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Originally published In Press as doi:10.1074/jbc.M602732200 on July 18, 2006

J. Biol. Chem., Vol. 281, Issue 39, 28529-28535, September 29, 2006
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A Tyrosine-sulfated Peptide Derived from the Heavy-chain CDR3 Region of an HIV-1-neutralizing Antibody Binds gp120 and Inhibits HIV-1 Infection*

Tatyana Dorfman{ddagger}, Michael J. Moore{ddagger}, Alexander C. Guth{ddagger}, Hyeryun Choe§, and Michael Farzan{ddagger}1

From the {ddagger}Department of Microbiology and Molecular Genetics, Harvard Medical School, New England Primate Research Center, Southborough, Massachusetts 01772 and the §Pulmonary Division, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Received for publication, March 23, 2006 , and in revised form, July 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 entry. Human antibodies that recognize the CCR5-binding region of gp120 are also modified by tyrosine sulfation, which is necessary for their ability to neutralize HIV-1. Here we demonstrate that a sulfated peptide derived from the CDR3 region of one of these antibodies, E51, can efficiently bind gp120. Association of this peptide, pE51, with gp120 requires tyrosine sulfation and is enhanced by, but not dependent on, CD4. Alteration of any of four pE51 tyrosines, or alteration of gp120 residues 420, 421, or 422, critical for association with CCR5, prevents gp120 association with pE51. pE51 neutralizes HIV-1 more effectively than peptides based on the CCR5 amino terminus and may be useful as a fusion partner with other protein inhibitors of HIV-1 entry. Our data provide further insight into the association of the CCR5 amino terminus with gp120, show that a conserved, sulfate-binding region of gp120 is accessible to inhibitors in the absence of CD4, and suggest that soluble mimetics of CCR5 can be more effective than previously appreciated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The entry of HIV-12 into target cells requires expression of the cellular receptor CD4 and a coreceptor, principally the chemokine receptors CCR5 or CXCR4 (1–7). Virus association with the primary receptor, CD4, triggers conformational changes in the envelope glycoprotein, gp120, that allow high affinity binding to the coreceptor (8, 9). HIV-1 variants that utilize CCR5 as a coreceptor, R5 isolates, mediate transmission and predominate throughout most of the asymptomatic phase of infection. In many cases, coincident with the decline in immune function, variants emerge that can utilize a range of coreceptors in addition to CCR5, in particular CXCR4 (10, 11). Some of these variants (R5X4 isolates) are dual tropic and utilize both principal coreceptors. Other variants (X4 isolates), including those that have been extensively passaged on tissue-culture cell lines, lose the ability to use CCR5 and enter cells primarily through CXCR4 (7, 12).

The ectodomains of CCR5 and CXCR4 consist of an amino terminus and three extracellular loops held in position by seven transmembrane helices (13). Entry of R5 HIV-1 isolates depends largely on the amino terminus and second extracellular loop of CCR5 (14–17). An acidic, tyrosine-rich sequence in the CCR5 amino terminus (residues 10–18) is especially important for virus entry (18–22). The tyrosines of this region are post-translationally modified by the addition of sulfate (23), and all R5 isolates examined to date are sensitive to the loss of one or more of these sulfates. Sulfated peptides corresponding in sequence to the CCR5 amino terminus can slow infection of R5 isolates (24, 25) and also can reconstitute the ability of a CCR5 variant lacking this region to support infection (26). In contrast, most X4 isolates are substantially less dependent on the amino terminus of CXCR4 or on the tyrosine sulfate moieties present there (27–29).

The HIV-1 envelope glycoprotein is composed of a surface (gp120) and a transmembrane (gp41) protein, processed from a single precursor (gp160) (30). Several groups of antibodies neutralize HIV-1 infection by binding gp120 or gp41 (31–34). These include antibodies whose association with gp120 is enhanced by CD4 (35). The epitopes of these CD4-induced (CD4i) antibodies overlap the coreceptor-binding region of gp120 (8, 9). The prototypes of these antibodies, 17b and 48d, are only weakly neutralizing (35, 36) and neutralize T-cell line-adapted X4 isolates more potently than primary R5 isolates (31, 35). However, the heavy-chain complementarity-determining region 3 (CDR3) of a subset of CD4i antibodies are tyrosine-rich and tyrosine-sulfated. Like the CCR5 amino terminus, sulfation of these CDR3 regions is critical for the ability of these antibodies to bind gp120 and neutralize infection. Unlike most anti-gp120-neutralizing antibodies, these tyrosine-sulfated antibodies preferentially neutralize R5 rather than X4 isolates.

