or CXCR4, induces conformational changes in SU, probably leading to its dissociation from TM. This causes TM to refold, exposing a hydrophobic N-terminal peptide that is believed to insert into the target cell membrane and then retract to pull viral and target cell membranes together. The retraction mechanism involves formation of a thermodynamically stable trimer of antiparallel alpha-helices (hairpins) derived from heptad repeats located just downstream of the fusion peptide (N-heptad repeats) and just upstream of where TM traverses the viral membrane (C-heptad repeats) (19, 21, 34).
The segment of TM between the C-heptad repeats and the transmembrane anchor, designated the membrane-proximal region (MPR), consists of 20 amino acids that are highly conserved among different clades of HIV-1. Mutation of amino acids in this region can impair fusion without altering surface expression of Env, suggesting that the region has a role in fusion (16, 35, 48). Amazingly, MPR is the target for three broadly reactive, neutralizing antibodies to HIV-1: 2F5, 4E10, and Z13 (3, 36, 41, 63). This region might be a "hot spot" for such antibodies because of constraints on sequence variability due to a role in membrane fusion that is sensitive to antibody binding. However, immunization with peptides from this region resulted in antibodies that bound well but did not block fusion (25, 31), suggesting that neutralization potency is influenced by special properties of some antibodies, possibly related to the membrane-proximal microenvironment. Both 2F5 and 4E10 have an unusually long, hydrophobic, third heavy-chain complementarity-determining region (CDR H3), which prompted the hypothesis that neutralization involves the interaction of this region with neighboring lipid membranes (7, 12, 23, 38, 57, 62). Given the dearth of broadly neutralizing antibodies to HIV-1 and their potential importance for therapy and vaccine development, it is important to understand whether membrane proximity of the epitope or some unrelated, intrinsic property of certain antibodies makes them neutralizing.
We used Moloney murine leukemia virus (Mo-MLV) Env-mediated fusion as a tool to investigate this question. MLV uses the mouse cationic amino acid transporter 1 (mCAT1) as receptor (1). Like HIV-1, MLV Env forms a homotrimer of SU-TM heterodimers. Only the amino-terminal 240 amino acids of MLV SU have been crystallized. Downstream of the crystallized portion is a proline-rich region that is thought to form a flexible "hinge" in SU. This hinge tolerates insertions without impairing Env function (26, 47, 55). Like HIV-1, MLV TM has an N-terminal hydrophobic fusion peptide followed by an N-heptad repeat that trimerizes. It is not known if MLV has an analogous C-heptad repeat region that folds back to form "hairpins" during fusion. The MPR of MLV has not been extensively studied for the effect of mutations, but it is likely that the mechanism of fusion for MLV is closely related to that of HIV-1 given the overall structural similarities of their Envs (8, 17, 18). We inserted the 2F5 epitope or control epitopes into MLV Env, in the TM at a position comparable to that of the 2F5 epitope in gp41, or in SU in the proline-rich region and characterized the effect of the 2F5 antibody on virus infection and MLV Env-mediated cell fusion.
MATERIALS AND METHODS
Constructs. To insert 2F5" (QELLELDKWASLWNW) or hemagglutinin (HA; YPYDVPDYA) into the SU of MLV Env, sense and antisense oligonucleotides encoding the epitope tag with SgrAI overhangs were synthesized, annealed, and ligated into pCEETR (44) cut with SgrAI. To facilitate inserting 2F5" and His6 epitope tags into the TM of MLV Env, a unique BstBI restriction site was introduced into pCEETR (with HA in SU) by site-directed mutagenesis 14 amino acids upstream of the transmembrane domain. This mutation does not change the amino acid sequence of MLV Env protein. Sense and antisense oligonucleotides encoding 2F5" or His6 (HHHHHH) with BstBI overhangs were synthesized, annealed, and ligated into pCEETR-BstBI (SU-HA) cut with BstBI. These Env mutations were transferred into pNCA (11) as a BspEI-ClaI restriction fragment.
Cell fusion assay. HEK293 or HeLa cells were seeded in a six-well plate at a density that resulted in their being 90 to 95% confluent the next day. Cells were cotransfected with 2 μg each of wild-type or mutant pCEETR plus pTet-Off (Clontech) per well using Lipofectamine 2000. Twenty-four hours later, the cotransfected cells were trypsinized and preincubated with or without antibody and then mixed in triplicate with human osteosarcoma cells (U2OS) stably expressing MLV receptor mCAT1 and a luciferase reporter gene under the control of the tetracycline response element (U2OS-mCAT1-TRELuc). Luciferase activity was assayed about 16 h after coculture using the Luciferase Assay System from Promega (Madison, Wis.) and a Victor 3 luminometer (PerkinElmer, Boston, Mass.). For cell fusion inhibition assays, transfected cells were premixed with twice the indicated concentration of antibody for 30 min before adding an equal volume of indicator cells; antibody was left in the coculture medium. Anti-HA antibody was monoclonal antibody HA-7 from Sigma (catalog number H9658), anti-His6 was monoclonal antibody H-3 from Santa Cruz Biotechnology (catalog number sc-8036x), and 2F5 (catalog number AB001) was from Polymun Scientific (Vienna, Austria).
Surface protein labeling and Western blot analysis. An aliquot of cells transfected for cell fusion was rinsed with phosphate-buffered saline (PBS) and labeled in PBS supplemented with 1 mg/ml membrane-impermeable biotinylation reagent (catalog number 20338; Pierce, Rockford, Ill.) for 1 h on ice. The free biotin was quenched with 100 mM glycine in PBS buffer. Following two washes with PBS, the cells were lysed using ice-cold radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate) supplemented with a protease inhibitor cocktail (catalog number 1836153; Roche, Indianapolis, IN). Biotinylated proteins were purified using streptavidin beads and eluted by heating in 1x SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer (catalog number NP0007; Invitrogen, Carlsbad, CA) at 98°C for 5 min. Samples were resolved on SDS-4 to 12% PAGE gels, and proteins were transferred to a polyvinylidene difluoride membrane. The membrane was blocked and blotted as described previously (39).
Virus infection and neutralization assay. Wild-type and mutant MLVs were made by cotransfecting HEK293T cells with pNCA plus pFB-Luc by using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, supernatant virus was filtered using a 0.45-μm filter and incubated with or without antibody for 30 min at 4°C and then added to plain NIH 3T3 cells in a 24-well plate in the presence of 4 μg/ml Polybrene (Sigma). Luciferase activity was assayed 60 h after infection as described for cell fusion.
Flow cytometry analysis of antibody affinity and virus binding. NIH 3T3 cells chronically infected by wild-type Mo-MLV or Mo-MLV containing 2F5" or HA in SU were detached at 37°C using PBS supplemented with 2 mM EDTA and 1% fetal calf serum (FCS) and resuspended in staining buffer (0.2% bovine serum albumin and 0.09% sodium azide in PBS at pH 7.4). HEK293 and 293mCAT1 cells were detached in the same way and mixed with free or antibody-bound virus in a total volume of 200 μl. Primary antibody was incubated with cells for 30 min on ice. The cells were washed once using 2 ml cold wash buffer (PBS, 2 mM EDTA, 1% fetal bovine serum [FBS]) and then incubated for 30 min on ice with fluorescein isothiocyanate (FITC) anti-mouse immunoglobulin G (IgG; catalog number F9137, lot number 015k4793; Sigma) or FITC-anti-human IgG (catalog number F4512, lot number 024k4811; Sigma) diluted 1:100 in wash buffer. After two washes, the cells were resuspended in 0.5 ml staining buffer supplemented with 20 μl 7-amino actinomycin D viability probe (catalog number 555816; BD Pharmingen) to identify dead cells. Data were acquired on a BD FACSCalibur flow cytometer and analyzed using FlowJo software (Tree Star, Ashland, OR).
RESULTS
Constructing chimeric MLV Env with the 2F5 epitope in TM or SU. The core epitope sequence for 2F5 antibody is ELDKWA. However, studies have shown that peptides with extra amino acids on each end of the epitope bind 2F5 better (2, 10, 31-33, 41, 53, 63). We extended the epitope by including an extra 4 or 5 amino acids on either end (QELLELDKWASLWNW); we designated this epitope 2F5". To investigate if the position of the 2F5 epitope affected neutralization potency, we inserted 2F5" in MLV TM or SU in a vector pCEETR that expressed a fusogenic form of Mo-MLV Env whose function was quantifiable in a cell fusion assay (39, 40, 44). In TM, 2F5" was inserted 14 amino acids upstream from the membrane anchor, the same position as in HIV-1 TM. In SU, 2F5" was inserted at a unique SgrAI site in the proline-rich region. For controls, we inserted an influenza virus HA epitope (YPYDVPDYA) at the same positions in SU and TM, but HA abrogated fusion when inserted in TM, so we replaced it with a six-His tag (HHHHHH).
We assessed the function of these chimeric Envs using a cell fusion assay (40) in which HEK 293 or HeLa cells were transfected with wild-type or epitope-tagged MLV Env in pCEETR plus a transcriptional transactivator (tTA, encoded by pTet-Off from Clontech). Twenty-four hours later, the transfected cells were cocultivated with human osteosarcoma cells (U2OS) stably expressing the MLV receptor mCAT1 and a luciferase reporter gene under the control of a tetracycline response element (TRE), a promoter that is activated by tTA. Luciferase activity was assayed 16 h after coculture. Insertion of 2F5" or HA in SU did not affect cell fusion in this assay, whereas insertion of 2F5" or a six-His tag in TM decreased cell fusion 50 to 75% (Fig. 1A).
To understand why insertions in TM reduced cell fusion, we checked the expression level of Env with 2F5" in TM by Western blot analysis of total cell lysates or purified cell surface proteins. The latter were prepared by biotinylating cells with a membrane-impermeable biotinylation reagent, binding the lysate to streptavidin beads, and eluting bound proteins by boiling in gel loading buffer. The Western blots were analyzed with anti-HA antibody since the Env with 2F5" in TM was also labeled with HA in SU. Lysates of cells transfected with singly tagged Env (HA in SU) contained the gp85 Env precursor and gp70 SU (Fig. 1B, lane 1); this pattern was the same as that of Env in cells transfected with wild-type Env, detected with anti-SU antibody (data not shown). Cells transfected with doubly tagged Env (HA in SU, 2F5" in TM) contained roughly the same amount of Env precursor, but no gp70 SU (lane 2). This suggests that insertion of 2F5" in the TM inhibited transport to the late Golgi apparatus, where cleavage into SU and TM normally occurs. Cell surface Env was also dramatically reduced in cells transfected with doubly tagged Env (HA in SU, 2F5" in TM) compared to singly tagged Env (HA in SU) (Fig. 1C, lane 2 versus lane 1), consistent with transport being blocked before Env reached the plasma membrane. Loading controls showed that approximately the same amounts of total protein (-actin) and surface protein (integrin 5) were loaded in all lanes (Fig. 1B and C, lower panels).
