An Unrelated Monoclonal Antibody Neutralizes Human
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病菌学杂志 2005年第9期
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute
Department of Pathology, Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115
ABSTRACT
Neutralizing antibodies often recognize regions of viral envelope glycoproteins that play a role in receptor binding or other aspects of virus entry. To address whether this is a necessary feature of a neutralizing antibody, we identified the V4 region of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) as a sequence that is tolerant of drastic change and thus appears to play a negligible role in envelope glycoprotein function. An artificial epitope tag was inserted into the V4 region without a significant effect on virus entry or neutralization by antibodies that recognize HIV-1 envelope glycoprotein sequences. An antibody directed against the artificial epitope tag was able to neutralize the modified, but not the wild-type, HIV-1. Thus, the specific target of a neutralizing antibody need not contribute functionally to the process of virus entry.
INTRODUCTION
The entry of human immunodeficiency virus type 1 (HIV-1) into the host cell is mediated by the viral envelope glycoproteins. The HIV-1 envelope glycoproteins are derived from a roughly 850-residue precursor that is heavily glycosylated and subsequently cleaved into the mature gp120 and gp41 subunits (72). The envelope glycoprotein spike on HIV-1 virions functions as a homotrimer containing three gp120 exterior envelope glycoproteins and three gp41 transmembrane envelope glycoproteins (14, 72). The HIV-1 gp41 glycoprotein is a type I membrane protein, and its ectodomain interacts noncovalently with gp120 to retain the latter on the virion surface (19, 45). The gp120 glycoprotein comprises most of the exposed surface of the envelope glycoprotein complex and is responsible for binding the CD4 and CCR5/CXCR4 target cell receptors (1-3, 9-13, 30, 31, 50). Receptor binding triggers conformational changes that allow the gp41 glycoprotein to mediate the fusion of the viral and target cell membrane (18, 23, 34, 56, 58), a process that is essential for virus entry into the host (8).
Structural and mutagenic analyses, as well as studies of inhibitory ligands, have provided insight into the functionally important regions of HIV-1 gp120 and gp41. The gp120 sequences of numerous HIV-1 strains exhibit five conserved (C1 to C5) and five variable (V1 to V5) regions; the gp41 ectodomain is well conserved among HIV-1 variants (21, 36, 46, 48, 61, 68). The conserved gp120 regions form a core, which consists of an inner, gp41-interacting domain, an outer domain, and a bridging sheet (37, 38). The outer domain of gp120 is heavily glycosylated and is thought to be exposed on the surface of the assembled envelope glycoprotein trimer (70). Elements of the inner domain, outer domain, and bridging sheet contribute to the ability of gp120 to bind the CD4 receptor. The gp120 variable regions are surface-exposed loops (20, 41, 52). The V3 loop and the 19 strand, which is located in the outer domain near the bridging sheet, are thought to comprise the binding site for the CCR5/CXCR4 chemokine receptors (4, 38, 54). The gp120 inner domain contributes to post-receptor binding events that allow efficient membrane fusion (16, 58, 75). Conserved elements of the gp41 ectodomain are essential for the interaction with the target cell membrane and for conformational changes that result in the creation of a six-helix bundle (7, 44, 67). The latter process is thought to provide the energy required to fuse the viral and target cell membranes.
The HIV-1 envelope glycoproteins represent the only available targets for antibodies capable of neutralizing the virus. Strain-restricted neutralizing antibodies bind the V2 and V3 loops of gp120; V3-directed antibodies block CCR5/CXCR4 binding (63, 69). More broadly reactive neutralizing antibodies are the CD4-binding site antibodies and the CD4-induced epitope antibodies, which recognize conserved elements of the gp120 binding regions for CD4 and CCR5/CXCR4, respectively (70, 72). Less frequently elicited neutralizing antibodies are directed against a carbohydrate-rich, outer domain epitope on gp120 or against a gp41 segment near the viral membrane (47, 64, 65).
Recently, we studied the stoichiometry of antibody-mediated neutralization of HIV-1, using heterotrimers composed of wild-type (wt) and neutralization escape mutant envelope glycoproteins (74). Fifteen combinations of different antibodies and HIV-1 strains were studied. The data suggested that binding of one antibody molecule is sufficient to neutralize the envelope glycoprotein trimer, regardless of the particular monoclonal antibody or HIV-1 strain studied. The antibodies used in this study bind distinct regions of the HIV-1 envelope glycoproteins, including those involved in receptor binding. These results hint that the ability of an antibody to bind the functional envelope glycoprotein trimer may be more important for achieving HIV-1 neutralization than the specific site of binding. Such a model predicts that even an antibody that recognizes a nonfunctional element on the functional envelope glycoprotein complex should be capable of neutralizing HIV-1.
To test this prediction, we considered regions of the HIV-1 envelope glycoproteins that might tolerate the insertion of an artificial peptide epitope without resulting in loss of envelope glycoprotein function. A second requirement for these experiments is that the inserted sequence be accessible to antibody on the assembled HIV-1 envelope glycoprotein trimer. The conserved regions of the HIV-1 envelope glycoproteins are critical for the folding, assembly, and function of these proteins in virus entry and, furthermore, are minimally exposed on the trimer (39, 70). The gp120 variable regions are surface exposed and naturally exhibit variation, indicating potential suitability for our purposes. Previous studies have demonstrated the effects of changes in the V2 and V3 variable loop on envelope glycoprotein receptor binding and virus entry (5, 17, 32, 60, 71, 73). The V5 region is naturally short and physically proximal to the CD4-binding region of gp120 (26, 38). Therefore, we focused on the V4 region of gp120. There are several features of V4 that make it attractive for these studies: (i) accessibility, as demonstrated by the X-ray crystal structures of gp120 and suggested by the natural addition of N-linked glycans to the region (15, 37, 38, 66); (ii) flexibility, as demonstrated by the disorder or lack of secondary structure in this region in current gp120 structures (37, 38); (iii) distance from known functional regions of the gp120 envelope glycoprotein (37, 38); (iv) natural length and amino acid variation, suggesting tolerance to insertions and significant changes in sequence (21, 35, 36); and (v) lack of any associated function (72). Here, we investigate the consequence of changes in the HIV-1 gp120 V4 loop on envelope glycoprotein function. We introduce an artificial epitope into this region and study whether an antibody directed against the inserted peptide can neutralize HIV-1.
MATERIALS AND METHODS
Expression plasmids and constructs. The envelope glycoproteins of HIV-1HXBc2 and HIV-1YU2 were expressed from a pSVIIIenv vector as previously described (62). The mutants were constructed using the PCR-based QuikChange protocol (Stratagene). The HXBc2400-405 mutant contains a deletion encompassing residues 400 to 405; this mutant contains the amino acid sequence W395FNSTNNTEG410. In the N406T mutant, the asparagine at position 406 in the HIV-1HXBc2 envelope glycoprotein is changed to threonine.