Here we demonstrate that the CDR3 region of one such antibody, expressed as peptide, is capable of binding and precipitating HIV-1 gp120 and of inhibiting HIV-1 infection more efficiently than peptides based on the amino terminus of CCR5. This antibody-derived peptide is sensitive to alterations in gp120 that disrupt association with CCR5. Association of this peptide with gp120 is sulfate-dependent and enhanced by, but not dependent on, the presence of CD4. Our data provide further structural insight into the association of gp120 with the CCR5 amino terminus and raise the possibility that the tyrosine sulfate-binding domain of HIV-1 gp120 can be a useful target of anti-viral therapies.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids and Cells—The CDR3 regions of the sulfated CD4i antibodies 412d, C12, 47e, E51, the unsulfated CD4i antibody 17b, and amino-terminal domains of CCR5 were generated by overlapping PCR and ligated between the NheI and BamHI restriction sites of a previously described pcDM8-derived plasmid expressing the signal sequence of CD5 and the Fc domain of human IgG1 (23). These plasmids express proteins described in the text as p412d-Ig, p17b-Ig, pC12-Ig, p47e-Ig, pE51-Ig, and pR5-Ig. Plasmids expressing {alpha}-defensin 1 or the first two immunoglobulin domains of human CD4 (defensin-Ig and CD4-Ig, respectively), fused to the Fc domain of human IgG1 were generated by PCR amplication and ligated into the same pcDM8-derived plasmid. Plasmids encoding tyrosylprotein sulfotransferase 2 (TPST-2) and single-chain variants of the 412d and E51 antibodies have been described previously (37). Plasmids encoding gp120 envelope glycoproteins of the clade B HIV-1 isolates YU2, JR-FL, ADA, and HXB2 have been described previously (3). p{Delta}E51-Ig and its variants were generated using the QuikChange method (Stratagene). All plasmids generated were fully sequenced in their coding regions. Plasmid encoding the NL4-3 HIV-1 genome in which the NL4-3 env gene has been replaced by that of the R5X4 isolate 89.6 and the nef gene replaced by a gene encoding green fluorescent protein (GFP) has been previously described (18, 38). Human embryonic kidney 293T cells and the human T cell line PM1, which expresses CCR5, were obtained from the American Type Culture Collection.

Immunoprecipitation of gp120—293T cells transfected with plasmids expressing peptide-Ig variants and, unless noted, a plasmid expressing TPST-2, were metabolically labeled with [35S]methionine and -cysteine. Labeled supernatants were harvested 2 days post-transfection. In parallel, 293T cells were transfected with plasmids expressing the gp120 molecules of the HIV-1 isolates ADA, JR-FL, YU2, and HXB2 and metabolically labeled in the same manner. Protein was quantified by SDS-PAGE and diluted to equivalent concentrations. 500 µlof gp120-containing supernatant was then incubated for 30 min at room temperature with 300 µl of diluted supernatants containing peptide-Ig protein. Protein A-Sepharose (Amersham Biosciences) was added to the mixture, which was subsequently incubated overnight at 4 °C. Protein A-Sepharose was washed twice with PBS and 1% CHAPSO (Anatrace) and once with PBS and analyzed by SDS-PAGE. In some experiments (Fig. 1B, for example), CD4-Ig was conjugated to CNBr-activated Sepharose beads (Amersham Biosciences) according to the manufacturer's instructions, incubated with supernatants containing radiolabeled gp120 and supernatants containing radiolabeled pE51-Ig, p412d-Ig, p17b-Ig, or radiolabeled single-chain variants of the E51 or 412d antibodies (E51 scFv, 413 scFv (37)).

Purification of Peptide-Ig Proteins—293T cells were transfected with plasmids encoding p17b-Ig, CD4-Ig, nR5-Ig, p{Delta}E51-Ig, or variants thereof, and, except if noted otherwise, cotransfected with a plasmid encoding TPST-2. One day post-transfection, cells were washed and subsequently incubated in 293 SFM II medium (Invitrogen). Medium was harvested after 48 h and proteins precipitated with protein A-Sepharose at 4 °C for 16 h. Beads were washed in PBS with 0.5 M NaCl, eluted with 50 mM sodium citrate/50 mM glycine (pH 2), and adjusted with NaOH to pH 7.4. Purified proteins were concentrated with Centricon filters (Amicon) and dialyzed in PBS. Protein amounts were quantified by comparison to bovine serum albumin standards in a Coomassie-stained SDS-PAGE gel.