Despite the very low levels of processed and surface SU detectable by Western blotting when 2F5" was inserted in TM, cell fusion detected by luciferase induction was reduced only two- to fourfold to a level well above the background in indicator cells cocultivated with cells transfected with tTA but no Env (Fig. 1, bar 6). This implies that only a small amount of cell surface Env is required for Env-mediated cell fusion. We obtained similar results with other mutant Envs, both in MLV and in HIV (data not shown), which showed marked decreases in cell surface Env detected by Western blotting but only moderate decreases in cell fusion measured by the luciferase induction assay. This suggests that wild-type Env protein is present in large excess for cell fusion, possibly because large areas of cell surface can contact one another, with fusion at any single site being sufficient to transfer transcriptional activator.
2F5 antibody specifically inhibits cell fusion mediated by Env with 2F5" in either SU or TM. We compared the ability of 2F5, anti-HA, and anti-His6 antibodies to inhibit Env-mediated cell fusion when the epitopes were inserted in SU or TM. Twenty-four hours after transfection, Env-expressing cells were trypsinized and preincubated with antibody at double the indicated concentrations for 30 min at 4°C and then cocultivated with an equal volume of U2OS-mCAT1-TRELuc indicator cells; antibody was left in the medium during the coculture. Luciferase activity was assayed 16 h later. Luciferase activity in the presence of antibody is shown as the percentage of luciferase activity in the absence of antibody (Fig. 2). The 2F5 antibody inhibited fusion mediated by Env with 2F5" in SU up to 80% (Fig. 2, group 4); inhibited fusion mediated by Env with 2F5" in TM up to 97% (group 5), which was approximately equal to background; and did not inhibit fusion mediated by Env with HA in SU, showing that the inhibition was specific (group 2). Anti-HA and anti-six-His antibodies did not inhibit fusion mediated by their corresponding Envs (groups 1 and 6).
Dose response of 2F5 inhibition of cell fusion is different for Env with 2F5" in SU versus TM. 2F5 inhibition of cell fusion was dose dependent and essentially complete at high concentrations of antibody (113 μg/ml) when 2F5" was in TM (Fig. 2, group 5), but little dose dependency was seen when 2F5" was in SU (group 4). To see if this was due to the limited range of antibody concentrations tested, we repeated the experiment with a more extensive, twofold serial dilution of antibody. The results (Fig. 3) show that cell fusion is inhibited at lower concentrations of 2F5 antibody when 2F5" is in SU than when it is in TM and that inhibition plateaus at about 80% inhibition when 2F5" is in SU, whereas it goes essentially to completion when 2F5" is in TM. These results are consistent with and extend the findings shown in Fig. 2. The antibody concentration for 50% inhibition (IC50) of cell fusion for Env with 2F5" in SU was 0.4 μg/ml, more than 10 times lower than that for Env with 2F5" in TM (6.4 μg/ml).
2F5 antibody specifically neutralizes MLV with 2F5" in SU. Cell fusion and virus infection do not always correlate (13, 14, 51, 54, 61). To see if 2F5 antibody also neutralized MLV with 2F5" in Env, we substituted epitope-tagged Envs for the wild-type Env in pNCA, a vector that encodes full-length, infectious Mo-MLV (11). HEK293T cells were cotransfected with wild-type or epitope-tagged pNCA vectors plus pFB-Luc (Stratagene, La Jolla, CA), a Mo-MLV retroviral vector encoding firefly luciferase as a reporter. Forty-eight hours after transfection, cell supernatants containing wild-type or epitope-tagged viruses were filtered and mixed with 80 μg/ml antibody at 4°C and then added to NIH 3T3 cells in the presence of 4 μg/ml Polybrene. Luciferase activity was assayed 60 h after infection. 2F5 antibody neutralized infection by the pseudotyped vector when Env contained 2F5" in SU (Fig. 4, bar 7) but not when it was pseudotyped with wild-type Env (bar 6). Anti-HA antibody did not neutralize infection when Env contained HA in SU (bar 5). The small decrease of luciferase activity in the presence of anti-HA antibody was nonspecific, since the same decrease was observed for wild-type virus (bar 4 versus bar 1). Infectious virus was not obtained from constructs with epitope tags in TM. This is probably because cell surface levels of Env with these epitopes were too low to make infectious virus particles. Virus infectivity may be more sensitive to reductions in surface Env than cell fusion because virus particles contain limited numbers of Env proteins (9, 60), whereas cell surfaces in contact with one another are much larger than individual viruses and therefore should contain a larger number of Env molecules. We cannot rule out the possibility that epitope tags in TM also impaired Env incorporation into viral particles.
Serial dilution of 2F5 antibody showed that virus containing SU-2F5" Env was neutralized with an IC50 of less than 0.2 μg/ml, and neutralization reached a plateau at about 90% neutralization (Fig. 5). The luciferase signal at this plateau level was well above background (mock bar). Thus, neutralization of virus with 2F5" in SU resembled inhibition of cell fusion mediated by the same Env in having a low IC50 and being only partially neutralized by 2F5 (Fig. 3).
2F5 and anti-HA antibody have comparable affinities for their epitopes in SU expressed on the cell surface and on virions. To see if the ability of 2F5 antibody, but not anti-HA, to block fusion was due to higher affinity for its epitope, we measured binding affinity by flow cytometry. NIH 3T3 cells that were chronically infected with wild-type virus or viruses with 2F5" or HA tags in SU were stained with serial dilutions of each antibody, followed by fluorescently labeled secondary antibodies. The fluorescence histograms (Fig. 6A) and mean fluorescence intensities at each antibody concentration (Fig. 6B) were very close for the two antibodies. The signal-to-noise ratio (fluorescence intensity of tagged-virus-infected cells versus wild-type-virus-infected cells) was highest for both antibodies at 10 μg/ml, and the concentration of antibody that gave half this fluorescence intensity was about 6 μg/ml for both antibodies. The 2F5 antibody is of human origin, and anti-HA is of mouse origin. The secondary antibodies, FITC-labeled anti-human IgG and FITC-labeled anti-mouse IgG, had the same fluorescein-to-protein ratios and were used at the same concentrations. The results imply that the ability of 2F5, but not anti-HA, to block fusion is not due to a difference in affinity for their epitopes.
We also checked the binding of 2F5 and anti-HA to virions. Epitope-tagged viruses from chronically infected cells were concentrated by centrifugation and incubated with HEK293 cells that stably express the mCAT1 receptor (293mCAT1; kindly provided by Christian Kozak, National Institute of Allergy and Infectious Diseases), versus control HEK293 cells that do not have this receptor. After 90 min on ice, 2F5 or anti-HA antibodies were added at 10 μg/ml. After 30 min on ice, the cells were washed and stained with equal amounts of corresponding secondary antibodies. 293mCAT1 cells fell into two populations (Fig. 7, panels A and C, green curves): slightly more than half of the cells bound a large amount of virus (mean fluorescence intensity, 2,000 RLU), while a second population bound virus very weakly, if at all (mean fluorescence intensity, 60 to 100 RLU). Control HEK293 cells lacking the receptor (panels B and D) defined the level of nonspecific virus binding (mean fluorescence intensity, 20 to 30 RLU). The "dim" population in 293mCAT1 cells presumably does not express receptor or expresses only small amounts of it. This is not unexpected, as the 293mCAT1 cells were not cloned but derived from a bulk culture of cells selected with Geneticin after transfection with a vector encoding mCAT1 and Neor. The mean fluorescence intensity of the "bright" peak of 293mCAT1 cells binding virus with 2F5" in SU was the same as that of cells binding virus with HA in SU (Fig. 7, panels A and C). This shows that the ability of 2F5—but not anti-HA—to neutralize virus is not due to a difference in the amounts of their respective Envs on virions.
The virus-binding assay allowed us to ask if 2F5 antibody blocked binding of virions with 2F5" in SU to the mCAT1 receptor. An aliquot of each concentrated, epitope-tagged virus was premixed with 10 μg/ml of corresponding antibody on ice for 30 min and then added to 293mCAT1 cells. After 90 min on ice, the cells were washed and stained with secondary antibody. Preincubation with 2F5 inhibited binding of the 2F5"-tagged virus by 53 to 61%, while anti-HA inhibited binding of the HA-tagged virus by 36 to 39%, based on mean fluorescence measurements in two independent experiments. These results suggest that inhibition of binding might marginally contribute to neutralization by 2F5 when its epitope is in SU, but it does not account for the full extent of neutralization.
DISCUSSION
Of the few potent neutralizing antibodies against HIV-1 virus, 2F5 is one of the best studied. However, its neutralization mechanism is still far from clear. To find out whether the location of its epitope is critical, as suggested by models in which the CDR H3 of 2F5 interacts with the viral membrane, we artificially expressed an extended 2F5 epitope in Mo-MLV SU or TM and found that 2F5 inhibited fusion regardless of the position of its epitope, whereas anti-HA or anti-His6 antibody did not inhibit fusion mediated by Env with the corresponding epitope in either SU or TM.
Most regions of MLV Env cannot tolerate insertions (47). Here, we inserted 2F5" or an HA epitope into the proline-rich region of SU without affecting fusion activity. However, insertion of 2F5" or the six-His tag in the MPR caused large reductions in SU/TM cleavage and transport to the cell surface, precluding production of infectious virus. Surprisingly, Env-mediated cell fusion was only modestly reduced so that we could still use these insertions to study the ability of the respective antibodies to inhibit cell fusion. Despite functioning in cell fusion, the TM-tagged Env molecules could be abnormal in other ways. We also inserted a FLAG epitope at the TM MPR site, but like HA, it completely abrogated fusion even in the cell fusion assay. These data suggest that the MPR is important for MLV Env folding and maturation, which could be related to the need for a critical degree of hydrophobicity in this region; in HIV-1, this region exists partly in and partly out of the membrane (52). Inhibition of Env processing, trafficking, and incorporation into virions as a result of the insertion of epitope tags in the MPR has also been reported for HIV-1 and vesicular stomatitis virus (VSV) (48, 50).
Why were the anti-HA and anti-His antibodies unable to block Env-mediated fusion, whereas 2F5 was a potent inhibitor A large body of data supports the idea that antibody binding to functional Env molecules is sufficient for neutralization (6). In the case of the anti-His antibody, it is possible that its epitope was not exposed when inserted in TM. We could not detect binding of the anti-His antibody to the surface of transfected cells by flow cytometry or by Western blotting. However, this could be because of low levels of surface Env or masking by SU, since we could not detect 2F5 binding to its epitope in MLV TM in parallel assays. Epitopes in this region of TM may be exposed transiently during the fusion process. In the case of the SU-tagged Envs, the anti-HA antibody bound its epitope as well as 2F5 bound 2F5", but we cannot rule out the possibility that exposure of the HA epitope was limited mainly to SU molecules that did not contribute to fusion. Since physical particles vastly outnumber infectious particles for retroviruses, the bulk of Env molecules detected immunologically or biochemically may be defective for fusion. In the case of HIV-1, experiments have shown that a nonneutralizing antibody fails to bind to a subset of virion SU molecules that binds a neutralizing antibody (24). However, this explanation requires the HA epitope, but not the 2F5 epitope, to be masked on functional Envs. In our view, a more straightforward interpretation is that there is some structural difference between 2F5 and HA epitopes that, upon antibody binding, leads to different functional consequences for fusion or that some special characteristic of the 2F5 antibody not shared by the anti-HA antibody is responsible for its potent neutralization capacity.