The mutant versions of the HIV-1YU2 envelope glycoproteins were named as follows, using the numbering scheme of the prototypic HIV-1HXBc2 sequence, as recommended previously (33). In the mutant designated "Insert," the sequence WSTEGS is inserted between leucine 406 and asparagine 407 to yield the sequence DTKRL406WSTEGSN407NTGRN, where the inserted sequence is underlined and leucine 406 and asparagine 407 are shown in bold type. The "FLAG" mutant of the HIV-1YU2 envelope glycoproteins contains the FLAG sequence DYKDDDDK(Sigma-Aldrich) inserted between leucine 406 and asparagine 407. The N407K derivatives of the wt, Insert, and FLAG HIV-1YU2 envelope glycoproteins have asparagine 407 converted to lysine and are designated N407K, Insert-N407K, and FLAG-N407K, respectively. The open reading frames of the mutants were sequenced in their entirety to verify the presence of the intended changes and the absence of unintended mutations.
Transfections and immunoprecipitations. The env-expressing plasmid was transfected into 293T cells using Lipofectamine reagent (Invitrogen) according to the manufacturer's recommendations. Beginning at approximately 24 h after transfection, the cells were labeled with 200 μCi [35S]methionine/cysteine in 5 ml methionine/cysteine-free medium overnight. The culture medium was then harvested after centrifuging at 2,500 rpm for 10 min. After a quick wash with 1x phosphate-buffered saline (PBS), the cells were lysed in 0.5% NP-40, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.4, on ice and clarified by centrifugation at 14,000 rpm for 30 min at 4°C. For immunoprecipitations, 400 μl of medium was incubated overnight at 4°C with 3 μl of pooled sera from HIV-1-infected individuals or 1 μg of the M2 antibody (Sigma) and 50 μl of protein A-Sepharose (10% in PBS) (Pharmacia) that had been preincubated with 5% bovine serum albumin in PBS. After three washes with 1x PBS, the beads were boiled for 5 min in 1x Lamelli sample buffer with 2% -mercaptoethanol. The protein samples were then analyzed on 8% SDS-polyacrylamide gels. For the CD4-Ig (fusion protein between the ectodomains of human CD4 and the Fc domain of human immunoglobulin G1 [IgG1]) binding assay, 2 μg of purified CD4-Ig was used by following the same protocol.
Assays measuring single-round infection and neutralization. Recombinant HIV-1 encoding luciferase and pseudotyped with the envelope glycoproteins was produced as previously described (25). Briefly, 293T cells in 100-mm-diameter tissue culture dishes were cotransfected by the Lipofectamine reagent with 2 μg pSVIIIenv plasmid expressing the HIV-1 envelope glycoprotein variants, 2 μg of the pCMV-PACK plasmid expressing the Gag/Pol and Tat proteins of HIV-1, and 6 μg of pHIV-1-Luc, which expresses an HIV-1 vector with a firefly luciferase reporter gene. Two days after transfection, the recombinant virions in the cell supernatants were harvested and quantitated by measuring the reverse transcriptase activity by [3H]TTP incorporation. Approximately 6 x 103 Cf2Th-CD4/CCR5 cells were seeded into each well of a 96-well luciferase assay plate (EG&G Wallac) and cultured at 37°C with 5% CO2 overnight. For each assay, 100-μl portions of the diluted viruses were added to each well of the target cells in three parallel wells, and the cells were cultured for another 48 h, lysed, and used for measurement of luciferase activity. For the neutralization assays, different amounts of the antibodies were first mixed with the same amounts of the virus stocks, which were normalized according to reverse transcriptase activity, and incubated at 37°C with 5% CO2 for 4 h before being added to the target cells for residual infectivity measurements. For the neutralization assay with HIV-1HXBc2, the target cells were Cf2Th-CD4/CXCR4 cells, and the incubation time of the antibody/virus mixture was 2 h. The residual infectivity was normalized to that of virus incubated with tissue culture medium only. Both the mean and the range of variation of the residual activities are reported.
CCR5 binding assay. The binding of radiolabeled HIV-1 gp120 to CCR5-expressing cells was studied as previously described (32). Briefly, the gp120 glycoprotein in the supernatant of [35S]Met/Cys-labeled cells was concentrated 10-fold with Centriprep YM-30. Then, 150 μl of the concentrated solution was incubated with 2 μg of soluble CD4 and 2 x 106 Cf2Th-CCR5 cells for 1 h in a 37°C water bath. After three washes with 1 ml 1 x PBS, the cells were lysed with lysis buffer, and the bound envelope proteins were detected using a standard immunoprecipitation assay, as described above. To investigate whether the M2 anti-FLAG antibody affects the ability of the gp120 envelope glycoprotein to bind CD4 and CCR5, 150 μl of the concentrated 35S-labeled envelope glycoproteins were first incubated with 100 μg/ml of M2 antibody at 37°C for 1 h, and the resulting mixtures were then studied by the CCR5 binding assay.
RESULTS
Functional effects of insertions in the gp120 V4 region. Comparison of the gp120 sequences from several HIV-1 strains reveals that, in addition to variation in individual amino acid residues, significant length polymorphism in the V4 region is tolerated among functional envelope glycoproteins (21, 36). The 23-residue V4 region of HIV-1YU2 gp120 is one of the shortest among clade B HIV-1 strains and thus was chosen as a good candidate for the ability to tolerate length expansion in this region (76). The HIV-1YU2 provirus was originally cloned directly from the brain tissue of an AIDS patient, and this primary HIV-1 isolate has been shown to be relatively resistant to antibody neutralization (42, 43). The peptide sequence WSTEGS from the V4 region of the HIV-1HXBC2 gp120 envelope glycoprotein was inserted into the corresponding region of the HIV-1YU2 gp120 glycoprotein. This mutant was designated Insert (Fig. 1A). Because the intended insertion site was immediately adjacent to a predicted N-glycosylation site, the effect of removing this site was investigated by converting the asparagine residue at position 407 to lysine (N407K in Fig. 1A). The N407K change was studied alone and in combination with the insert. The three V4 mutants and the wt HIV-1YU2 envelope glycoproteins were expressed in 293T cells, which were radiolabeled and used for immunoprecipitation. The wt and mutant envelope glycoproteins were expressed and processed efficiently; a slight diminution in the proteolytic processing of the Insert envelope glycoproteins was noted (Fig. 1B, top panel). On the basis of the ratios of gp120 glycoproteins shed into the culture medium and present in the cell lysates, the association of the gp120 and gp41 subunits was comparable for the wt and mutant envelope glycoproteins (Fig. 1B, bottom panel). The slight increase in the migration of the N407K and Insert-N407K glycoproteins compared with those of the wild-type and Insert glycoproteins, respectively, suggests that the potential glycosylation site at asparagine 407 is utilized in 293T cells.