Infection Assay—293T cells were transfected with a plasmid encoding the genome of the NL4-3 HIV-1 isolate, modified to express the R5X4 envelope glycoprotein of the 89.6 isolate and GFP, or with an NL4-3 variant with a deletion in the env gene and cotransfected with the envelope glyocproteins of the ADA, YU2, NL4-3, JR-FL, SG3, or HXB2 isolates, or with the G protein of the vesicular stomatitis virus (18, 38). Supernatants of transfected cells were harvested, and reverse transcriptase activity was measured, as described previously (3). These supernatants, diluted to 20,000 reverse transcriptase counts per ml, were added to PM1, GHOST-CXCR4, or GHOST-CCR5 cells in the presence of the indicated concentrations of p17b-Ig, nCCR5-Ig, p{Delta}E51-Ig, and CD4-Ig. Media were changed the following day and GFP expression in infected cells was measured 2 days later by flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The amino terminus of CCR5 includes several sulfated tyrosines that are critical to its role as an HIV-1 coreceptor (23). Tyrosine-sulfated peptides based on sequences from the CCR5 amino terminus bind HIV-1 gp120 and inhibit HIV-1 infection (24, 25, 39). However, although specific, these peptides inhibit infection at concentrations of 50 to 100 µM (24, 25). A subset of CD4-inducible antibodies also utilize tyrosine sulfate to recognize the CCR5-binding region of the HIV-1 envelope glycoprotein (37). We therefore sought to determine whether peptides deriving from the sulfated heavy-chain CDR3 regions of these antibodies bound gp120 and slowed infection more efficiently than peptides based on CCR5 sequences.

A series of plasmids was generated that express the heavychain CDR3 regions of the CD4-inducible antibodies 412d, 17b, C12, 47e, and E51 fused to the Fc region of human IgG1 (p412d-Ig, p17b-Ig, pC12-Ig p47e-Ig, and pE51-Ig; see Table 1). These plasmids, as well as one expressing the amino terminus of CCR5 also fused to the same Fc region (pR5-Ig), were cotransfected into 293T cells with a plasmid expressing human TPST-2. We have previously shown (37) that expression of exogenous TPST-2 enhances the sulfation of overexpressed single-chain antibody (scFv; all subsequent scFv and Fc-fusion proteins were generated in the presence of exogenous TPST-2 unless otherwise indicated). Transfected cells were metabolically labeled with [35S]methionine and -cysteine. Cell supernatants, or sera from HIV-1-infected patients, were incubated with metabolically labeled gp120 (derived from the ADA isolate) and protein A-Sepharose, immunoprecipitated, and analyzed by SDS-PAGE. Of the CDR3-Ig fusion proteins, only pE51-Ig detectably precipitated gp120 (Fig. 1A). Of note, pR5-Ig did not immunoprecipitate gp120, consistent with the relatively low affinity of sulfated CCR5-derived peptides for the HIV-1 envelope glycoprotein.


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  		TABLE 1
Peptide sequences characterized in this study

The amino acid sequences of peptides characterized as fusion proteins with the Fc domain of human IgG1. Cysteine 20 of CCR5 has been converted to a serine, to prevent disulfide bond formation and for consistency with previous studies (23,26,41). Tyrosines are shown in bold. Except for the 17b CDR3, all sequences shown are tyrosine sulfated in their native contexts (23,37,42).

 


Figure 1
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  		FIGURE 1.
The CDR3 region of antibody E51 precipitates HIV-1 gp120. A, radiolabeled HIV-1 gp120 (ADA isolate) was incubated with radiolabeled supernatants from 293T cells transfected with plasmid expressing the indicated peptide-Ig fusion proteins or with patient sera. Mixtures were precipitated with protein A-Sepharose and analyzed by SDS-PAGE. Numbers to the left of the panel indicate molecular mass in kilodaltons (kDa). Bands that migrate with the 67-kDa marker indicate residual dimeric forms of the peptide-Ig proteins. B, Sepharose beads conjugated to CD4-Ig were incubated in the presence and absence of radiolabeled gp120 (indicated) and radiolabeled supernatants of 293T cells transfected with vector alone or with plasmid expressing the indicated peptide-Ig fusion protein or single-chain antibody (scFv). C, an experiment similar to A except that the indicated pE51-Ig variants and CD4-ig were characterized.

 
To confirm a specific association between pE51-Ig and gp120, we used an alternative strategy in which CD4-bound gp120 was used to precipitate various CDR3-Ig and single-chain antibody proteins (Fig. 1B). Radiolabeled pE51-Ig, p412d-Ig, p17b-Ig, and single-chain variants of the sulfated antibodies E51 and 412d (E51 scFV, 412d scFv) were incubated with Sepharose beads conjugated to CD4-Ig and radiolabeled gp120. CD4-bound gp120 precipitated both single-chain antibodies. However, CD4-bound gp120 precipitated only pE51-Ig but not p412d-Ig or p17b-Ig. Together with the data of Fig. 1A, these data demonstrate a specific association between gp120 and pE51-Ig.