What property of 2F5 might make it potently neutralizing Previous reports suggested that neutralization might be a consequence of CDR H3, which extends away from the peptide epitope in a crystal structure. More specifically, it was proposed that neutralization might entail CDR H3 interacting with the adjacent viral membrane (7, 23, 38, 62). The strongest experimental evidence for this hypothesis is that adjacent lipids enhance 2F5 binding to its epitope, that 2F5 by itself binds to some phospholipids such as cardiolipin, and that CDR H3 mutations decrease 2F5 neutralization efficiency more than they reduce binding to the peptide epitope (22, 23, 38, 62). But CDR H3 is unlikely to be near a membrane when its epitope is in the proline-rich region of MLV SU. Furthermore, consistent with our results, Schlehuber and Rose (50) recently reported that the 2F5 antibody neutralized VSV when the 2F5 epitope was inserted into VSV-G protein, again at a site not known to be near a membrane. Taking these results into account, we propose a modification of the CDR H3 hypothesis, namely that the CDR H3 loop, because of its length and hydrophobicity, is particularly disruptive to the structure of proteins brought into proximity with it when 2F5 binds to its epitope. Exchanging the CDR H3 region between 2F5 and other antibodies might allow a test of this hypothesis. Parts of 2F5 other than the CDR H3 loop could also contribute to neutralization potency. Such parts would not likely involve constant regions of the heavy chain, since class-switched versions of 2F5 with IgG1, IgM, and IgA heavy chains are neutralizing like the original IgG3 antibody (30, 56). In addition to a property of 2F5 antibody itself, our data show that epitope position also affects neutralization potency. Two features of the fusion inhibition curves as a function of antibody concentration were clearly affected by epitope location: IC50 and fraction of fusion events not inhibited by high concentrations of antibody. The IC50 was lowest for virus neutralization, intermediate for cell fusion with 2F5 in SU, and highest for cell fusion with 2F5 in TM. Virus infection may require the least antibody because Env molecules are likely limiting for virus fusion but redundant for cell fusion, as discussed above. We speculate that inhibition of cell fusion requires more antibody when 2F5" is in TM due to steric hindrance by membrane or SU partially blocking antibody access to the epitope in TM. The environment of the 2F5 epitope may also affect parameters of 2F5 antibody binding such as on and off rates, with consequences for neutralization potency. Exposure of the epitope in TM may be triggered by interaction of SU with receptor, leaving the antibody only a short time window to bind to block fusion; thus, kinetics may be more important than equilibrium binding when the epitope is in TM. The step in cell fusion that is blocked by antibody may also be different for 2F5" in SU versus TM. For example, 2F5 is more likely to block an early step such as attachment when its epitope is in SU and a late step related to pulling membranes together when in TM (15, 20), paralleling the known roles of SU and TM in fusion. Consistent with this possibility, 2F5 does not inhibit HIV-1 binding to receptor (in which case 2F5" is in TM), whereas we found slight inhibition of MLV binding when 2F5" was in SU.
The inability of high concentrations of antibody to inhibit the last 10 to 20% of virus or cell fusion events when 2F5" was in SU (but not when in TM) could be explained by the heterogeneity of SU molecules, e.g., in glycosylation, resulting in a fraction of SU molecules being resistant to 2F5 binding or to a consequence of that binding (43). The site in SU chosen for epitope insertion might be tolerant of insertions because it is naturally unstructured, which could contribute to the heterogeneity of epitope exposure. Insertion of a FLAG epitope in HIV-1 SU was recently shown to lead to a neutralization-resistant fraction (45). Foreign epitope insertion might for unknown reasons be associated with multiple folding states, some of which hide the epitope, or with greater heterogeneity in the quantity of Env molecules among viruses, with viruses bearing the fewest Env molecules being resistant to neutralization. The nonneutralized fraction could also be due to the reversibility of antibody binding to virions (28).
Taken together, our data show that both epitope position and an as yet undefined property of 2F5 distinct from binding affinity influence its neutralization potency. These results contrast with those of Ren et al. (45), who found that inserting a FLAG epitope in the V4 loop of gp120 leads to the neutralization of HIV-1 by M2 anti-FLAG antibody; on the basis of their results, they suggested that the binding ability of an antibody to Env is more important than the location of its binding site for neutralization.
Finally, we note that the IC50 for neutralization of virus containing 2F5" in SU (<0.2 μg/ml) was more than 10 times lower than the Kd for 2F5 binding to the same Env on virus-infected cells (6 μg/ml). While for most anti-SU neutralizing antibodies, the IC50 is approximately equal to the Kd, for some monoclonal antibodies, a low IC50/Kd ratio has been reported (4, 42). A low IC50/Kd ratio would result if one antibody molecule inactivated a fusion complex composed of several trimers or if one antibody molecule inactivated several Env molecules in succession, such as by inducing shedding of SU, dissociating from the shed SU, and binding to another spike. More complex mathematical models of neutralization also predict extensive neutralization at less than the Kd under certain circumstances (27). The meaning of the low ratio in our case requires more investigation.
ACKNOWLEDGMENTS
We thank C. Kozak for 293mCAT1 cells, J. Ragheb for the pCEETR plasmid, and S. Goff for the pNCA plasmid.
REFERENCES
Albritton, L. M., L. Tseng, D. Scadden, and J. M. Cunningham. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659-666.
Barbato, G., E. Bianchi, P. Ingallinella, W. H. Hurni, M. D. Miller, G. Ciliberto, R. Cortese, R. Bazzo, J. W. Shiver, and A. Pessi. 2003. Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion. J. Mol. Biol. 330:1101-1115.
Buchacher, A., R. Predl, K. Strutzenberger, W. Steinfellner, A. Trkola, M. Purtscher, G. Gruber, C. Tauer, F. Steindl, and A. Jungbauer. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retrovir. 10:359-369.
Burkhart, M. D., S. C. Kayman, Y. He, and A. Pinter. 2003. Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU. J. Virol. 77:3993-4003.
Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B. Thornton, P. W. Parren, L. S. Sawyer, R. M. Hendry, N. Dunlop, and P. L. Nara. 1994. Efficient neutralization of primary isolates of HIV- 1 by a recombinant human monoclonal antibody. Science 266:1024-1027.
Burton, D. R., E. O. Saphire, and P. W. Parren. 2001. A model for neutralization of viruses based on antibody coating of the virion surface. Curr. Top. Microbiol. Immunol. 260:109-143.
Cardoso, R. M., M. B. Zwick, R. L. Stanfield, R. Kunert, J. M. Binley, H. Katinger, D. R. Burton, and I. A. Wilson. 2005. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 22:163-173.
Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263-273.
Chertova, E., J. J. Bess, Jr., B. J. Crise, R. C. Sowder, I. I., T. M. Schaden, J. M. Hilburn, J. A. Hoxie, R. E. Benveniste, J. D. Lifson, L. E. Henderson, and L. O. Arthur. 2002. Envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/SU), is the primary determinant of SU content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus. J. Virol. 76:5315-5325.
Coeffier, E., J. M. Clement, V. Cussac, N. Khodaei-Boorane, M. Jehanno, M. Rojas, A. Dridi, M. Latour, H. R. El, F. Barre-Sinoussi, M. Hofnung, and C. Leclerc. 2000. Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein. Vaccine 19:684-693.
Colicelli, J., and S. P. Goff. 1988. Sequence and spacing requirements of a retrovirus integration site. J. Mol. Biol. 199:47-59.
Collis, A. V., A. P. Brouwer, and A. C. Martin. 2003. Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable loops and the nature of the antigen. J. Mol. Biol. 325:337-354.
Dedera, D., and L. Ratner. 1991. Demonstration of two distinct cytopathic effects with syncytium formation-defective human immunodeficiency virus type 1 mutants. J. Virol. 65:6129-6136.
Denesvre, C., C. Carrington, A. Corbin, Y. Takeuchi, F. L. Cosset, T. Schulz, M. Sitbon, and P. Sonigo. 1996. TM domain swapping of murine leukemia virus and human T-cell leukemia virus envelopes confers different infectious abilities despite similar incorporation into virions. J. Virol. 70:4380-4386.
de Rosny, E., R. Vassell, S. Jiang, R. Kunert, and C. D. Weiss. 2004. Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes. J. Virol. 78:2627-2631.
Dimitrov, A. S., S. S. Rawat, S. Jiang, and R. Blumenthal. 2003. Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion. Biochemistry 42:14150-14158.
Fass, D., S. C. Harrison, and P. S. Kim. 1996. Retrovirus envelope domain at 1.7 angstrom resolution. Nat. Struct. Biol. 3:465-469.
Fass, D., and P. S. Kim. 1995. Dissection of a retrovirus envelope protein reveals structural similarity to influenza hemagglutinin. Curr. Biol. 5:1377-1383.
Gallo, S. A., C. M. Finnegan, M. Viard, Y. Raviv, A. Dimitrov, S. S. Rawat, A. Puri, S. Durell, and R. Blumenthal. 2003. The HIV Env-mediated fusion reaction. Biochim. Biophys. Acta 1614:36-50.
Gorny, M. K., and S. Zolla-Pazner. 2000. Recognition by human monoclonal antibodies of free and complexed peptides representing the prefusogenic and fusogenic forms of human immunodeficiency virus type 1 gp41. J. Virol. 74:6186-6192.
Greenberg, M., N. Cammack, M. Salgo, and L. Smiley. 2004. HIV fusion and its inhibition in antiretroviral therapy. Rev. Med. Virol. 14:321-337.
Grundner, C., T. Mirzabekov, J. Sodroski, and R. Wyatt. 2002. Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins. J. Virol. 76:3511-3521.
Haynes, B. F., J. Fleming, E. W. St Clair, H. Katinger, G. Stiegler, R. Kunert, J. Robinson, R. M. Scearce, K. Plonk, H. F. Staats, T. L. Ortel, H. X. Liao, and S. M. Alam. 2005. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308:1906-1908.
Herrera, C., C. Spenlehauer, M. S. Fung, D. R. Burton, S. Beddows, and J. P. Moore. 2003. Nonneutralizing antibodies to the CD4-binding site on the gp120 subunit of human immunodeficiency virus type 1 do not interfere with the activity of a neutralizing antibody against the same site. J. Virol. 77:1084-1091.
Ho, J., R. A. Uger, M. B. Zwick, M. A. Luscher, B. H. Barber, and K. S. MacDonald. 2005. Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in increased antigenicity but not neutralizing response. Vaccine 23:1559-1573.
Kayman, S. C., H. Park, M. Saxon, and A. Pinter. 1999. The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system. J. Virol. 73:1802-1808.
Klasse, P. J., and J. P. Moore. 1996. Quantitative model of antibody- and soluble CD4-mediated neutralization of primary isolates and T-cell line-adapted strains of human immunodeficiency virus type 1. J. Virol. 70:3668-3677.
Klasse, P. J., and Q. J. Sattentau. 2001. Mechanisms of virus neutralization by antibody. Curr. Top. Microbiol. Immunol. 260:87-108.