The effects of the V4 changes on the ability of the HIV-1YU2 envelope glycoproteins to support HIV-1 entry were examined in a single-round infectivity assay. As shown in Fig. 1C, reporter viruses with the mutant envelope glycoproteins infected Cf2Th-CD4/CCR5 cells with an efficiency similar to that of the wt HIV-1YU2 envelope glycoproteins. The infection efficiency associated with the Insert mutant was slightly reduced than that of the wt HIV-1YU2 envelope glycoproteins but still was within 70% of the latter.
The sensitivities of the viruses with the wt and mutant HIV-1YU2 envelope glycoproteins to neutralization by several anti-HIV-1 antibodies were examined. As expected, the viruses with the wt HIV-1YU2 envelope glycoproteins were neutralized only by IgG1b12 and 2F5, two of the more potent neutralizing antibodies tested (Fig. 1D and data not shown). The neutralization curves of the mutant and wt viruses for these two antibodies were almost identical (Fig. 1D).
In a reciprocal experiment, we introduced a deletion into the V4 region of the envelope glycoproteins of the T-cell-adapted HIV-1HXBc2, which is very sensitive to neutralization by antibodies (48). The WSTEGS sequence was deleted in a mutant designated HXBc2400-405 (Fig. 2A). A site of potential N-linked glycosylation in the HIV-1HXBC2 V4 region was altered in the N406T mutant. The wt and mutant envelope glycoproteins of HIV-1HXBC2 were expressed and processed similarly in transfected 293T cells (Fig. 2B). The amounts of gp120 shed into the tissue culture medium were similar for all three envelope glycoproteins. The infectivities of viruses containing the wt or mutant HIV-1HXBC2 envelope glycoproteins were within twofold of one another (Fig. 2C). The sensitivities of these viruses to neutralization by a variety of antibodies directed against either gp120 or gp41 were comparable (Fig. 2D and data not shown).
These results indicate that substantial alteration of the HIV-1 gp120 V4 region, including a large deletion, a large insertion, and removal of a carbohydrate moiety, exerted minimal effects on the function and neutralization sensitivity of envelope glycoproteins from HIV-1YU2 and HIV-1HXBc2.
Ability of an epitope inserted into the V4 region to act as a neutralization target. Encouraged by the above data on the tolerance of HIV-1 envelope glycoprotein function to V4 modification, we inserted a FLAG epitope tag in the V4 region of the HIV-1YU2 gp120 envelope glycoprotein (Fig. 3A). The N407K change was also introduced into this construct to assess the effect of removing the N-linked glycosylation site immediately carboxy terminal to the inserted epitope. The wt and mutant envelope glycoproteins were transiently expressed in 293T cells, which were radiolabeled and used for immunoprecipitation with pooled sera from HIV-1-infected individuals. The mutant proteins were expressed, processed, and secreted into the culture medium at levels similar to those of the wt HIV-1YU2 envelope glycoproteins (Fig. 3B, top panel). The FLAG-N407K gp120 glycoprotein migrated appreciably faster than the wt or FLAG gp120 glycoproteins, consistent with the expected lack of carbohydrate modification of asparagine 407 (Fig. 3B, middle panel). The M2 antibody, which is directed against the FLAG epitope (59), successfully precipitated the FLAG and FLAG-N407K glycoproteins but did not recognize the wt HIV-1YU2 envelope glycoproteins (Fig. 3B, lower panel). Thus, the FLAG epitope is accessible on the surfaces of the FLAG and FLAG-N407K envelope glycoproteins.
Recombinant, luciferase-expressing HIV-1 vectors with the wt, FLAG, and FLAG-N407K envelope glycoproteins were equally infectious for Cf2Th-CD4/CCR5 target cells (Fig. 3C). The three viruses were neutralized by the IgG1b12 and 2F5 antibodies equivalently (Fig. 3D). Thus, the insertion of the FLAG sequence, either alone or in combination with the removal of the glycosylation site at asparagine 407, in the gp120 V4 region exerted no significant effects on the expression, function in virus entry, or general neutralization sensitivity of the HIV-1YU2 envelope glycoproteins.
The luciferase-expressing viruses with the wt, FLAG, and FLAG-N407K envelope glycoproteins were tested for sensitivity to neutralization by the M2 antibody directed against the FLAG epitope. The M2 antibody neutralized the viruses with the FLAG and FLAG-N407K envelope glycoproteins equivalently, with a 50% effective concentration between 10 and 20 nM (Fig. 3E). By contrast, the viruses with the wt YU2 envelope glycoproteins were resistant to neutralization by the M2 antibody. These experiments were repeated with different preparations of the M2 antibody, different amounts of input virus, reduction in the preincubation time of antibody and virus to as little as 10 min, and reduction in the exposure of the target cells to the antibody to as little as 2 h. Results similar to those described above were obtained in these independent experiments (data not shown). Of note, a small fraction (10 to 20%) of the FLAG and FLAG-N407K viruses remained infectious despite incubation with high concentrations of the M2 antibody. As further increases in M2 antibody concentration led to less specific virus inhibition, as evidenced by some effects on the viruses with wild-type envelope glycoproteins, we could not assess whether this residual fraction of infectious virus could be neutralized at higher doses of M2 antibody. We conclude that HIV-1 bearing an extraneous epitope in the gp120 V4 region can be neutralized by an antibody that recognizes that foreign epitope.
Effects of M2 antibody binding on gp120-receptor interactions. The V4 region is spatially distant from the gp120 regions involved in CD4 and CCR5/CXCR4 binding (38, 55). Thus, the binding of the M2 antibody is not expected to interfere with gp120-receptor interaction. To examine this, we studied the precipitation of radiolabeled gp120 from the HIV-1YU2 strain by CD4-Ig. The precipitation of the wt, FLAG, and FLAG-N407K gp120 envelope glycoproteins by CD4-Ig was equivalent (Fig. 4). This result suggests that the three gp120 glycoproteins bind CD4 comparably. The gp120 supernatants were concentrated about 10-fold using CentriprepYM-30. Then, 30-μl portions of the concentrated gp120s were precipitated with pooled sera from HIV-1-infected individuals, indicating that similar levels of gp120 were present in the three gp120 preparations (Fig. 4, middle panel). Next, 150 μl of the concentrated gp120 solutions were preincubated with 100 μg/ml of the M2 antibody prior to incubation with soluble CD4 and target cells expressing CCR5. Preincubation with 100 μg/ml M2 had no detectable effect on the binding of the wt, FLAG, or FLAG-N407K gp120 glycoproteins to CCR5 on the surfaces of Cf2Th-CCR5 cells (Fig. 4), a process that is dependent upon initial binding of gp120 to CD4 (50, 54). We conclude that both CD4 binding and CCR5 binding by gp120 are not impeded by the M2 antibody to the V4 FLAG sequence.