Figure 2
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  		FIGURE 2.
pE51-Ig and p{Delta}E51-Ig bind gp120 of R5 and X4 isolates. An experiment similar to those shown in Fig. 1, A and C, except that the indicated radiolabeled pE51-Ig variants were incubated with radiolabeled gp120 of the indicated HIV-1 isolates in the presence and absence of soluble CD4, as indicated.

 
We also assayed two variants of pE51-Ig for their ability to bind HIV-1 gp120. In one, p{Delta}E51-Ig, the first nine uncharged residues of pE51-Ig were deleted. In a second, the final three (of five) tyrosines were altered to glutamic acid. This pE51-Ig variant is described as YYEEE-Ig to indicate the amino acids present at the five tyrosines of unmodified pE51-Ig (see Table 2 for pE51-Ig variant names and peptide sequences). As shown in Fig. 1C, p{Delta}E51-Ig immunoprecipitated gp120 with an efficiency comparable with pE51-Ig, whereas YYEEE-Ig could not detectably bind gp120.


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  		TABLE 2
Characterized pE51-Ig variants

Sequences of pE51 variants, fused to the Fc domain of human IgG1, are shown, as is the name of each peptide. Pluses indicate the efficiency of gp120 immunoprecipitation by each variant. Bold indicates differences from pE51 sequence.

 
We next assayed the ability of pE51-Ig, p{Delta}E51-Ig, YYEEE-Ig, and patient sera to immunoprecipitate the gp120 proteins of clade B isolates YU2, JR-FL, ADA, and HXBc2 (Fig. 2). YU2, JR-FL, and ADA envelope glycoproteins utilize CCR5 to infect cells, whereas HXBc2 utilizes the coreceptor CXCR4. YU2 gp120 bound pE51-Ig and p{Delta}E51-Ig as efficiently as ADA gp120, whereas that of JR-FL and HXBc2 bound pE51-Ig and p{Delta}E51-Ig detectably, but less efficiently. In each case the addition of soluble CD4 enhanced association. YYEEE-Ig did not precipitate the gp120 of any isolate assayed. These data indicate some variation in affinity of the gp120 of clade B isolates for pE51-Ig and p{Delta}E51-Ig and suggest that, like the E51 antibody, these fusion proteins bind a CD4-inducible epitope on gp120.


Figure 3
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  		FIGURE 3.
Tyrosines contribute to p{Delta}E51-Ig association with gp120. Experiments similar to those in Fig. 1, A and C, except that variants in which the indicated individual tyrosines were altered to phenylalanine (A) or aspartic acid (B). p{Delta}E51-Ig expressed in the absence of exogenous TPST-2 is also characterized in B as indicated.

 
We further defined the determinants on p{Delta}E51-Ig essential for association with gp120 (Fig. 3). We characterized a series of p{Delta}E51-Ig variants, in which each of the five tyrosines were individually altered to phenylalanine or to aspartic acid. Alteration of any of the five tyrosines to phenylalanine abolished detectable gp120 association. Alteration of the first four tyrosines to aspartic acid similarly abolished gp120 binding, whereas alteration of the fifth tyrosine diminished, but did not abolish, gp120 association. In this experiment we also assayed the contribution of p{Delta}E51-Ig sulfation to its association with gp120 by expressing p{Delta}E51-Ig in the absence of exogenous TPST-2. We have previously shown that protein overexpressed in 293T cells is not efficiently sulfated without the coexpression of exogenous TPST-2 (37). p{Delta}E51-Ig expressed in the absence of exogenous TPST-2 could not detectably precipitate gp120 (Fig. 3). These data suggest a critical role for tyrosines, and for sulfate groups on these tyrosines, in the ability of p{Delta}E51-Ig to immunoprecipate gp120.


Figure 4
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  		FIGURE 4.
p{Delta}E51-Ig association with gp120 depends on conserved CCR5-binding residues. A, radiolabeled ADA gp120, and variants in which isoleucine 420, lysine 421, or glutamine 422 are altered to alanine, were immunoprecipitated with patient sera or with radiolabeled CD4-Ig and analyzed by SDS-PAGE. B, the same gp120 variants were incubated with radiolabeled p{Delta}E51-Ig expressed in the presence or absence of exogenous TPST-2, as indicated.