Kunert, R., F. Ruker, and H. Katinger. 1998. Molecular characterization of five neutralizing anti-HIV type 1 antibodies: identification of nonconventional D segments in the human monoclonal antibodies 2G12 and 2F5. AIDS Res. Hum. Retrovir. 14:1115-1128.
Kunert, R., W. Steinfellner, M. Purtscher, A. Assadian, and H. Katinger. 2000. Stable recombinant expression of the anti HIV-1 monoclonal antibody 2F5 after IgG3/IgG1 subclass switch in CHO cells. Biotechnol. Bioeng. 67:97-103.
McGaughey, G. B., G. Barbato, E. Bianchi, R. M. Freidinger, V. M. Garsky, W. M. Hurni, J. G. Joyce, X. Liang, M. D. Miller, A. Pessi, J. W. Shiver, and M. J. Bogusky. 2004. Progress towards the development of a HIV-1 gp41-directed vaccine. Curr. HIV Res. 2:193-204.
McGaughey, G. B., M. Citron, R. C. Danzeisen, R. M. Freidinger, V. M. Garsky, W. M. Hurni, J. G. Joyce, X. Liang, M. Miller, J. Shiver, and M. J. Bogusky. 2003. HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb. Biochemistry 42:3214-3223.
Menendez, A., K. C. Chow, O. C. Pan, and J. K. Scott. 2004. Human immunodeficiency virus type 1-neutralizing monoclonal antibody 2F5 is multispecific for sequences flanking the DKW core epitope. J. Mol. Biol. 338:311-327.
Moore, J. P., and R. W. Doms. 2003. The entry of entry inhibitors: a fusion of science and medicine. Proc. Natl. Acad. Sci. USA 100:10598-10602.
Munoz-Barroso, I., K. Salzwedel, E. Hunter, and R. Blumenthal. 1999. Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion. J. Virol. 73:6089-6092.
Muster, T., F. Steindl, M. Purtscher, A. Trkola, A. Klima, G. Himmler, F. Ruker, and H. Katinger. 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67:6642-6647.
Nabel, G. J. 2005. Immunology. Close to the edge: neutralizing the HIV-1 envelope. Science 308:1878-1879.
Ofek, G., M. Tang, A. Sambor, H. Katinger, J. R. Mascola, R. Wyatt, and P. D. Kwong. 2004. Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J. Virol. 78:10724-10737.
Ou, W., and J. Silver. 2005. Inhibition of murine leukemia virus envelope protein (Env) processing by intracellular expression of the Env N-terminal heptad repeat region. J. Virol. 79:4782-4792.
Ou, W., Y. Xiong, and J. Silver. 2004. Quantification of virus-envelope-mediated cell fusion using a tetracycline transcriptional transactivator: fusion does not correlate with syncytium formation. Virology 324:263-272.
Parker, C. E., L. J. Deterding, C. Hager-Braun, J. M. Binley, N. Schulke, H. Katinger, J. P. Moore, and K. B. Tomer. 2001. Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5. J. Virol. 75:10906-10911.
Parren, P. W., I. Mondor, D. Naniche, H. J. Ditzel, P. J. Klasse, D. R. Burton, and Q. J. Sattentau. 1998. Neutralization of human immunodeficiency virus type 1 by antibody to gp120 is determined primarily by occupancy of sites on the virion irrespective of epitope specificity. J. Virol. 72:3512-3519.
Poignard, P., M. Moulard, E. Golez, V. Vivona, M. Franti, S. Venturini, M. Wang, P. W. Parren, and D. R. Burton. 2003. Heterogeneity of envelope molecules expressed on primary human immunodeficiency virus type 1 particles as probed by the binding of neutralizing and nonneutralizing antibodies. J. Virol. 77:353-365.
Ragheb, J. A., and W. F. Anderson. 1994. pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry. J. Virol. 68:3220-3231.
Ren, X., J. Sodroski, and X. Yang. 2005. An unrelated monoclonal antibody neutralizes human immunodeficiency virus type 1 by binding to an artificial epitope engineered in a functionally neutral region of the viral envelope glycoproteins. J. Virol. 79:5616-5624.
Roben, P., J. P. Moore, M. Thali, J. Sodroski, C. F. Barbas III, and D. R. Burton. 1994. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J. Virol. 68:4821-4828.
Rothenberg, S. M., M. N. Olsen, L. C. Laurent, R. A. Crowley, and P. O. Brown. 2001. Comprehensive mutational analysis of the Moloney murine leukemia virus envelope protein. J. Virol. 75:11851-11862.
Salzwedel, K., J. T. West, and E. Hunter. 1999. A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for Env-mediated fusion and virus infectivity. J. Virol. 73:2469-2480.
Scanlan, C. N., R. Pantophlet, M. R. Wormald, S. E. Ollmann, R. Stanfield, I. A. Wilson, H. Katinger, R. A. Dwek, P. M. Rudd, and D. R. Burton. 2002. The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha12 mannose residues on the outer face of gp120. J. Virol. 76:7306-7321.
Schlehuber, L. D., and J. K. Rose. 2004. Prediction and identification of a permissive epitope insertion site in the vesicular stomatitis virus glycoprotein. J. Virol. 78:5079-5087.
Schmid, E., A. Zurbriggen, U. Gassen, B. Rima, V. ter Meulen, and J. Schneider-Schaulies. 2000. Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion. J. Virol. 74:7554-7561.
Suarez, T., W. R. Gallaher, A. Agirre, F. M. Goni, and J. L. Nieva. 2000. Membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion. J. Virol. 74:8038-8047.
Tian, Y., C. V. Ramesh, X. Ma, S. Naqvi, T. Patel, T. Cenizal, M. Tiscione, K. Diaz, T. Crea, E. Arnold, G. F. Arnold, and J. W. Taylor. 2002. Structure-affinity relationships in the gp41 ELDKWA epitope for the HIV-1 neutralizing monoclonal antibody 2F5: effects of side-chain and backbone modifications and conformational constraints. J. Pept. Res. 59:264-276.
von Messling, V., G. Zimmer, G. Herrler, L. Haas, and R. Cattaneo. 2001. The hemagglutinin of canine distemper virus determines tropism and cytopathogenicity. J. Virol. 75:6418-6427.
Weimin, W. B., P. M. Cannon, E. M. Gordon, F. L. Hall, and W. F. Anderson. 1998. Characterization of the proline-rich region of murine leukemia virus envelope protein. J. Virol. 72:5383-5391.
Wolbank, S., R. Kunert, G. Stiegler, and H. Katinger. 2003. Characterization of human class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12. J. Virol. 77:4095-4103.
Wu, T. T., G. Johnson, and E. A. Kabat. 1993. Length distribution of CDRH3 in antibodies. Proteins 16:1-7.
Zhang, M. Y., Y. Shu, I. Sidorov, and D. S. Dimitrov. 2004. Identification of a novel CD4i human monoclonal antibody Fab that neutralizes HIV-1 primary isolates from different clades. Antivir. Res. 61:161-164.
Zhang, M. Y., X. Xiao, I. A. Sidorov, V. Choudhry, F. Cham, P. F. Zhang, P. Bouma, M. Zwick, A. Choudhary, D. C. Montefiori, C. C. Broder, D. R. Burton, G. V. Quinnan, Jr., and D. S. Dimitrov. 2004. Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody. J. Virol. 78:9233-9242.
Zhu, P., E. Chertova, J. Bess, Jr., J. D. Lifson, L. O. Arthur, J. Liu, K. A. Taylor, and K. H. Roux. 2003. Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions. Proc. Natl. Acad. Sci. USA 100:15812-15817.
Zimmer, G., K. K. Conzelmann, and G. Herrler. 2002. Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture. J. Virol. 76:9218-9224.
Zwick, M. B., H. K. Komori, R. L. Stanfield, S. Church, M. Wang, P. W. Parren, R. Kunert, H. Katinger, I. A. Wilson, and D. R. Burton. 2004. The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5. J. Virol. 78:3155-3161.
Zwick, M. B., A. F. Labrijn, M. Wang, C. Spenlehauer, E. O. Saphire, J. M. Binley, J. P. Moore, G. Stiegler, H. Katinger, D. R. Burton, and P. W. Parren. 2001. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J. Virol. 75:10892-10905.(Wu Ou, Ning Lu, Sloane S.)
ABSTRACT
The membrane-proximal region of the human immunodeficiency virus type 1 (HIV-1) transmembrane protein (TM) is critical for envelope (Env)-mediated membrane fusion and contains the target for broadly reactive neutralizing antibody 2F5. It has been proposed that 2F5 neutralization might involve interaction of its long, hydrophobic, complementarity-determining region (CDR) H3, with adjacent viral membrane. Using Moloney murine leukemia virus (MLV) as a tool, we examined the effect of epitope position on 2F5 neutralization. When the 2F5 epitope was inserted in the proline-rich region of MLV Env surface protein (SU), 2F5 blocked cell fusion and virus infection, whereas MLV with a hemagglutinin (HA) epitope at the same position was not neutralized by anti-HA, even though the antibodies bound their respective Envs on the surface of infected cells and viruses equally well. When the 2F5 epitope was inserted in the MLV Env TM at a position comparable to its natural position in HIV-1 TM, 2F5 antibody blocked Env-mediated cell fusion. Epitope position had subtle effects on neutralization by 2F5: the antibody concentration for 50% inhibition of cell fusion was more than 10-fold lower when the 2F5 epitope was in SU than in TM, and inhibition was less complete at high concentrations of antibody; we discuss possible explanations for these effects of epitope position. Since membrane proximity was not required for neutralization by 2F5 antibody, we speculate that the CDR H3 of 2F5 contributes to neutralization by destabilizing an adjacent protein rather than by inserting into an adjacent membrane.
INTRODUCTION
Human immunodeficiency virus type 1 (HIV-1), the cause of AIDS, is highly immunogenic but notoriously poor at generating broadly reactive, neutralizing antibodies. This is a crucial problem for vaccine development. The major target for neutralizing antibodies is the envelope glycoprotein (Env). Although the Env sequence is highly variable between viruses and over time in infected individuals, some regions of Env are highly conserved. Nevertheless, only a few broadly reactive, anti-Env neutralizing antibodies have been identified (5, 29, 36, 37, 41, 46, 49, 58, 59, 63).
HIV-1 Env is translated as a precursor (gp160) that undergoes posttranslational modification including trimerization, glycosylation, and proteolytic processing to form surface protein (SU; gp120) and transmembrane protein (TM; gp41) as it travels from the endoplasmic reticulum (ER) to the cell surface. SU and TM remain associated through noncovalent interactions, forming a trimer of heterodimers. The process of Env-mediated membrane fusion has been extensively analyzed. SU binding to the HIV-1 receptor CD4 and a coreceptor, usually CCR5 or CXCR4, induces conformational changes in SU, probably leading to its dissociation from TM. This causes TM to refold, exposing a hydrophobic N-terminal peptide that is believed to insert into the target cell membrane and then retract to pull viral and target cell membranes together. The retraction mechanism involves formation of a thermodynamically stable trimer of antiparallel alpha-helices (hairpins) derived from heptad repeats located just downstream of the fusion peptide (N-heptad repeats) and just upstream of where TM traverses the viral membrane (C-heptad repeats) (19, 21, 34).