DISCUSSION
The HIV-1 gp120 variable regions exhibit considerable diversity among naturally occurring viral strains. The V4 region in particular tolerates substantial changes in length, amino acid composition, and glycosylation without compromising HIV-1 envelope glycoprotein function (21, 36, 66). This is consistent with its exposed status on the gp120 outer domain, far removed from envelope glycoprotein regions known to be important for virus entry (4, 37, 38). The V4 surface loop connects the 18 and 19 strands of the outer domain; in various HIV-1 strains, this connection is accomplished by as few as 2 or as many as 19 amino acid residues (36). Although most HIV-1 strains have glycosylated V4 regions, some do not (36). The impressive structural heterogeneity of the V4 region (36-38) almost precludes a specific contribution of this element to HIV-1 entry. Indeed, reminiscent of results seen in previous mutagenic studies (35), alteration of the V4 region in the envelope glycoproteins from two very different strains, HIV-1YU2 and HIV-1HXBc2, exerted little effect on the ability to mediate virus entry. Regions of hypervariability in the envelope glycoproteins of other viruses, such as those of Friend murine leukemia virus and vesicular stomatitis virus, have likewise been shown to accept large inserts without compromising virus replication (27, 57). Thus, natural variation in a viral envelope glycoprotein can provide guidance in identifying surface-exposed elements that can be manipulated for a variety of purposes.
The minimal apparent contribution of the V4 region to HIV-1 envelope glycoprotein function, its expected exposure on the assembled envelope glycoprotein trimer, and its hypervariability in naturally occurring HIV-1 strains all suggest that V4 contributes to immune evasion. Indeed, it has been shown that changes in V4 glycosylation can affect the sensitivity of an HIV-1 isolate to strain-specific neutralizing antibodies present in the serum of an acutely infected individual (66). The V4 variant virus also exhibited altered susceptibility to neutralization by a monoclonal antibody directed against the V3 variable loop (66). The ability of changes in V4 glycosylation to influence neutralization by an antibody that recognizes a distant gp120 structure suggested the concept of a "glycan shield." In this model, a densely packed array of carbohydrate structures on gp120 creates a steric impediment to the binding of antibodies to more distantly situated gp120 structures. One prediction of this model is that loss of glycosylation sites in V4 might result in an increased sensitivity to neutralization by antibodies directed against distant gp120 or gp41 elements. In this study, alteration of the V4 glycosylation site had no detectable effect on the sensitivities of viruses with the HIV-1HXBc2 or HIV-1YU2 envelope glycoproteins to neutralization by a variety of anti-HIV-1 monoclonal antibodies. Moreover, in the context of both HIV-1HXBc2 and HIV-1YU2 envelope glycoproteins, addition or removal of a glycan at amino acid 362, which is adjacent to the V4 region on native gp120, alone or in combination with the V4 carbohydrate alteration did not alter HIV-1 neutralization sensitivity (data not shown). Thus, simple removal of some gp120 outer domain glycans is not sufficient to increase the general susceptibility of HIV-1 to antibody neutralization.
Whether antibody binding to HIV-1 envelope glycoprotein spikes can occur without neutralizing spike function has been the subject of much discussion and has not been resolved yet (6, 24, 49, 53). The M2 antibody neutralized viruses with the two HIV-1 envelope glycoproteins carrying the FLAG epitope in the V4 region. The tolerance of functional HIV-1 envelope glycoproteins to change in the V4 region indicates that this region per se does not play a significant role in the virus entry process. Thus, the neutralizing activity of the M2 antibody is unlikely to result from a disruption of a function intrinsic to the V4 region of gp120. Furthermore, the M2 antibody exhibited no detrimental effect on gp120-CCR5 binding, which is dependent upon prior interaction with CD4 and CD4-induced conformational changes in gp120 (63, 69). Thus, there is no evidence of an indirect effect of M2 antibody binding on the known functions of gp120. Taken together, virus neutralization in this case may result simply from the binding and continued presence of the M2 antibody during the attempted entry process. Mechanistically, steric hindrance may account for the reduced efficiency with which virus attachment and/or membrane fusion events occur in the presence of the bound antibody, given the considerable size of an antibody molecule in comparison to that of the HIV-1 envelope glycoprotein trimer (28, 29, 40). Our results provide further support for the notion that all antibodies that bind with high affinity to the functional HIV-1 envelope glycoprotein spike should, at a minimum, exhibit some degree of neutralizing activity simply as a consequence of this steric hindrance, irrespective of epitope specificity (51).
Neutralization by antibodies that bind functionally unimportant regions of the envelope glycoproteins will probably apply to viruses other than HIV-1. Recently, Schlehuber and Rose introduced an HIV-1 gp41 peptide epitope into a naturally variable site on the vesicular stomatitis virus G glycoprotein (57). The resulting virus was replication competent and was neutralized by a monoclonal antibody directed against the inserted peptide.
A small fraction of the HIV-1 FLAG preparations was not neutralized even by high concentrations of the M2 antibody. The reason for this is not clear. The potency of neutralization by steric hindrance is expected to be largely determined by the efficiency of antibody binding to the epitope in the context of the virion spike (22). Heterogeneity in the virions due to factors, such as differential glycosylation or the formation of virus aggregates, could limit the accessibility of the M2 antibody to a subset of viruses in the stock. Alternatively, if the binding of the M2 antibody to the envelope glycoprotein trimer were reversible, a fraction of viruses may have an opportunity to proceed down the entry pathway before rebinding of the antibody occurs. The latter scenario allows the possibility that greater potency of neutralization results not only from an increased affinity of trimer binding but also from the induction of irreversible inhibitory consequences in the envelope glycoproteins. Such a mechanism may explain the exquisite sensitivity of CD4-independent HIV-1 to neutralization by a wide range of antibodies (15, 32).
The results in this report indicate that the gp120 V4 region is a receptive site for introducing exogenous peptide tags without affecting the intrinsic function and neutralization sensitivity of the HIV-1 glycoproteins. This establishes an experimental system to study antibody-mediated neutralization in a controlled environment, in which the immunoepitopes can be manipulated without interfering with normal structure and function of the carrier envelope glycoproteins. Such studies may provide further insights into mechanisms of HIV-1 neutralization by antibodies and other molecules.
ACKNOWLEDGMENTS
We thank Yvette McLaughlin and Sheri Farnum for manuscript preparation.
This work was supported in part by NIH grants (AI24755, AI31783, and AI39420), by a Center for AIDS research grant (AI42848), by an unrestricted research grant from the Bristol-Myers Squibb Foundation, by a gift from the late William F. McCarty-Cooper, and by funds from the International AIDS Vaccine Initiative and the Pendleton Trust.