 
To identify the region of gp120 bound by pE51-Ig, gp120 variants were generated in which each of three gp120 residues (residues 420, 421, and 422), previously demonstrated to be critical for association of gp120 to CCR5, were altered to alanine (40). As described, each of these variants bound CD4-Ig and was recognized by patient sera with comparable efficiency (Fig. 4A). However, none of these gp120 variants detectably bound p{Delta}E51-Ig (Fig. 4B). These data suggest that p{Delta}E51-Ig specifically binds gp120 in a region critical for its association with CCR5. If so, they also localize the tyrosine sulfate-binding region of gp120 to residues 420 through 422.


Figure 5
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  		FIGURE 5.
p{Delta}E51-Ig neutralizes HIV-1 infection more efficiently than a CCR5-derived peptide-Ig variant. A, PM1 cells were incubated with an HIV-1 variant expressing the envelope glycoprotein of the R5X4 89.6 isolate and GFP in the presence of the indicated concentrations of purified peptide-Ig variants. Viral entry, as measured by flow cytometry, was determined 2 days post-infection. B, HIV-1 viruses pseudotyped with the envelope glycoproteins of ADA, JR-FL, or HXB2 HIV-1 isolates were incubated with GHOST-CCR5 (ADA, JR-FL) or GHOST-CXCR4 (HXB2) cells in the presence of the indicated concentrations of the purified peptide-Ig variants. Viral entry, measured by flow cytometry, was determined 2 days post infection. C, an experiment similar to that in B except that viruses were pseudotyped with the envelope glyoproteins of the indicated HIV-1 isolates or with the vesicular stomatitis virus G protein (VSV-G). Viruses pseudotyped with the YU2, ADA, and JR-FL envelope glycoproteins were incubated with GHOST-CCR5 cells in the presence of a 3 µM concentration of the indicated peptide-Ig variants. All other pseudotyped viruses were incubated with GHOST-CXCR4 cells, again with a 3 µM concentration of the indicated peptide-Ig variants.

 
Sulfated peptides based on the amino terminus of CCR5 specifically block HIV-1 infection, but only at 50–100 µM concentrations (24, 41). The ability of p{Delta}E51-Ig, but not pR5-Ig, to precipitate gp120 in the presence and absence of CD4 suggested that p{Delta}E51-Ig may be more effective at inhibiting HIV-1 infection. Accordingly, CD4-Ig, p{Delta}E51-Ig, pR5-Ig, and p17b-Ig were compared for their ability to inhibit infection of an HIV-1 variant expressing GFP and the envelope glycoprotein of the R5X4 isolate 89.6 (Fig. 5A). Virus was incubated with the CD4-positive T-cell line PM1 and the indicated fusion proteins for 1 h and then washed. As we and others have previously reported for sulfated CCR5-derived peptides (24, 41), pR5-Ig partially inhibited infection at a concentration of ~40 µM, whereas p17b-Ig had no detectable effect on HIV-1 entry. Consistent with its ability to bind gp120, p{Delta}E51-Ig inhibited infection markedly more efficiently than did pR5-Ig, with 50% inhibition observed at ~2 µM. Expectedly, CD4-Ig blocked infection in the nanomolar range. Similar results were obtained using a broader range of envelope glycoproteins of clade B isolates (Fig. 5, B and C). Infection mediated by the envelope glycoproteins of the R5 isolates ADA, YU2, JR-FL, and of the X4 isolates HXB2 and NL4-3 was inhibited by p{Delta}E51-Ig, comparably that of the 89.6 isolate, and for all isolates p{Delta}E51 inhibited entry more efficiently than pR5-Ig. However, the clade C isolate SG3 was only modestly inhibited by p{Delta}E51. In addition, HIV-1 pseudotyped with the vesicular stomatitis virus G protein was not substantially inhibited by p{Delta}E51 (Fig. 5C). These data indicate that p{Delta}E51-Ig neutralizes a broad range of HIV-1 isolates more efficiently than does pR5-Ig.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we demonstrate that a peptide based on the tyrosine-sulfated CDR3 region of the antibody E51, fused to the Fc region of human IgG1, could immunoprecipitate gp120 of several clade B HIV-1 isolates. In contrast, an analogous fusion protein based on the tyrosine-sulfated amino terminus of CCR5 could not detectable precipitate gp120. This latter construct inhibited HIV-1 infection at approximately the same high concentration required for synthetic sulfated peptides, whereas the IC50 (50% inhibitory concentration) of p{Delta}E51-Ig was at least 10-fold lower. p{Delta}E51-Ig has several notable properties. First, although its binding to gp120 appears to be enhanced by the presence of CD4, its association did not require CD4, suggesting that a tyrosine sulfate-binding pocket on gp120 is exposed in the absence of CD4. Second, most of its five tyrosines contribute to gp120 association. Third, as is the case with CCR5 and the E51 antibody, association of p{Delta}E51-Ig with gp120 depends on sulfation of one or more tyrosines. Fourth, binding of p{Delta}E51-Ig was abolished by gp120 alterations that most potently interfered with CCR5 association, localizing the interaction of this peptide, and presumably the sulfated region of CCR5, to this conserved region of gp120.