The segment of TM between the C-heptad repeats and the transmembrane anchor, designated the membrane-proximal region (MPR), consists of 20 amino acids that are highly conserved among different clades of HIV-1. Mutation of amino acids in this region can impair fusion without altering surface expression of Env, suggesting that the region has a role in fusion (16, 35, 48). Amazingly, MPR is the target for three broadly reactive, neutralizing antibodies to HIV-1: 2F5, 4E10, and Z13 (3, 36, 41, 63). This region might be a "hot spot" for such antibodies because of constraints on sequence variability due to a role in membrane fusion that is sensitive to antibody binding. However, immunization with peptides from this region resulted in antibodies that bound well but did not block fusion (25, 31), suggesting that neutralization potency is influenced by special properties of some antibodies, possibly related to the membrane-proximal microenvironment. Both 2F5 and 4E10 have an unusually long, hydrophobic, third heavy-chain complementarity-determining region (CDR H3), which prompted the hypothesis that neutralization involves the interaction of this region with neighboring lipid membranes (7, 12, 23, 38, 57, 62). Given the dearth of broadly neutralizing antibodies to HIV-1 and their potential importance for therapy and vaccine development, it is important to understand whether membrane proximity of the epitope or some unrelated, intrinsic property of certain antibodies makes them neutralizing.
We used Moloney murine leukemia virus (Mo-MLV) Env-mediated fusion as a tool to investigate this question. MLV uses the mouse cationic amino acid transporter 1 (mCAT1) as receptor (1). Like HIV-1, MLV Env forms a homotrimer of SU-TM heterodimers. Only the amino-terminal 240 amino acids of MLV SU have been crystallized. Downstream of the crystallized portion is a proline-rich region that is thought to form a flexible "hinge" in SU. This hinge tolerates insertions without impairing Env function (26, 47, 55). Like HIV-1, MLV TM has an N-terminal hydrophobic fusion peptide followed by an N-heptad repeat that trimerizes. It is not known if MLV has an analogous C-heptad repeat region that folds back to form "hairpins" during fusion. The MPR of MLV has not been extensively studied for the effect of mutations, but it is likely that the mechanism of fusion for MLV is closely related to that of HIV-1 given the overall structural similarities of their Envs (8, 17, 18). We inserted the 2F5 epitope or control epitopes into MLV Env, in the TM at a position comparable to that of the 2F5 epitope in gp41, or in SU in the proline-rich region and characterized the effect of the 2F5 antibody on virus infection and MLV Env-mediated cell fusion.
MATERIALS AND METHODS
Constructs. To insert 2F5" (QELLELDKWASLWNW) or hemagglutinin (HA; YPYDVPDYA) into the SU of MLV Env, sense and antisense oligonucleotides encoding the epitope tag with SgrAI overhangs were synthesized, annealed, and ligated into pCEETR (44) cut with SgrAI. To facilitate inserting 2F5" and His6 epitope tags into the TM of MLV Env, a unique BstBI restriction site was introduced into pCEETR (with HA in SU) by site-directed mutagenesis 14 amino acids upstream of the transmembrane domain. This mutation does not change the amino acid sequence of MLV Env protein. Sense and antisense oligonucleotides encoding 2F5" or His6 (HHHHHH) with BstBI overhangs were synthesized, annealed, and ligated into pCEETR-BstBI (SU-HA) cut with BstBI. These Env mutations were transferred into pNCA (11) as a BspEI-ClaI restriction fragment.
Cell fusion assay. HEK293 or HeLa cells were seeded in a six-well plate at a density that resulted in their being 90 to 95% confluent the next day. Cells were cotransfected with 2 μg each of wild-type or mutant pCEETR plus pTet-Off (Clontech) per well using Lipofectamine 2000. Twenty-four hours later, the cotransfected cells were trypsinized and preincubated with or without antibody and then mixed in triplicate with human osteosarcoma cells (U2OS) stably expressing MLV receptor mCAT1 and a luciferase reporter gene under the control of the tetracycline response element (U2OS-mCAT1-TRELuc). Luciferase activity was assayed about 16 h after coculture using the Luciferase Assay System from Promega (Madison, Wis.) and a Victor 3 luminometer (PerkinElmer, Boston, Mass.). For cell fusion inhibition assays, transfected cells were premixed with twice the indicated concentration of antibody for 30 min before adding an equal volume of indicator cells; antibody was left in the coculture medium. Anti-HA antibody was monoclonal antibody HA-7 from Sigma (catalog number H9658), anti-His6 was monoclonal antibody H-3 from Santa Cruz Biotechnology (catalog number sc-8036x), and 2F5 (catalog number AB001) was from Polymun Scientific (Vienna, Austria).
Surface protein labeling and Western blot analysis. An aliquot of cells transfected for cell fusion was rinsed with phosphate-buffered saline (PBS) and labeled in PBS supplemented with 1 mg/ml membrane-impermeable biotinylation reagent (catalog number 20338; Pierce, Rockford, Ill.) for 1 h on ice. The free biotin was quenched with 100 mM glycine in PBS buffer. Following two washes with PBS, the cells were lysed using ice-cold radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate) supplemented with a protease inhibitor cocktail (catalog number 1836153; Roche, Indianapolis, IN). Biotinylated proteins were purified using streptavidin beads and eluted by heating in 1x SDS-polyacrylamide gel electrophoresis (PAGE) sample loading buffer (catalog number NP0007; Invitrogen, Carlsbad, CA) at 98°C for 5 min. Samples were resolved on SDS-4 to 12% PAGE gels, and proteins were transferred to a polyvinylidene difluoride membrane. The membrane was blocked and blotted as described previously (39).
Virus infection and neutralization assay. Wild-type and mutant MLVs were made by cotransfecting HEK293T cells with pNCA plus pFB-Luc by using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, supernatant virus was filtered using a 0.45-μm filter and incubated with or without antibody for 30 min at 4°C and then added to plain NIH 3T3 cells in a 24-well plate in the presence of 4 μg/ml Polybrene (Sigma). Luciferase activity was assayed 60 h after infection as described for cell fusion.
Flow cytometry analysis of antibody affinity and virus binding. NIH 3T3 cells chronically infected by wild-type Mo-MLV or Mo-MLV containing 2F5" or HA in SU were detached at 37°C using PBS supplemented with 2 mM EDTA and 1% fetal calf serum (FCS) and resuspended in staining buffer (0.2% bovine serum albumin and 0.09% sodium azide in PBS at pH 7.4). HEK293 and 293mCAT1 cells were detached in the same way and mixed with free or antibody-bound virus in a total volume of 200 μl. Primary antibody was incubated with cells for 30 min on ice. The cells were washed once using 2 ml cold wash buffer (PBS, 2 mM EDTA, 1% fetal bovine serum [FBS]) and then incubated for 30 min on ice with fluorescein isothiocyanate (FITC) anti-mouse immunoglobulin G (IgG; catalog number F9137, lot number 015k4793; Sigma) or FITC-anti-human IgG (catalog number F4512, lot number 024k4811; Sigma) diluted 1:100 in wash buffer. After two washes, the cells were resuspended in 0.5 ml staining buffer supplemented with 20 μl 7-amino actinomycin D viability probe (catalog number 555816; BD Pharmingen) to identify dead cells. Data were acquired on a BD FACSCalibur flow cytometer and analyzed using FlowJo software (Tree Star, Ashland, OR).
RESULTS
Constructing chimeric MLV Env with the 2F5 epitope in TM or SU. The core epitope sequence for 2F5 antibody is ELDKWA. However, studies have shown that peptides with extra amino acids on each end of the epitope bind 2F5 better (2, 10, 31-33, 41, 53, 63). We extended the epitope by including an extra 4 or 5 amino acids on either end (QELLELDKWASLWNW); we designated this epitope 2F5". To investigate if the position of the 2F5 epitope affected neutralization potency, we inserted 2F5" in MLV TM or SU in a vector pCEETR that expressed a fusogenic form of Mo-MLV Env whose function was quantifiable in a cell fusion assay (39, 40, 44). In TM, 2F5" was inserted 14 amino acids upstream from the membrane anchor, the same position as in HIV-1 TM. In SU, 2F5" was inserted at a unique SgrAI site in the proline-rich region. For controls, we inserted an influenza virus HA epitope (YPYDVPDYA) at the same positions in SU and TM, but HA abrogated fusion when inserted in TM, so we replaced it with a six-His tag (HHHHHH).
We assessed the function of these chimeric Envs using a cell fusion assay (40) in which HEK 293 or HeLa cells were transfected with wild-type or epitope-tagged MLV Env in pCEETR plus a transcriptional transactivator (tTA, encoded by pTet-Off from Clontech). Twenty-four hours later, the transfected cells were cocultivated with human osteosarcoma cells (U2OS) stably expressing the MLV receptor mCAT1 and a luciferase reporter gene under the control of a tetracycline response element (TRE), a promoter that is activated by tTA. Luciferase activity was assayed 16 h after coculture. Insertion of 2F5" or HA in SU did not affect cell fusion in this assay, whereas insertion of 2F5" or a six-His tag in TM decreased cell fusion 50 to 75% (Fig. 1A).
To understand why insertions in TM reduced cell fusion, we checked the expression level of Env with 2F5" in TM by Western blot analysis of total cell lysates or purified cell surface proteins. The latter were prepared by biotinylating cells with a membrane-impermeable biotinylation reagent, binding the lysate to streptavidin beads, and eluting bound proteins by boiling in gel loading buffer. The Western blots were analyzed with anti-HA antibody since the Env with 2F5" in TM was also labeled with HA in SU. Lysates of cells transfected with singly tagged Env (HA in SU) contained the gp85 Env precursor and gp70 SU (Fig. 1B, lane 1); this pattern was the same as that of Env in cells transfected with wild-type Env, detected with anti-SU antibody (data not shown). Cells transfected with doubly tagged Env (HA in SU, 2F5" in TM) contained roughly the same amount of Env precursor, but no gp70 SU (lane 2). This suggests that insertion of 2F5" in the TM inhibited transport to the late Golgi apparatus, where cleavage into SU and TM normally occurs. Cell surface Env was also dramatically reduced in cells transfected with doubly tagged Env (HA in SU, 2F5" in TM) compared to singly tagged Env (HA in SU) (Fig. 1C, lane 2 versus lane 1), consistent with transport being blocked before Env reached the plasma membrane. Loading controls showed that approximately the same amounts of total protein (-actin) and surface protein (integrin 5) were loaded in all lanes (Fig. 1B and C, lower panels).
Despite the very low levels of processed and surface SU detectable by Western blotting when 2F5" was inserted in TM, cell fusion detected by luciferase induction was reduced only two- to fourfold to a level well above the background in indicator cells cocultivated with cells transfected with tTA but no Env (Fig. 1, bar 6). This implies that only a small amount of cell surface Env is required for Env-mediated cell fusion. We obtained similar results with other mutant Envs, both in MLV and in HIV (data not shown), which showed marked decreases in cell surface Env detected by Western blotting but only moderate decreases in cell fusion measured by the luciferase induction assay. This suggests that wild-type Env protein is present in large excess for cell fusion, possibly because large areas of cell surface can contact one another, with fusion at any single site being sufficient to transfer transcriptional activator.