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Department of Pathology, Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115
Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115
ABSTRACT
Neutralizing antibodies often recognize regions of viral envelope glycoproteins that play a role in receptor binding or other aspects of virus entry. To address whether this is a necessary feature of a neutralizing antibody, we identified the V4 region of the gp120 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) as a sequence that is tolerant of drastic change and thus appears to play a negligible role in envelope glycoprotein function. An artificial epitope tag was inserted into the V4 region without a significant effect on virus entry or neutralization by antibodies that recognize HIV-1 envelope glycoprotein sequences. An antibody directed against the artificial epitope tag was able to neutralize the modified, but not the wild-type, HIV-1. Thus, the specific target of a neutralizing antibody need not contribute functionally to the process of virus entry.
INTRODUCTION
The entry of human immunodeficiency virus type 1 (HIV-1) into the host cell is mediated by the viral envelope glycoproteins. The HIV-1 envelope glycoproteins are derived from a roughly 850-residue precursor that is heavily glycosylated and subsequently cleaved into the mature gp120 and gp41 subunits (72). The envelope glycoprotein spike on HIV-1 virions functions as a homotrimer containing three gp120 exterior envelope glycoproteins and three gp41 transmembrane envelope glycoproteins (14, 72). The HIV-1 gp41 glycoprotein is a type I membrane protein, and its ectodomain interacts noncovalently with gp120 to retain the latter on the virion surface (19, 45). The gp120 glycoprotein comprises most of the exposed surface of the envelope glycoprotein complex and is responsible for binding the CD4 and CCR5/CXCR4 target cell receptors (1-3, 9-13, 30, 31, 50). Receptor binding triggers conformational changes that allow the gp41 glycoprotein to mediate the fusion of the viral and target cell membrane (18, 23, 34, 56, 58), a process that is essential for virus entry into the host (8).
Structural and mutagenic analyses, as well as studies of inhibitory ligands, have provided insight into the functionally important regions of HIV-1 gp120 and gp41. The gp120 sequences of numerous HIV-1 strains exhibit five conserved (C1 to C5) and five variable (V1 to V5) regions; the gp41 ectodomain is well conserved among HIV-1 variants (21, 36, 46, 48, 61, 68). The conserved gp120 regions form a core, which consists of an inner, gp41-interacting domain, an outer domain, and a bridging sheet (37, 38). The outer domain of gp120 is heavily glycosylated and is thought to be exposed on the surface of the assembled envelope glycoprotein trimer (70). Elements of the inner domain, outer domain, and bridging sheet contribute to the ability of gp120 to bind the CD4 receptor. The gp120 variable regions are surface-exposed loops (20, 41, 52). The V3 loop and the 19 strand, which is located in the outer domain near the bridging sheet, are thought to comprise the binding site for the CCR5/CXCR4 chemokine receptors (4, 38, 54). The gp120 inner domain contributes to post-receptor binding events that allow efficient membrane fusion (16, 58, 75). Conserved elements of the gp41 ectodomain are essential for the interaction with the target cell membrane and for conformational changes that result in the creation of a six-helix bundle (7, 44, 67). The latter process is thought to provide the energy required to fuse the viral and target cell membranes.
The HIV-1 envelope glycoproteins represent the only available targets for antibodies capable of neutralizing the virus. Strain-restricted neutralizing antibodies bind the V2 and V3 loops of gp120; V3-directed antibodies block CCR5/CXCR4 binding (63, 69). More broadly reactive neutralizing antibodies are the CD4-binding site antibodies and the CD4-induced epitope antibodies, which recognize conserved elements of the gp120 binding regions for CD4 and CCR5/CXCR4, respectively (70, 72). Less frequently elicited neutralizing antibodies are directed against a carbohydrate-rich, outer domain epitope on gp120 or against a gp41 segment near the viral membrane (47, 64, 65).
Recently, we studied the stoichiometry of antibody-mediated neutralization of HIV-1, using heterotrimers composed of wild-type (wt) and neutralization escape mutant envelope glycoproteins (74). Fifteen combinations of different antibodies and HIV-1 strains were studied. The data suggested that binding of one antibody molecule is sufficient to neutralize the envelope glycoprotein trimer, regardless of the particular monoclonal antibody or HIV-1 strain studied. The antibodies used in this study bind distinct regions of the HIV-1 envelope glycoproteins, including those involved in receptor binding. These results hint that the ability of an antibody to bind the functional envelope glycoprotein trimer may be more important for achieving HIV-1 neutralization than the specific site of binding. Such a model predicts that even an antibody that recognizes a nonfunctional element on the functional envelope glycoprotein complex should be capable of neutralizing HIV-1.
To test this prediction, we considered regions of the HIV-1 envelope glycoproteins that might tolerate the insertion of an artificial peptide epitope without resulting in loss of envelope glycoprotein function. A second requirement for these experiments is that the inserted sequence be accessible to antibody on the assembled HIV-1 envelope glycoprotein trimer. The conserved regions of the HIV-1 envelope glycoproteins are critical for the folding, assembly, and function of these proteins in virus entry and, furthermore, are minimally exposed on the trimer (39, 70). The gp120 variable regions are surface exposed and naturally exhibit variation, indicating potential suitability for our purposes. Previous studies have demonstrated the effects of changes in the V2 and V3 variable loop on envelope glycoprotein receptor binding and virus entry (5, 17, 32, 60, 71, 73). The V5 region is naturally short and physically proximal to the CD4-binding region of gp120 (26, 38). Therefore, we focused on the V4 region of gp120. There are several features of V4 that make it attractive for these studies: (i) accessibility, as demonstrated by the X-ray crystal structures of gp120 and suggested by the natural addition of N-linked glycans to the region (15, 37, 38, 66); (ii) flexibility, as demonstrated by the disorder or lack of secondary structure in this region in current gp120 structures (37, 38); (iii) distance from known functional regions of the gp120 envelope glycoprotein (37, 38); (iv) natural length and amino acid variation, suggesting tolerance to insertions and significant changes in sequence (21, 35, 36); and (v) lack of any associated function (72). Here, we investigate the consequence of changes in the HIV-1 gp120 V4 loop on envelope glycoprotein function. We introduce an artificial epitope into this region and study whether an antibody directed against the inserted peptide can neutralize HIV-1.
MATERIALS AND METHODS
Expression plasmids and constructs. The envelope glycoproteins of HIV-1HXBc2 and HIV-1YU2 were expressed from a pSVIIIenv vector as previously described (62). The mutants were constructed using the PCR-based QuikChange protocol (Stratagene). The HXBc2400-405 mutant contains a deletion encompassing residues 400 to 405; this mutant contains the amino acid sequence W395FNSTNNTEG410. In the N406T mutant, the asparagine at position 406 in the HIV-1HXBc2 envelope glycoprotein is changed to threonine.