The conservation of a tyrosine sulfate-binding pocket, and its exposure in the absence of CD4, suggest that this region of gp120 may be a useful therapeutic target. The present study shows that it is possible to enhance the antiviral activities of peptides that are similar to the CCR5 amino terminus. The basis of this enhancement is unclear, but replacement of hydrophobic residues, which may point into the body of CCR5 (Table 3), with glycines likely increases both the solubility and flexibility of the peptide. The elimination of additional residues separating tyrosine 3 and tyrosine 10 of CCR5 may permit binding of an additional sulfated tyrosine to gp120 without the entropic penalty of folding the small intervening loop.


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  		TABLE 3
Alignment of p{Delta}E51 and the CCR5 amino terminus

An alignment of residues suggesting that the basis of the activity of p{Delta}E51 may be its homology with CCR5 in residues that contact gp120 (18,19). Higher affinity of p{Delta}E51 for gp120 may be due to the absence of hydrophobic residues present in the CCR5 amino terminus that presumably interact with CCR5 transmembrane regions. Homologous residues are shown in bold.

 
Although more efficient than CCR5-based peptides in inhibiting HIV-1 infection, p{Delta}E51-Ig is still not sufficiently potent for therapeutic use. These studies nonetheless suggest that the sulfate-binding pocket, which is conserved both in R5 and X4 isolates, may be an important therapeutic target. Peptides like p{Delta}E51 may also be useful when fused to soluble CD4 or other protein inhibitors of HIV-1 entry. Such fusion proteins would likely exhibit higher affinity than their unmodified counterparts and may limit viral escape. Finally, these studies suggest that work identifying additional sulfated antibodies may yield more potent entry inhibitors and provide additional insight into the CCR5 determinants necessary for its association with gp120.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Microbiology and Molecular Genetics, Harvard Medical School, New England Primate Research Center, 1 Pine Hill Dr., Southborough, MA 01772-9102. Tel.: 508-624-8109; Fax: 508-786-3317; E-mail: farzan{at}hms.harvard.edu.