2F5 antibody specifically inhibits cell fusion mediated by Env with 2F5" in either SU or TM. We compared the ability of 2F5, anti-HA, and anti-His6 antibodies to inhibit Env-mediated cell fusion when the epitopes were inserted in SU or TM. Twenty-four hours after transfection, Env-expressing cells were trypsinized and preincubated with antibody at double the indicated concentrations for 30 min at 4°C and then cocultivated with an equal volume of U2OS-mCAT1-TRELuc indicator cells; antibody was left in the medium during the coculture. Luciferase activity was assayed 16 h later. Luciferase activity in the presence of antibody is shown as the percentage of luciferase activity in the absence of antibody (Fig. 2). The 2F5 antibody inhibited fusion mediated by Env with 2F5" in SU up to 80% (Fig. 2, group 4); inhibited fusion mediated by Env with 2F5" in TM up to 97% (group 5), which was approximately equal to background; and did not inhibit fusion mediated by Env with HA in SU, showing that the inhibition was specific (group 2). Anti-HA and anti-six-His antibodies did not inhibit fusion mediated by their corresponding Envs (groups 1 and 6).
Dose response of 2F5 inhibition of cell fusion is different for Env with 2F5" in SU versus TM. 2F5 inhibition of cell fusion was dose dependent and essentially complete at high concentrations of antibody (113 μg/ml) when 2F5" was in TM (Fig. 2, group 5), but little dose dependency was seen when 2F5" was in SU (group 4). To see if this was due to the limited range of antibody concentrations tested, we repeated the experiment with a more extensive, twofold serial dilution of antibody. The results (Fig. 3) show that cell fusion is inhibited at lower concentrations of 2F5 antibody when 2F5" is in SU than when it is in TM and that inhibition plateaus at about 80% inhibition when 2F5" is in SU, whereas it goes essentially to completion when 2F5" is in TM. These results are consistent with and extend the findings shown in Fig. 2. The antibody concentration for 50% inhibition (IC50) of cell fusion for Env with 2F5" in SU was 0.4 μg/ml, more than 10 times lower than that for Env with 2F5" in TM (6.4 μg/ml).
2F5 antibody specifically neutralizes MLV with 2F5" in SU. Cell fusion and virus infection do not always correlate (13, 14, 51, 54, 61). To see if 2F5 antibody also neutralized MLV with 2F5" in Env, we substituted epitope-tagged Envs for the wild-type Env in pNCA, a vector that encodes full-length, infectious Mo-MLV (11). HEK293T cells were cotransfected with wild-type or epitope-tagged pNCA vectors plus pFB-Luc (Stratagene, La Jolla, CA), a Mo-MLV retroviral vector encoding firefly luciferase as a reporter. Forty-eight hours after transfection, cell supernatants containing wild-type or epitope-tagged viruses were filtered and mixed with 80 μg/ml antibody at 4°C and then added to NIH 3T3 cells in the presence of 4 μg/ml Polybrene. Luciferase activity was assayed 60 h after infection. 2F5 antibody neutralized infection by the pseudotyped vector when Env contained 2F5" in SU (Fig. 4, bar 7) but not when it was pseudotyped with wild-type Env (bar 6). Anti-HA antibody did not neutralize infection when Env contained HA in SU (bar 5). The small decrease of luciferase activity in the presence of anti-HA antibody was nonspecific, since the same decrease was observed for wild-type virus (bar 4 versus bar 1). Infectious virus was not obtained from constructs with epitope tags in TM. This is probably because cell surface levels of Env with these epitopes were too low to make infectious virus particles. Virus infectivity may be more sensitive to reductions in surface Env than cell fusion because virus particles contain limited numbers of Env proteins (9, 60), whereas cell surfaces in contact with one another are much larger than individual viruses and therefore should contain a larger number of Env molecules. We cannot rule out the possibility that epitope tags in TM also impaired Env incorporation into viral particles.
Serial dilution of 2F5 antibody showed that virus containing SU-2F5" Env was neutralized with an IC50 of less than 0.2 μg/ml, and neutralization reached a plateau at about 90% neutralization (Fig. 5). The luciferase signal at this plateau level was well above background (mock bar). Thus, neutralization of virus with 2F5" in SU resembled inhibition of cell fusion mediated by the same Env in having a low IC50 and being only partially neutralized by 2F5 (Fig. 3).
2F5 and anti-HA antibody have comparable affinities for their epitopes in SU expressed on the cell surface and on virions. To see if the ability of 2F5 antibody, but not anti-HA, to block fusion was due to higher affinity for its epitope, we measured binding affinity by flow cytometry. NIH 3T3 cells that were chronically infected with wild-type virus or viruses with 2F5" or HA tags in SU were stained with serial dilutions of each antibody, followed by fluorescently labeled secondary antibodies. The fluorescence histograms (Fig. 6A) and mean fluorescence intensities at each antibody concentration (Fig. 6B) were very close for the two antibodies. The signal-to-noise ratio (fluorescence intensity of tagged-virus-infected cells versus wild-type-virus-infected cells) was highest for both antibodies at 10 μg/ml, and the concentration of antibody that gave half this fluorescence intensity was about 6 μg/ml for both antibodies. The 2F5 antibody is of human origin, and anti-HA is of mouse origin. The secondary antibodies, FITC-labeled anti-human IgG and FITC-labeled anti-mouse IgG, had the same fluorescein-to-protein ratios and were used at the same concentrations. The results imply that the ability of 2F5, but not anti-HA, to block fusion is not due to a difference in affinity for their epitopes.
We also checked the binding of 2F5 and anti-HA to virions. Epitope-tagged viruses from chronically infected cells were concentrated by centrifugation and incubated with HEK293 cells that stably express the mCAT1 receptor (293mCAT1; kindly provided by Christian Kozak, National Institute of Allergy and Infectious Diseases), versus control HEK293 cells that do not have this receptor. After 90 min on ice, 2F5 or anti-HA antibodies were added at 10 μg/ml. After 30 min on ice, the cells were washed and stained with equal amounts of corresponding secondary antibodies. 293mCAT1 cells fell into two populations (Fig. 7, panels A and C, green curves): slightly more than half of the cells bound a large amount of virus (mean fluorescence intensity, 2,000 RLU), while a second population bound virus very weakly, if at all (mean fluorescence intensity, 60 to 100 RLU). Control HEK293 cells lacking the receptor (panels B and D) defined the level of nonspecific virus binding (mean fluorescence intensity, 20 to 30 RLU). The "dim" population in 293mCAT1 cells presumably does not express receptor or expresses only small amounts of it. This is not unexpected, as the 293mCAT1 cells were not cloned but derived from a bulk culture of cells selected with Geneticin after transfection with a vector encoding mCAT1 and Neor. The mean fluorescence intensity of the "bright" peak of 293mCAT1 cells binding virus with 2F5" in SU was the same as that of cells binding virus with HA in SU (Fig. 7, panels A and C). This shows that the ability of 2F5—but not anti-HA—to neutralize virus is not due to a difference in the amounts of their respective Envs on virions.
The virus-binding assay allowed us to ask if 2F5 antibody blocked binding of virions with 2F5" in SU to the mCAT1 receptor. An aliquot of each concentrated, epitope-tagged virus was premixed with 10 μg/ml of corresponding antibody on ice for 30 min and then added to 293mCAT1 cells. After 90 min on ice, the cells were washed and stained with secondary antibody. Preincubation with 2F5 inhibited binding of the 2F5"-tagged virus by 53 to 61%, while anti-HA inhibited binding of the HA-tagged virus by 36 to 39%, based on mean fluorescence measurements in two independent experiments. These results suggest that inhibition of binding might marginally contribute to neutralization by 2F5 when its epitope is in SU, but it does not account for the full extent of neutralization.
DISCUSSION
Of the few potent neutralizing antibodies against HIV-1 virus, 2F5 is one of the best studied. However, its neutralization mechanism is still far from clear. To find out whether the location of its epitope is critical, as suggested by models in which the CDR H3 of 2F5 interacts with the viral membrane, we artificially expressed an extended 2F5 epitope in Mo-MLV SU or TM and found that 2F5 inhibited fusion regardless of the position of its epitope, whereas anti-HA or anti-His6 antibody did not inhibit fusion mediated by Env with the corresponding epitope in either SU or TM.
Most regions of MLV Env cannot tolerate insertions (47). Here, we inserted 2F5" or an HA epitope into the proline-rich region of SU without affecting fusion activity. However, insertion of 2F5" or the six-His tag in the MPR caused large reductions in SU/TM cleavage and transport to the cell surface, precluding production of infectious virus. Surprisingly, Env-mediated cell fusion was only modestly reduced so that we could still use these insertions to study the ability of the respective antibodies to inhibit cell fusion. Despite functioning in cell fusion, the TM-tagged Env molecules could be abnormal in other ways. We also inserted a FLAG epitope at the TM MPR site, but like HA, it completely abrogated fusion even in the cell fusion assay. These data suggest that the MPR is important for MLV Env folding and maturation, which could be related to the need for a critical degree of hydrophobicity in this region; in HIV-1, this region exists partly in and partly out of the membrane (52). Inhibition of Env processing, trafficking, and incorporation into virions as a result of the insertion of epitope tags in the MPR has also been reported for HIV-1 and vesicular stomatitis virus (VSV) (48, 50).
Why were the anti-HA and anti-His antibodies unable to block Env-mediated fusion, whereas 2F5 was a potent inhibitor A large body of data supports the idea that antibody binding to functional Env molecules is sufficient for neutralization (6). In the case of the anti-His antibody, it is possible that its epitope was not exposed when inserted in TM. We could not detect binding of the anti-His antibody to the surface of transfected cells by flow cytometry or by Western blotting. However, this could be because of low levels of surface Env or masking by SU, since we could not detect 2F5 binding to its epitope in MLV TM in parallel assays. Epitopes in this region of TM may be exposed transiently during the fusion process. In the case of the SU-tagged Envs, the anti-HA antibody bound its epitope as well as 2F5 bound 2F5", but we cannot rule out the possibility that exposure of the HA epitope was limited mainly to SU molecules that did not contribute to fusion. Since physical particles vastly outnumber infectious particles for retroviruses, the bulk of Env molecules detected immunologically or biochemically may be defective for fusion. In the case of HIV-1, experiments have shown that a nonneutralizing antibody fails to bind to a subset of virion SU molecules that binds a neutralizing antibody (24). However, this explanation requires the HA epitope, but not the 2F5 epitope, to be masked on functional Envs. In our view, a more straightforward interpretation is that there is some structural difference between 2F5 and HA epitopes that, upon antibody binding, leads to different functional consequences for fusion or that some special characteristic of the 2F5 antibody not shared by the anti-HA antibody is responsible for its potent neutralization capacity.