The mutant versions of the HIV-1YU2 envelope glycoproteins were named as follows, using the numbering scheme of the prototypic HIV-1HXBc2 sequence, as recommended previously (33). In the mutant designated "Insert," the sequence WSTEGS is inserted between leucine 406 and asparagine 407 to yield the sequence DTKRL406WSTEGSN407NTGRN, where the inserted sequence is underlined and leucine 406 and asparagine 407 are shown in bold type. The "FLAG" mutant of the HIV-1YU2 envelope glycoproteins contains the FLAG sequence DYKDDDDK(Sigma-Aldrich) inserted between leucine 406 and asparagine 407. The N407K derivatives of the wt, Insert, and FLAG HIV-1YU2 envelope glycoproteins have asparagine 407 converted to lysine and are designated N407K, Insert-N407K, and FLAG-N407K, respectively. The open reading frames of the mutants were sequenced in their entirety to verify the presence of the intended changes and the absence of unintended mutations.
Transfections and immunoprecipitations. The env-expressing plasmid was transfected into 293T cells using Lipofectamine reagent (Invitrogen) according to the manufacturer's recommendations. Beginning at approximately 24 h after transfection, the cells were labeled with 200 μCi [35S]methionine/cysteine in 5 ml methionine/cysteine-free medium overnight. The culture medium was then harvested after centrifuging at 2,500 rpm for 10 min. After a quick wash with 1x phosphate-buffered saline (PBS), the cells were lysed in 0.5% NP-40, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.4, on ice and clarified by centrifugation at 14,000 rpm for 30 min at 4°C. For immunoprecipitations, 400 μl of medium was incubated overnight at 4°C with 3 μl of pooled sera from HIV-1-infected individuals or 1 μg of the M2 antibody (Sigma) and 50 μl of protein A-Sepharose (10% in PBS) (Pharmacia) that had been preincubated with 5% bovine serum albumin in PBS. After three washes with 1x PBS, the beads were boiled for 5 min in 1x Lamelli sample buffer with 2% -mercaptoethanol. The protein samples were then analyzed on 8% SDS-polyacrylamide gels. For the CD4-Ig (fusion protein between the ectodomains of human CD4 and the Fc domain of human immunoglobulin G1 [IgG1]) binding assay, 2 μg of purified CD4-Ig was used by following the same protocol.
Assays measuring single-round infection and neutralization. Recombinant HIV-1 encoding luciferase and pseudotyped with the envelope glycoproteins was produced as previously described (25). Briefly, 293T cells in 100-mm-diameter tissue culture dishes were cotransfected by the Lipofectamine reagent with 2 μg pSVIIIenv plasmid expressing the HIV-1 envelope glycoprotein variants, 2 μg of the pCMV-PACK plasmid expressing the Gag/Pol and Tat proteins of HIV-1, and 6 μg of pHIV-1-Luc, which expresses an HIV-1 vector with a firefly luciferase reporter gene. Two days after transfection, the recombinant virions in the cell supernatants were harvested and quantitated by measuring the reverse transcriptase activity by [3H]TTP incorporation. Approximately 6 x 103 Cf2Th-CD4/CCR5 cells were seeded into each well of a 96-well luciferase assay plate (EG&G Wallac) and cultured at 37°C with 5% CO2 overnight. For each assay, 100-μl portions of the diluted viruses were added to each well of the target cells in three parallel wells, and the cells were cultured for another 48 h, lysed, and used for measurement of luciferase activity. For the neutralization assays, different amounts of the antibodies were first mixed with the same amounts of the virus stocks, which were normalized according to reverse transcriptase activity, and incubated at 37°C with 5% CO2 for 4 h before being added to the target cells for residual infectivity measurements. For the neutralization assay with HIV-1HXBc2, the target cells were Cf2Th-CD4/CXCR4 cells, and the incubation time of the antibody/virus mixture was 2 h. The residual infectivity was normalized to that of virus incubated with tissue culture medium only. Both the mean and the range of variation of the residual activities are reported.
CCR5 binding assay. The binding of radiolabeled HIV-1 gp120 to CCR5-expressing cells was studied as previously described (32). Briefly, the gp120 glycoprotein in the supernatant of [35S]Met/Cys-labeled cells was concentrated 10-fold with Centriprep YM-30. Then, 150 μl of the concentrated solution was incubated with 2 μg of soluble CD4 and 2 x 106 Cf2Th-CCR5 cells for 1 h in a 37°C water bath. After three washes with 1 ml 1 x PBS, the cells were lysed with lysis buffer, and the bound envelope proteins were detected using a standard immunoprecipitation assay, as described above. To investigate whether the M2 anti-FLAG antibody affects the ability of the gp120 envelope glycoprotein to bind CD4 and CCR5, 150 μl of the concentrated 35S-labeled envelope glycoproteins were first incubated with 100 μg/ml of M2 antibody at 37°C for 1 h, and the resulting mixtures were then studied by the CCR5 binding assay.
RESULTS
Functional effects of insertions in the gp120 V4 region. Comparison of the gp120 sequences from several HIV-1 strains reveals that, in addition to variation in individual amino acid residues, significant length polymorphism in the V4 region is tolerated among functional envelope glycoproteins (21, 36). The 23-residue V4 region of HIV-1YU2 gp120 is one of the shortest among clade B HIV-1 strains and thus was chosen as a good candidate for the ability to tolerate length expansion in this region (76). The HIV-1YU2 provirus was originally cloned directly from the brain tissue of an AIDS patient, and this primary HIV-1 isolate has been shown to be relatively resistant to antibody neutralization (42, 43). The peptide sequence WSTEGS from the V4 region of the HIV-1HXBC2 gp120 envelope glycoprotein was inserted into the corresponding region of the HIV-1YU2 gp120 glycoprotein. This mutant was designated Insert (Fig. 1A). Because the intended insertion site was immediately adjacent to a predicted N-glycosylation site, the effect of removing this site was investigated by converting the asparagine residue at position 407 to lysine (N407K in Fig. 1A). The N407K change was studied alone and in combination with the insert. The three V4 mutants and the wt HIV-1YU2 envelope glycoproteins were expressed in 293T cells, which were radiolabeled and used for immunoprecipitation. The wt and mutant envelope glycoproteins were expressed and processed efficiently; a slight diminution in the proteolytic processing of the Insert envelope glycoproteins was noted (Fig. 1B, top panel). On the basis of the ratios of gp120 glycoproteins shed into the culture medium and present in the cell lysates, the association of the gp120 and gp41 subunits was comparable for the wt and mutant envelope glycoproteins (Fig. 1B, bottom panel). The slight increase in the migration of the N407K and Insert-N407K glycoproteins compared with those of the wild-type and Insert glycoproteins, respectively, suggests that the potential glycosylation site at asparagine 407 is utilized in 293T cells.