2 The abbreviations used are: HIV-1, human immunodeficiency virus type 1; CCR5, CC-chemokine receptor 5; CXCR4, CXC-chemokine receptor 4; CDR3, complementarity-determining region 3; CD4i, CD4 inducible; TPST, tyrosylprotein sulfotransferase; scFv, single-chain antibody fragment; R5, CCR5-using; X4, CXCR4-using; R5X4, both CCR5 and CXCR4 using; GFP, green fluorescent protein; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonic acid; PBS, phosphate-buffered saline. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Dragic, T., Litwin, V., Allaway, G. P., Martin, S. R., Huang, Y., Nagashima, K. A., Cayanan, C., Maddon, P. J., Koup, R. A., Moore, J. P., and Paxton, W. A. (1996) Nature 381, 667–673[CrossRef][Medline] [Order article via Infotrieve]
  2. Doranz, B. J., Rucker, J., Yi, Y., Smyth, R. J., Samson, M., Peiper, S. C., Parmentier, M., Collman, R. G., and Doms, R. W. (1996) Cell 85, 1149–1158[CrossRef][Medline] [Order article via Infotrieve]
  3. Choe, H., Farzan, M., Sun, Y., Sullivan, N., Rollins, B., Ponath, P. D., Wu, L., Mackay, C. R., LaRosa, G., Newman, W., Gerard, N., Gerard, C., and Sodroski, J. (1996) Cell 85, 1135–1148[CrossRef][Medline] [Order article via Infotrieve]
  4. Deng, H., Liu, R., Ellmeier, W., Choe, S., Unutmaz, D., Burkhart, M., Di Marzio, P., Marmon, S., Sutton, R. E., Hill, C. M., Davis, C. B., Peiper, S. C., Schall, T. J., Littman, D. R., and Landau, N. R. (1996) Nature 381, 661–666[CrossRef][Medline] [Order article via Infotrieve]
  5. Alkhatib, G., Combadiere, C., Broder, C. C., Feng, Y., Kennedy, P. E., Murphy, P. M., and Berger, E. A. (1996) Science 272, 1955–1958[Abstract]
  6. Dalgleish, A. G., Beverley, P. C., Clapham, P. R., Crawford, D. H., Greaves, M. F., and Weiss, R. A. (1984) Nature 312, 763–767[CrossRef][Medline] [Order article via Infotrieve]
  7. Feng, Y., Broder, C. C., Kennedy, P. E., and Berger, E. A. (1996) Science 272, 872–877[Abstract]
  8. Trkola, A., Dragic, T., Arthos, J., Binley, J. M., Olson, W. C., Allaway, G. P., Cheng-Mayer, C., Robinson, J., Maddon, P. J., and Moore, J. P. (1996) Nature 384, 184–187[CrossRef][Medline] [Order article via Infotrieve]
  9. Wu, L., Gerard, N. P., Wyatt, R., Choe, H., Paralin, C., Ruffing, N., Borsetti, A., Cardosa, A. A., Desjardin, E., Newman, W., Gerard, C., and Sodroski, J. (1996) Nature 184, 179–183[CrossRef]
  10. Connor, R. I., Sheridan, K. E., Ceradini, D., Choe, S., and Landau, N. R. (1997) J. Exp. Med. 185, 621–628[Abstract/Free Full Text]
  11. Bleul, C. C., Wu, L., Hoxie, J. A., Springer, T. A., and Mackay, C. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1925–1930[Abstract/Free Full Text]
  12. Berger, E. A., Doms, R. W., Fenyo, E. M., Korber, B. T., Littman, D. R., Moore, J. P., Sattentau, Q. J., Schuitemaker, H., Sodroski, J., and Weiss, R. A. (1998) Nature 391, 240[CrossRef][Medline] [Order article via Infotrieve]
  13. Berger, E. A., Murphy, P. M., and Farber, J. M. (1999) Annu. Rev. Immunol. 17, 657–700[CrossRef][Medline] [Order article via Infotrieve]
  14. Rucker, J., Samson, M., Doranz, B. J., Libert, F., Berson, J. F., Yi, Y., Smyth, R. J., Collman, R. G., Broder, C. C., Vassart, G., Doms, R. W., and Parmentier, M. (1996) Cell 87, 437–446[CrossRef][Medline] [Order article via Infotrieve]
  15. Atchison, R. E., Gosling, J., Monteclaro, F. S., Franci, C., Digilio, L., Charo, I. F., and Goldsmith, M. A. (1996) Science 274, 1924–1926[Abstract/Free Full Text]
  16. Doranz, B. J., Lu, Z. H., Rucker, J., Zhang, T. Y., Sharron, M., Cen, Y. H., Wang, Z. X., Guo, H. H., Du, J. G., Accavitti, M. A., Doms, R. W., and Peiper, S. C. (1997) J. Virol. 71, 6305–6314[Abstract]
  17. Farzan, M., Choe, H., Martin, K. A., Sun, Y., Sidelko, M., Mackay, C. R., Gerard, N. P., Sodroski, J., and Gerard, C. (1997) J. Biol. Chem. 272, 6854–6857[Abstract/Free Full Text]
  18. Farzan, M., Choe, H., Vaca, L., Martin, K., Sun, Y., Desjardins, E., Ruffing, N., Wu, L., Wyatt, R., Gerard, N., Gerard, C., and Sodroski, J. (1998) J. Virol. 72, 1160–1164[Abstract/Free Full Text]
  19. Dragic, T., Trkola, A., Lin, S. W., Nagashima, K. A., Kajumo, F., Zhao, L., Olson, W. C., Wu, L., Mackay, C. R., Allaway, G. P., Sakmar, T. P., Moore, J. P., and Maddon, P. J. (1998) J. Virol. 72, 279–285[Abstract/Free Full Text]