What property of 2F5 might make it potently neutralizing Previous reports suggested that neutralization might be a consequence of CDR H3, which extends away from the peptide epitope in a crystal structure. More specifically, it was proposed that neutralization might entail CDR H3 interacting with the adjacent viral membrane (7, 23, 38, 62). The strongest experimental evidence for this hypothesis is that adjacent lipids enhance 2F5 binding to its epitope, that 2F5 by itself binds to some phospholipids such as cardiolipin, and that CDR H3 mutations decrease 2F5 neutralization efficiency more than they reduce binding to the peptide epitope (22, 23, 38, 62). But CDR H3 is unlikely to be near a membrane when its epitope is in the proline-rich region of MLV SU. Furthermore, consistent with our results, Schlehuber and Rose (50) recently reported that the 2F5 antibody neutralized VSV when the 2F5 epitope was inserted into VSV-G protein, again at a site not known to be near a membrane. Taking these results into account, we propose a modification of the CDR H3 hypothesis, namely that the CDR H3 loop, because of its length and hydrophobicity, is particularly disruptive to the structure of proteins brought into proximity with it when 2F5 binds to its epitope. Exchanging the CDR H3 region between 2F5 and other antibodies might allow a test of this hypothesis. Parts of 2F5 other than the CDR H3 loop could also contribute to neutralization potency. Such parts would not likely involve constant regions of the heavy chain, since class-switched versions of 2F5 with IgG1, IgM, and IgA heavy chains are neutralizing like the original IgG3 antibody (30, 56). In addition to a property of 2F5 antibody itself, our data show that epitope position also affects neutralization potency. Two features of the fusion inhibition curves as a function of antibody concentration were clearly affected by epitope location: IC50 and fraction of fusion events not inhibited by high concentrations of antibody. The IC50 was lowest for virus neutralization, intermediate for cell fusion with 2F5 in SU, and highest for cell fusion with 2F5 in TM. Virus infection may require the least antibody because Env molecules are likely limiting for virus fusion but redundant for cell fusion, as discussed above. We speculate that inhibition of cell fusion requires more antibody when 2F5" is in TM due to steric hindrance by membrane or SU partially blocking antibody access to the epitope in TM. The environment of the 2F5 epitope may also affect parameters of 2F5 antibody binding such as on and off rates, with consequences for neutralization potency. Exposure of the epitope in TM may be triggered by interaction of SU with receptor, leaving the antibody only a short time window to bind to block fusion; thus, kinetics may be more important than equilibrium binding when the epitope is in TM. The step in cell fusion that is blocked by antibody may also be different for 2F5" in SU versus TM. For example, 2F5 is more likely to block an early step such as attachment when its epitope is in SU and a late step related to pulling membranes together when in TM (15, 20), paralleling the known roles of SU and TM in fusion. Consistent with this possibility, 2F5 does not inhibit HIV-1 binding to receptor (in which case 2F5" is in TM), whereas we found slight inhibition of MLV binding when 2F5" was in SU.
The inability of high concentrations of antibody to inhibit the last 10 to 20% of virus or cell fusion events when 2F5" was in SU (but not when in TM) could be explained by the heterogeneity of SU molecules, e.g., in glycosylation, resulting in a fraction of SU molecules being resistant to 2F5 binding or to a consequence of that binding (43). The site in SU chosen for epitope insertion might be tolerant of insertions because it is naturally unstructured, which could contribute to the heterogeneity of epitope exposure. Insertion of a FLAG epitope in HIV-1 SU was recently shown to lead to a neutralization-resistant fraction (45). Foreign epitope insertion might for unknown reasons be associated with multiple folding states, some of which hide the epitope, or with greater heterogeneity in the quantity of Env molecules among viruses, with viruses bearing the fewest Env molecules being resistant to neutralization. The nonneutralized fraction could also be due to the reversibility of antibody binding to virions (28).
Taken together, our data show that both epitope position and an as yet undefined property of 2F5 distinct from binding affinity influence its neutralization potency. These results contrast with those of Ren et al. (45), who found that inserting a FLAG epitope in the V4 loop of gp120 leads to the neutralization of HIV-1 by M2 anti-FLAG antibody; on the basis of their results, they suggested that the binding ability of an antibody to Env is more important than the location of its binding site for neutralization.
Finally, we note that the IC50 for neutralization of virus containing 2F5" in SU (<0.2 μg/ml) was more than 10 times lower than the Kd for 2F5 binding to the same Env on virus-infected cells (6 μg/ml). While for most anti-SU neutralizing antibodies, the IC50 is approximately equal to the Kd, for some monoclonal antibodies, a low IC50/Kd ratio has been reported (4, 42). A low IC50/Kd ratio would result if one antibody molecule inactivated a fusion complex composed of several trimers or if one antibody molecule inactivated several Env molecules in succession, such as by inducing shedding of SU, dissociating from the shed SU, and binding to another spike. More complex mathematical models of neutralization also predict extensive neutralization at less than the Kd under certain circumstances (27). The meaning of the low ratio in our case requires more investigation.
ACKNOWLEDGMENTS
We thank C. Kozak for 293mCAT1 cells, J. Ragheb for the pCEETR plasmid, and S. Goff for the pNCA plasmid.
REFERENCES
Albritton, L. M., L. Tseng, D. Scadden, and J. M. Cunningham. 1989. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell 57:659-666.
Barbato, G., E. Bianchi, P. Ingallinella, W. H. Hurni, M. D. Miller, G. Ciliberto, R. Cortese, R. Bazzo, J. W. Shiver, and A. Pessi. 2003. Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion. J. Mol. Biol. 330:1101-1115.
Buchacher, A., R. Predl, K. Strutzenberger, W. Steinfellner, A. Trkola, M. Purtscher, G. Gruber, C. Tauer, F. Steindl, and A. Jungbauer. 1994. Generation of human monoclonal antibodies against HIV-1 proteins; electrofusion and Epstein-Barr virus transformation for peripheral blood lymphocyte immortalization. AIDS Res. Hum. Retrovir. 10:359-369.
Burkhart, M. D., S. C. Kayman, Y. He, and A. Pinter. 2003. Distinct mechanisms of neutralization by monoclonal antibodies specific for sites in the N-terminal or C-terminal domain of murine leukemia virus SU. J. Virol. 77:3993-4003.
Burton, D. R., J. Pyati, R. Koduri, S. J. Sharp, G. B. Thornton, P. W. Parren, L. S. Sawyer, R. M. Hendry, N. Dunlop, and P. L. Nara. 1994. Efficient neutralization of primary isolates of HIV- 1 by a recombinant human monoclonal antibody. Science 266:1024-1027.
Burton, D. R., E. O. Saphire, and P. W. Parren. 2001. A model for neutralization of viruses based on antibody coating of the virion surface. Curr. Top. Microbiol. Immunol. 260:109-143.
Cardoso, R. M., M. B. Zwick, R. L. Stanfield, R. Kunert, J. M. Binley, H. Katinger, D. R. Burton, and I. A. Wilson. 2005. Broadly neutralizing anti-HIV antibody 4E10 recognizes a helical conformation of a highly conserved fusion-associated motif in gp41. Immunity 22:163-173.
Chan, D. C., D. Fass, J. M. Berger, and P. S. Kim. 1997. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89:263-273.
Chertova, E., J. J. Bess, Jr., B. J. Crise, R. C. Sowder, I. I., T. M. Schaden, J. M. Hilburn, J. A. Hoxie, R. E. Benveniste, J. D. Lifson, L. E. Henderson, and L. O. Arthur. 2002. Envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp120/SU), is the primary determinant of SU content of purified human immunodeficiency virus type 1 and simian immunodeficiency virus. J. Virol. 76:5315-5325.
Coeffier, E., J. M. Clement, V. Cussac, N. Khodaei-Boorane, M. Jehanno, M. Rojas, A. Dridi, M. Latour, H. R. El, F. Barre-Sinoussi, M. Hofnung, and C. Leclerc. 2000. Antigenicity and immunogenicity of the HIV-1 gp41 epitope ELDKWA inserted into permissive sites of the MalE protein. Vaccine 19:684-693.
Colicelli, J., and S. P. Goff. 1988. Sequence and spacing requirements of a retrovirus integration site. J. Mol. Biol. 199:47-59.
Collis, A. V., A. P. Brouwer, and A. C. Martin. 2003. Analysis of the antigen combining site: correlations between length and sequence composition of the hypervariable loops and the nature of the antigen. J. Mol. Biol. 325:337-354.
Dedera, D., and L. Ratner. 1991. Demonstration of two distinct cytopathic effects with syncytium formation-defective human immunodeficiency virus type 1 mutants. J. Virol. 65:6129-6136.
Denesvre, C., C. Carrington, A. Corbin, Y. Takeuchi, F. L. Cosset, T. Schulz, M. Sitbon, and P. Sonigo. 1996. TM domain swapping of murine leukemia virus and human T-cell leukemia virus envelopes confers different infectious abilities despite similar incorporation into virions. J. Virol. 70:4380-4386.
de Rosny, E., R. Vassell, S. Jiang, R. Kunert, and C. D. Weiss. 2004. Binding of the 2F5 monoclonal antibody to native and fusion-intermediate forms of human immunodeficiency virus type 1 gp41: implications for fusion-inducing conformational changes. J. Virol. 78:2627-2631.
Dimitrov, A. S., S. S. Rawat, S. Jiang, and R. Blumenthal. 2003. Role of the fusion peptide and membrane-proximal domain in HIV-1 envelope glycoprotein-mediated membrane fusion. Biochemistry 42:14150-14158.
Fass, D., S. C. Harrison, and P. S. Kim. 1996. Retrovirus envelope domain at 1.7 angstrom resolution. Nat. Struct. Biol. 3:465-469.
Fass, D., and P. S. Kim. 1995. Dissection of a retrovirus envelope protein reveals structural similarity to influenza hemagglutinin. Curr. Biol. 5:1377-1383.
Gallo, S. A., C. M. Finnegan, M. Viard, Y. Raviv, A. Dimitrov, S. S. Rawat, A. Puri, S. Durell, and R. Blumenthal. 2003. The HIV Env-mediated fusion reaction. Biochim. Biophys. Acta 1614:36-50.
Gorny, M. K., and S. Zolla-Pazner. 2000. Recognition by human monoclonal antibodies of free and complexed peptides representing the prefusogenic and fusogenic forms of human immunodeficiency virus type 1 gp41. J. Virol. 74:6186-6192.
Greenberg, M., N. Cammack, M. Salgo, and L. Smiley. 2004. HIV fusion and its inhibition in antiretroviral therapy. Rev. Med. Virol. 14:321-337.
Grundner, C., T. Mirzabekov, J. Sodroski, and R. Wyatt. 2002. Solid-phase proteoliposomes containing human immunodeficiency virus envelope glycoproteins. J. Virol. 76:3511-3521.
Haynes, B. F., J. Fleming, E. W. St Clair, H. Katinger, G. Stiegler, R. Kunert, J. Robinson, R. M. Scearce, K. Plonk, H. F. Staats, T. L. Ortel, H. X. Liao, and S. M. Alam. 2005. Cardiolipin polyspecific autoreactivity in two broadly neutralizing HIV-1 antibodies. Science 308:1906-1908.
Herrera, C., C. Spenlehauer, M. S. Fung, D. R. Burton, S. Beddows, and J. P. Moore. 2003. Nonneutralizing antibodies to the CD4-binding site on the gp120 subunit of human immunodeficiency virus type 1 do not interfere with the activity of a neutralizing antibody against the same site. J. Virol. 77:1084-1091.