The effects of the V4 changes on the ability of the HIV-1YU2 envelope glycoproteins to support HIV-1 entry were examined in a single-round infectivity assay. As shown in Fig. 1C, reporter viruses with the mutant envelope glycoproteins infected Cf2Th-CD4/CCR5 cells with an efficiency similar to that of the wt HIV-1YU2 envelope glycoproteins. The infection efficiency associated with the Insert mutant was slightly reduced than that of the wt HIV-1YU2 envelope glycoproteins but still was within 70% of the latter.
The sensitivities of the viruses with the wt and mutant HIV-1YU2 envelope glycoproteins to neutralization by several anti-HIV-1 antibodies were examined. As expected, the viruses with the wt HIV-1YU2 envelope glycoproteins were neutralized only by IgG1b12 and 2F5, two of the more potent neutralizing antibodies tested (Fig. 1D and data not shown). The neutralization curves of the mutant and wt viruses for these two antibodies were almost identical (Fig. 1D).
In a reciprocal experiment, we introduced a deletion into the V4 region of the envelope glycoproteins of the T-cell-adapted HIV-1HXBc2, which is very sensitive to neutralization by antibodies (48). The WSTEGS sequence was deleted in a mutant designated HXBc2400-405 (Fig. 2A). A site of potential N-linked glycosylation in the HIV-1HXBC2 V4 region was altered in the N406T mutant. The wt and mutant envelope glycoproteins of HIV-1HXBC2 were expressed and processed similarly in transfected 293T cells (Fig. 2B). The amounts of gp120 shed into the tissue culture medium were similar for all three envelope glycoproteins. The infectivities of viruses containing the wt or mutant HIV-1HXBC2 envelope glycoproteins were within twofold of one another (Fig. 2C). The sensitivities of these viruses to neutralization by a variety of antibodies directed against either gp120 or gp41 were comparable (Fig. 2D and data not shown).
These results indicate that substantial alteration of the HIV-1 gp120 V4 region, including a large deletion, a large insertion, and removal of a carbohydrate moiety, exerted minimal effects on the function and neutralization sensitivity of envelope glycoproteins from HIV-1YU2 and HIV-1HXBc2.
Ability of an epitope inserted into the V4 region to act as a neutralization target. Encouraged by the above data on the tolerance of HIV-1 envelope glycoprotein function to V4 modification, we inserted a FLAG epitope tag in the V4 region of the HIV-1YU2 gp120 envelope glycoprotein (Fig. 3A). The N407K change was also introduced into this construct to assess the effect of removing the N-linked glycosylation site immediately carboxy terminal to the inserted epitope. The wt and mutant envelope glycoproteins were transiently expressed in 293T cells, which were radiolabeled and used for immunoprecipitation with pooled sera from HIV-1-infected individuals. The mutant proteins were expressed, processed, and secreted into the culture medium at levels similar to those of the wt HIV-1YU2 envelope glycoproteins (Fig. 3B, top panel). The FLAG-N407K gp120 glycoprotein migrated appreciably faster than the wt or FLAG gp120 glycoproteins, consistent with the expected lack of carbohydrate modification of asparagine 407 (Fig. 3B, middle panel). The M2 antibody, which is directed against the FLAG epitope (59), successfully precipitated the FLAG and FLAG-N407K glycoproteins but did not recognize the wt HIV-1YU2 envelope glycoproteins (Fig. 3B, lower panel). Thus, the FLAG epitope is accessible on the surfaces of the FLAG and FLAG-N407K envelope glycoproteins.
Recombinant, luciferase-expressing HIV-1 vectors with the wt, FLAG, and FLAG-N407K envelope glycoproteins were equally infectious for Cf2Th-CD4/CCR5 target cells (Fig. 3C). The three viruses were neutralized by the IgG1b12 and 2F5 antibodies equivalently (Fig. 3D). Thus, the insertion of the FLAG sequence, either alone or in combination with the removal of the glycosylation site at asparagine 407, in the gp120 V4 region exerted no significant effects on the expression, function in virus entry, or general neutralization sensitivity of the HIV-1YU2 envelope glycoproteins.
The luciferase-expressing viruses with the wt, FLAG, and FLAG-N407K envelope glycoproteins were tested for sensitivity to neutralization by the M2 antibody directed against the FLAG epitope. The M2 antibody neutralized the viruses with the FLAG and FLAG-N407K envelope glycoproteins equivalently, with a 50% effective concentration between 10 and 20 nM (Fig. 3E). By contrast, the viruses with the wt YU2 envelope glycoproteins were resistant to neutralization by the M2 antibody. These experiments were repeated with different preparations of the M2 antibody, different amounts of input virus, reduction in the preincubation time of antibody and virus to as little as 10 min, and reduction in the exposure of the target cells to the antibody to as little as 2 h. Results similar to those described above were obtained in these independent experiments (data not shown). Of note, a small fraction (10 to 20%) of the FLAG and FLAG-N407K viruses remained infectious despite incubation with high concentrations of the M2 antibody. As further increases in M2 antibody concentration led to less specific virus inhibition, as evidenced by some effects on the viruses with wild-type envelope glycoproteins, we could not assess whether this residual fraction of infectious virus could be neutralized at higher doses of M2 antibody. We conclude that HIV-1 bearing an extraneous epitope in the gp120 V4 region can be neutralized by an antibody that recognizes that foreign epitope.
Effects of M2 antibody binding on gp120-receptor interactions. The V4 region is spatially distant from the gp120 regions involved in CD4 and CCR5/CXCR4 binding (38, 55). Thus, the binding of the M2 antibody is not expected to interfere with gp120-receptor interaction. To examine this, we studied the precipitation of radiolabeled gp120 from the HIV-1YU2 strain by CD4-Ig. The precipitation of the wt, FLAG, and FLAG-N407K gp120 envelope glycoproteins by CD4-Ig was equivalent (Fig. 4). This result suggests that the three gp120 glycoproteins bind CD4 comparably. The gp120 supernatants were concentrated about 10-fold using CentriprepYM-30. Then, 30-μl portions of the concentrated gp120s were precipitated with pooled sera from HIV-1-infected individuals, indicating that similar levels of gp120 were present in the three gp120 preparations (Fig. 4, middle panel). Next, 150 μl of the concentrated gp120 solutions were preincubated with 100 μg/ml of the M2 antibody prior to incubation with soluble CD4 and target cells expressing CCR5. Preincubation with 100 μg/ml M2 had no detectable effect on the binding of the wt, FLAG, or FLAG-N407K gp120 glycoproteins to CCR5 on the surfaces of Cf2Th-CCR5 cells (Fig. 4), a process that is dependent upon initial binding of gp120 to CD4 (50, 54). We conclude that both CD4 binding and CCR5 binding by gp120 are not impeded by the M2 antibody to the V4 FLAG sequence.