  20. Blanpain, C., Doranz, B. J., Vakili, J., Rucker, J., Govaerts, C., Baik, S. S., Lorthioir, O., Migeotte, I., Libert, F., Baleux, F., Vassart, G., Doms, R. W., and Parmentier, M. (1999) J. Biol. Chem. 274, 34719–34727[Abstract/Free Full Text]
  21. Ross, T. M., Bieniasz, P. D., and Cullen, B. R. (1998) J. Virol. 72, 1918–1924[Abstract/Free Full Text]
  22. Rabut, G. E., Konner, J. A., Kajumo, F., Moore, J. P., and Dragic, T. (1998) J. Virol. 72, 3464–3468[Abstract/Free Full Text]
  23. Farzan, M., Mirzabekov, T., Kolchinsky, P., Wyatt, R., Cayabyab, M., Gerard, N. P., Gerard, C., Sodroski, J., and Choe, H. (1999) Cell 96, 667–676[CrossRef][Medline] [Order article via Infotrieve]
  24. Cormier, E. G., Persuh, M., Thompson, D. A., Lin, S. W., Sakmar, T. P., Olson, W. C., and Dragic, T. (2000) Proc. Natl. Acad. Sci. 97, 5762–5767[Abstract/Free Full Text]
  25. Farzan, M., Vasilieva, N., Schnitzler, C. E., Chung, S., Robinson, J., Gerard, N. P., Gerard, C., Choe, H., and Sodroski, J. (2000) J. Biol. Chem. 275, 33516–33521[Abstract/Free Full Text]
  26. Farzan, M., Chung, S., Li, W., Vasilieva, N., Wright, P. L., Schnitzler, C. E., Marchione, R. J., Gerard, C., Gerard, N. P., Sodroski, J., and Choe, H. (2002) J. Biol. Chem. 277, 40397–40402[Abstract/Free Full Text]
  27. Lu, Z., Berson, J. F., Chen, Y., Turner, J. D., Zhang, T., Sharron, M., Jenks, M. H., Wang, Z., Kim, J., Rucker, J., Hoxie, J. A., Peiper, S. C., and Doms, R. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6426–6431[Abstract/Free Full Text]
  28. Farzan, M., Babcock, G. J., Vasilieva, N., Wright, P. L., Kiprilov, E., Mirzabekov, T., and Choe, H. (2002) J. Biol. Chem. 277, 29484–29489[Abstract/Free Full Text]
  29. Picard, L., Wilkinson, D. A., McKnight, A., Gray, P. W., Hoxie, J. A., Clapham, P. R., and Weiss, R. A. (1997) Virology 231, 105–111[CrossRef][Medline] [Order article via Infotrieve]
  30. Earl, P. L., Doms, R. W., and Moss, B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 648–652[Abstract/Free Full Text]
  31. Poignard, P., Saphire, E. O., Parren, P. W., and Burton, D. R. (2001) Annu. Rev. Immunol 19, 253–274[CrossRef][Medline] [Order article via Infotrieve]
  32. Zwick, M. B., Labrijn, A. F., Wang, M., Spenlehauer, C., Saphire, E. O., Binley, J. M., Moore, J. P., Stiegler, G., Katinger, H., Burton, D. R., and Parren, P. W. (2001) J. Virol. 75, 10892–10905[Abstract/Free Full Text]
  33. Muster, T., Steindl, F., Purtscher, M., Trkola, A., Klima, A., Himmler, G., Ruker, F., and Katinger, H. (1993) J. Virol. 67, 6642–6647[Abstract/Free Full Text]
  34. Roben, P., Moore, J. P., Thali, M., Sodroski, J., Barbas, C. F. r., and Burton, D. R. (1994) J. Virol. 68, 4821–4828[Abstract/Free Full Text]
  35. Thali, M., Moore, J. P., Furman, C., Charles, M., Ho, D. D., Robinson, J., and Sodroski, J. (1993) J. Virol. 67, 3978–3988[Abstract/Free Full Text]
  36. Sullivan, N., Sun, Y., Sattentau, Q., Thali, M., Wu, D., Denisova, G., Gershoni, J., Robinson, J., Moore, J., and Sodroski, J. (1998) J. Virol. 72, 4694–4703[Abstract/Free Full Text]
  37. Choe, H., Li, W., Wright, P. L., Vasilieva, N., Venturi, M., Huang, C. C., Grundner, C., Dorfman, T., Zwick, M. B., Wang, L., Rosenberg, E. S., Kwong, P. D., Burton, D. R., Robinson, J. E., Sodroski, J. G., and Farzan, M. (2003) Cell 114, 161–170[CrossRef][Medline] [Order article via Infotrieve]
  38. Collman, R., Balliet, J. W., Gregory, S. A., Friedman, H., Kolson, D. L., Nathanson, N., and Srinivasan, A. (1992) J. Virol. 66, 7517–7521[Abstract/Free Full Text]
  39. Cormier, E. G., Tran, D. N., Yukhayeva, L., Olson, W. C., and Dragic, T. (2001) J. Virol. 75, 5541–5549[Abstract/Free Full Text]
  40. Rizzuto, C. D., Wyatt, R., Hernandez-Ramos, N., Sun, Y., Kwong, P. D., Hendrickson, W. A., and Sodroski, J. (1998) Science 280, 1949–1953[Abstract/Free Full Text]
  41. Farzan, M., Schnitzler, C. E., Vasilieva, N., Leung, D., Kuhn, J., Gerard, C., Gerard, N. P., and Choe, H. (2001) J. Exp. Med. 193, 1059–1066[Abstract/Free Full Text]
  42. Huang, C. C., Venturi, M., Majeed, S., Moore, M. J., Phogat, S., Zhang, M. Y., Dimitrov, D. S., Hendrickson, W. A., Robinson, J., Sodroski, J., Wyatt, R., Choe, H., Farzan, M., and Kwong, P. D. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2706–2711[Abstract/Free Full Text]

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