Ho, J., R. A. Uger, M. B. Zwick, M. A. Luscher, B. H. Barber, and K. S. MacDonald. 2005. Conformational constraints imposed on a pan-neutralizing HIV-1 antibody epitope result in increased antigenicity but not neutralizing response. Vaccine 23:1559-1573.
Kayman, S. C., H. Park, M. Saxon, and A. Pinter. 1999. The hypervariable domain of the murine leukemia virus surface protein tolerates large insertions and deletions, enabling development of a retroviral particle display system. J. Virol. 73:1802-1808.
Klasse, P. J., and J. P. Moore. 1996. Quantitative model of antibody- and soluble CD4-mediated neutralization of primary isolates and T-cell line-adapted strains of human immunodeficiency virus type 1. J. Virol. 70:3668-3677.
Klasse, P. J., and Q. J. Sattentau. 2001. Mechanisms of virus neutralization by antibody. Curr. Top. Microbiol. Immunol. 260:87-108.
Kunert, R., F. Ruker, and H. Katinger. 1998. Molecular characterization of five neutralizing anti-HIV type 1 antibodies: identification of nonconventional D segments in the human monoclonal antibodies 2G12 and 2F5. AIDS Res. Hum. Retrovir. 14:1115-1128.
Kunert, R., W. Steinfellner, M. Purtscher, A. Assadian, and H. Katinger. 2000. Stable recombinant expression of the anti HIV-1 monoclonal antibody 2F5 after IgG3/IgG1 subclass switch in CHO cells. Biotechnol. Bioeng. 67:97-103.
McGaughey, G. B., G. Barbato, E. Bianchi, R. M. Freidinger, V. M. Garsky, W. M. Hurni, J. G. Joyce, X. Liang, M. D. Miller, A. Pessi, J. W. Shiver, and M. J. Bogusky. 2004. Progress towards the development of a HIV-1 gp41-directed vaccine. Curr. HIV Res. 2:193-204.
McGaughey, G. B., M. Citron, R. C. Danzeisen, R. M. Freidinger, V. M. Garsky, W. M. Hurni, J. G. Joyce, X. Liang, M. Miller, J. Shiver, and M. J. Bogusky. 2003. HIV-1 vaccine development: constrained peptide immunogens show improved binding to the anti-HIV-1 gp41 MAb. Biochemistry 42:3214-3223.
Menendez, A., K. C. Chow, O. C. Pan, and J. K. Scott. 2004. Human immunodeficiency virus type 1-neutralizing monoclonal antibody 2F5 is multispecific for sequences flanking the DKW core epitope. J. Mol. Biol. 338:311-327.
Moore, J. P., and R. W. Doms. 2003. The entry of entry inhibitors: a fusion of science and medicine. Proc. Natl. Acad. Sci. USA 100:10598-10602.
Munoz-Barroso, I., K. Salzwedel, E. Hunter, and R. Blumenthal. 1999. Role of the membrane-proximal domain in the initial stages of human immunodeficiency virus type 1 envelope glycoprotein-mediated membrane fusion. J. Virol. 73:6089-6092.
Muster, T., F. Steindl, M. Purtscher, A. Trkola, A. Klima, G. Himmler, F. Ruker, and H. Katinger. 1993. A conserved neutralizing epitope on gp41 of human immunodeficiency virus type 1. J. Virol. 67:6642-6647.
Nabel, G. J. 2005. Immunology. Close to the edge: neutralizing the HIV-1 envelope. Science 308:1878-1879.
Ofek, G., M. Tang, A. Sambor, H. Katinger, J. R. Mascola, R. Wyatt, and P. D. Kwong. 2004. Structure and mechanistic analysis of the anti-human immunodeficiency virus type 1 antibody 2F5 in complex with its gp41 epitope. J. Virol. 78:10724-10737.
Ou, W., and J. Silver. 2005. Inhibition of murine leukemia virus envelope protein (Env) processing by intracellular expression of the Env N-terminal heptad repeat region. J. Virol. 79:4782-4792.
Ou, W., Y. Xiong, and J. Silver. 2004. Quantification of virus-envelope-mediated cell fusion using a tetracycline transcriptional transactivator: fusion does not correlate with syncytium formation. Virology 324:263-272.
Parker, C. E., L. J. Deterding, C. Hager-Braun, J. M. Binley, N. Schulke, H. Katinger, J. P. Moore, and K. B. Tomer. 2001. Fine definition of the epitope on the gp41 glycoprotein of human immunodeficiency virus type 1 for the neutralizing monoclonal antibody 2F5. J. Virol. 75:10906-10911.
Parren, P. W., I. Mondor, D. Naniche, H. J. Ditzel, P. J. Klasse, D. R. Burton, and Q. J. Sattentau. 1998. Neutralization of human immunodeficiency virus type 1 by antibody to gp120 is determined primarily by occupancy of sites on the virion irrespective of epitope specificity. J. Virol. 72:3512-3519.
Poignard, P., M. Moulard, E. Golez, V. Vivona, M. Franti, S. Venturini, M. Wang, P. W. Parren, and D. R. Burton. 2003. Heterogeneity of envelope molecules expressed on primary human immunodeficiency virus type 1 particles as probed by the binding of neutralizing and nonneutralizing antibodies. J. Virol. 77:353-365.
Ragheb, J. A., and W. F. Anderson. 1994. pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry. J. Virol. 68:3220-3231.
Ren, X., J. Sodroski, and X. Yang. 2005. An unrelated monoclonal antibody neutralizes human immunodeficiency virus type 1 by binding to an artificial epitope engineered in a functionally neutral region of the viral envelope glycoproteins. J. Virol. 79:5616-5624.
Roben, P., J. P. Moore, M. Thali, J. Sodroski, C. F. Barbas III, and D. R. Burton. 1994. Recognition properties of a panel of human recombinant Fab fragments to the CD4 binding site of gp120 that show differing abilities to neutralize human immunodeficiency virus type 1. J. Virol. 68:4821-4828.
Rothenberg, S. M., M. N. Olsen, L. C. Laurent, R. A. Crowley, and P. O. Brown. 2001. Comprehensive mutational analysis of the Moloney murine leukemia virus envelope protein. J. Virol. 75:11851-11862.
Salzwedel, K., J. T. West, and E. Hunter. 1999. A conserved tryptophan-rich motif in the membrane-proximal region of the human immunodeficiency virus type 1 gp41 ectodomain is important for Env-mediated fusion and virus infectivity. J. Virol. 73:2469-2480.
Scanlan, C. N., R. Pantophlet, M. R. Wormald, S. E. Ollmann, R. Stanfield, I. A. Wilson, H. Katinger, R. A. Dwek, P. M. Rudd, and D. R. Burton. 2002. The broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2G12 recognizes a cluster of alpha12 mannose residues on the outer face of gp120. J. Virol. 76:7306-7321.
Schlehuber, L. D., and J. K. Rose. 2004. Prediction and identification of a permissive epitope insertion site in the vesicular stomatitis virus glycoprotein. J. Virol. 78:5079-5087.
Schmid, E., A. Zurbriggen, U. Gassen, B. Rima, V. ter Meulen, and J. Schneider-Schaulies. 2000. Antibodies to CD9, a tetraspan transmembrane protein, inhibit canine distemper virus-induced cell-cell fusion but not virus-cell fusion. J. Virol. 74:7554-7561.
Suarez, T., W. R. Gallaher, A. Agirre, F. M. Goni, and J. L. Nieva. 2000. Membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion. J. Virol. 74:8038-8047.
Tian, Y., C. V. Ramesh, X. Ma, S. Naqvi, T. Patel, T. Cenizal, M. Tiscione, K. Diaz, T. Crea, E. Arnold, G. F. Arnold, and J. W. Taylor. 2002. Structure-affinity relationships in the gp41 ELDKWA epitope for the HIV-1 neutralizing monoclonal antibody 2F5: effects of side-chain and backbone modifications and conformational constraints. J. Pept. Res. 59:264-276.
von Messling, V., G. Zimmer, G. Herrler, L. Haas, and R. Cattaneo. 2001. The hemagglutinin of canine distemper virus determines tropism and cytopathogenicity. J. Virol. 75:6418-6427.
Weimin, W. B., P. M. Cannon, E. M. Gordon, F. L. Hall, and W. F. Anderson. 1998. Characterization of the proline-rich region of murine leukemia virus envelope protein. J. Virol. 72:5383-5391.
Wolbank, S., R. Kunert, G. Stiegler, and H. Katinger. 2003. Characterization of human class-switched polymeric (immunoglobulin M [IgM] and IgA) anti-human immunodeficiency virus type 1 antibodies 2F5 and 2G12. J. Virol. 77:4095-4103.
Wu, T. T., G. Johnson, and E. A. Kabat. 1993. Length distribution of CDRH3 in antibodies. Proteins 16:1-7.
Zhang, M. Y., Y. Shu, I. Sidorov, and D. S. Dimitrov. 2004. Identification of a novel CD4i human monoclonal antibody Fab that neutralizes HIV-1 primary isolates from different clades. Antivir. Res. 61:161-164.
Zhang, M. Y., X. Xiao, I. A. Sidorov, V. Choudhry, F. Cham, P. F. Zhang, P. Bouma, M. Zwick, A. Choudhary, D. C. Montefiori, C. C. Broder, D. R. Burton, G. V. Quinnan, Jr., and D. S. Dimitrov. 2004. Identification and characterization of a new cross-reactive human immunodeficiency virus type 1-neutralizing human monoclonal antibody. J. Virol. 78:9233-9242.
Zhu, P., E. Chertova, J. Bess, Jr., J. D. Lifson, L. O. Arthur, J. Liu, K. A. Taylor, and K. H. Roux. 2003. Electron tomography analysis of envelope glycoprotein trimers on HIV and simian immunodeficiency virus virions. Proc. Natl. Acad. Sci. USA 100:15812-15817.
Zimmer, G., K. K. Conzelmann, and G. Herrler. 2002. Cleavage at the furin consensus sequence RAR/KR(109) and presence of the intervening peptide of the respiratory syncytial virus fusion protein are dispensable for virus replication in cell culture. J. Virol. 76:9218-9224.
Zwick, M. B., H. K. Komori, R. L. Stanfield, S. Church, M. Wang, P. W. Parren, R. Kunert, H. Katinger, I. A. Wilson, and D. R. Burton. 2004. The long third complementarity-determining region of the heavy chain is important in the activity of the broadly neutralizing anti-human immunodeficiency virus type 1 antibody 2F5. J. Virol. 78:3155-3161.
Zwick, M. B., A. F. Labrijn, M. Wang, C. Spenlehauer, E. O. Saphire, J. M. Binley, J. P. Moore, G. Stiegler, H. Katinger, D. R. Burton, and P. W. Parren. 2001. Broadly neutralizing antibodies targeted to the membrane-proximal external region of human immunodeficiency virus type 1 glycoprotein gp41. J. Virol. 75:10892-10905.(Wu Ou, Ning Lu, Sloane S.)