DISCUSSION
The HIV-1 gp120 variable regions exhibit considerable diversity among naturally occurring viral strains. The V4 region in particular tolerates substantial changes in length, amino acid composition, and glycosylation without compromising HIV-1 envelope glycoprotein function (21, 36, 66). This is consistent with its exposed status on the gp120 outer domain, far removed from envelope glycoprotein regions known to be important for virus entry (4, 37, 38). The V4 surface loop connects the 18 and 19 strands of the outer domain; in various HIV-1 strains, this connection is accomplished by as few as 2 or as many as 19 amino acid residues (36). Although most HIV-1 strains have glycosylated V4 regions, some do not (36). The impressive structural heterogeneity of the V4 region (36-38) almost precludes a specific contribution of this element to HIV-1 entry. Indeed, reminiscent of results seen in previous mutagenic studies (35), alteration of the V4 region in the envelope glycoproteins from two very different strains, HIV-1YU2 and HIV-1HXBc2, exerted little effect on the ability to mediate virus entry. Regions of hypervariability in the envelope glycoproteins of other viruses, such as those of Friend murine leukemia virus and vesicular stomatitis virus, have likewise been shown to accept large inserts without compromising virus replication (27, 57). Thus, natural variation in a viral envelope glycoprotein can provide guidance in identifying surface-exposed elements that can be manipulated for a variety of purposes.
The minimal apparent contribution of the V4 region to HIV-1 envelope glycoprotein function, its expected exposure on the assembled envelope glycoprotein trimer, and its hypervariability in naturally occurring HIV-1 strains all suggest that V4 contributes to immune evasion. Indeed, it has been shown that changes in V4 glycosylation can affect the sensitivity of an HIV-1 isolate to strain-specific neutralizing antibodies present in the serum of an acutely infected individual (66). The V4 variant virus also exhibited altered susceptibility to neutralization by a monoclonal antibody directed against the V3 variable loop (66). The ability of changes in V4 glycosylation to influence neutralization by an antibody that recognizes a distant gp120 structure suggested the concept of a "glycan shield." In this model, a densely packed array of carbohydrate structures on gp120 creates a steric impediment to the binding of antibodies to more distantly situated gp120 structures. One prediction of this model is that loss of glycosylation sites in V4 might result in an increased sensitivity to neutralization by antibodies directed against distant gp120 or gp41 elements. In this study, alteration of the V4 glycosylation site had no detectable effect on the sensitivities of viruses with the HIV-1HXBc2 or HIV-1YU2 envelope glycoproteins to neutralization by a variety of anti-HIV-1 monoclonal antibodies. Moreover, in the context of both HIV-1HXBc2 and HIV-1YU2 envelope glycoproteins, addition or removal of a glycan at amino acid 362, which is adjacent to the V4 region on native gp120, alone or in combination with the V4 carbohydrate alteration did not alter HIV-1 neutralization sensitivity (data not shown). Thus, simple removal of some gp120 outer domain glycans is not sufficient to increase the general susceptibility of HIV-1 to antibody neutralization.
Whether antibody binding to HIV-1 envelope glycoprotein spikes can occur without neutralizing spike function has been the subject of much discussion and has not been resolved yet (6, 24, 49, 53). The M2 antibody neutralized viruses with the two HIV-1 envelope glycoproteins carrying the FLAG epitope in the V4 region. The tolerance of functional HIV-1 envelope glycoproteins to change in the V4 region indicates that this region per se does not play a significant role in the virus entry process. Thus, the neutralizing activity of the M2 antibody is unlikely to result from a disruption of a function intrinsic to the V4 region of gp120. Furthermore, the M2 antibody exhibited no detrimental effect on gp120-CCR5 binding, which is dependent upon prior interaction with CD4 and CD4-induced conformational changes in gp120 (63, 69). Thus, there is no evidence of an indirect effect of M2 antibody binding on the known functions of gp120. Taken together, virus neutralization in this case may result simply from the binding and continued presence of the M2 antibody during the attempted entry process. Mechanistically, steric hindrance may account for the reduced efficiency with which virus attachment and/or membrane fusion events occur in the presence of the bound antibody, given the considerable size of an antibody molecule in comparison to that of the HIV-1 envelope glycoprotein trimer (28, 29, 40). Our results provide further support for the notion that all antibodies that bind with high affinity to the functional HIV-1 envelope glycoprotein spike should, at a minimum, exhibit some degree of neutralizing activity simply as a consequence of this steric hindrance, irrespective of epitope specificity (51).
Neutralization by antibodies that bind functionally unimportant regions of the envelope glycoproteins will probably apply to viruses other than HIV-1. Recently, Schlehuber and Rose introduced an HIV-1 gp41 peptide epitope into a naturally variable site on the vesicular stomatitis virus G glycoprotein (57). The resulting virus was replication competent and was neutralized by a monoclonal antibody directed against the inserted peptide.
A small fraction of the HIV-1 FLAG preparations was not neutralized even by high concentrations of the M2 antibody. The reason for this is not clear. The potency of neutralization by steric hindrance is expected to be largely determined by the efficiency of antibody binding to the epitope in the context of the virion spike (22). Heterogeneity in the virions due to factors, such as differential glycosylation or the formation of virus aggregates, could limit the accessibility of the M2 antibody to a subset of viruses in the stock. Alternatively, if the binding of the M2 antibody to the envelope glycoprotein trimer were reversible, a fraction of viruses may have an opportunity to proceed down the entry pathway before rebinding of the antibody occurs. The latter scenario allows the possibility that greater potency of neutralization results not only from an increased affinity of trimer binding but also from the induction of irreversible inhibitory consequences in the envelope glycoproteins. Such a mechanism may explain the exquisite sensitivity of CD4-independent HIV-1 to neutralization by a wide range of antibodies (15, 32).
The results in this report indicate that the gp120 V4 region is a receptive site for introducing exogenous peptide tags without affecting the intrinsic function and neutralization sensitivity of the HIV-1 glycoproteins. This establishes an experimental system to study antibody-mediated neutralization in a controlled environment, in which the immunoepitopes can be manipulated without interfering with normal structure and function of the carrier envelope glycoproteins. Such studies may provide further insights into mechanisms of HIV-1 neutralization by antibodies and other molecules.
ACKNOWLEDGMENTS
We thank Yvette McLaughlin and Sheri Farnum for manuscript preparation.
This work was supported in part by NIH grants (AI24755, AI31783, and AI39420), by a Center for AIDS research grant (AI42848), by an unrestricted research grant from the Bristol-Myers Squibb Foundation, by a gift from the late William F. McCarty-Cooper, and by funds from the International AIDS Vaccine Initiative and the Pendleton Trust.
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