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Journal of Virology, September 2008, p. 9134-9142, Vol. 82, No. 18
0022-538X/08/$08.00+0     doi:10.1128/JVI.00394-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Mapping of the CXCR4 Binding Site within Variable Region 3 of the Feline Immunodeficiency Virus Surface Glycoprotein

Magnus Sundstrom,¹ Rebecca L. White,¹ Aymeric de Parseval,² K. Jagannadha Sastry,³ Garrett Morris,¹ Chris K. Grant,⁴ and John H. Elder^1*

The Scripps Research Institute, Department of Molecular Biology, La Jolla, California,¹ Public Health Research Institute, Newark, New Jersey,² The University of Texas M. D. Anderson Cancer Center, Department of Immunology, Houston, Texas,³ Custom Monoclonals International, Inc., West Sacramento, California⁴

Received 23 February 2008/ Accepted 25 June 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Feline immunodeficiency virus (FIV) shares with T-cell tropic strains of human immunodeficiency virus type 1 (HIV-1) the use of the chemokine receptor CXCR4 for cellular entry. In order to map the interaction of the FIV envelope surface unit (SU) with CXCR4, full-length FIV SU-Fc as well as constructs with deletions of extended loop L2, V3, V4, or V5 were produced in stable CHO cell lines. Binding studies were performed using these proteins on 3201 cells (CXCR4^hi CD134^–), with or without the CXCR4 inhibitor AMD3100. The findings established that SU binding to CXCR4 specifically requires the V3 region of SU. Synthetic peptides spanning the V3 region as well as a panel of monoclonal antibodies (MAbs) to SU were used to further map the site of CXCR4 interaction. Both the SU V3-specific antibodies and the full-length V3 peptide potently blocked binding of SU to CXCR4 and virus entry. By using a set of nested peptides overlapping a region of SU specifically recognized by CD134-dependent neutralizing V3 MAbs, we showed that the neutralizing epitope and the region required for CXCR4 binding are within the same contiguous nine-amino-acid sequence of V3. Site-directed mutagenesis was used to reveal that serine 393 and tryptophan 394 at the predicted tip of V3 are required to facilitate entry into the target cell via CXCR4. Although the amino acid sequences are not identical between FIV and HIV, the ability of FIV to bind and utilize both feline and human CXCR4 makes the feline model an attractive venue for development of broad-based entry antagonists.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Feline immunodeficiency virus (FIV) infection in cats is considered a valuable small-animal model of human immunodeficiency virus (HIV) vaccination strategies and drug development, as it is the only nonprimate lentivirus that causes a loss of CD4⁺ T cells followed by an AIDS-like disease in its natural host. Although FIV primarily targets CD4⁺ T cells (9, 12, 20), it uses CD134 instead of CD4 as its primary binding receptor (12, 40). CD134 is a member of the tumor necrosis factor receptor superfamily and is specifically expressed on activated CD4⁺ T cells, where it induces antiapoptotic signals to allow for survival and proliferation late in T-cell responses (7). The direct correlation between CD134 upregulation and the preferential targeting of FIV for activated T cells explains the FIV tropism for these cells (12).

Similar to T-cell-tropic HIV type 1 (HIV-1), FIV utilizes CXC chemokine receptor 4 (CXCR4) as an entry receptor for infection (48, 50), after interaction with a primary binding receptor (14). Also, the overall genetic structure of FIV is similar to that of HIV (33, 37, 45), and the FIV env gene encodes a surface glycoprotein (SU or gp95) and a transmembrane protein (TM or gp41). The SU protein exhibits considerable amino acid sequence variation, with five consensus major variable (V) regions (V1 to V5) (36, 37). Of these domains, the V3 loop has been identified as a major immunogenic domain in SU by epitope mapping with sera from infected cats (3, 18, 29, 34).

Studies of FIV-PPR interactions with CD134 have identified specific amino acids involved in SU binding in the outermost domain 1 of CD134 (11). Recent studies have indicated the involvement of additional residues in domain 2 in binding of certain other strains of FIV (49). For SU interaction with its entry receptor, CXCR4, involvement of the second extracellular loop of CXCR4 has been reported (5, 47). However, the domains of SU involved in direct interaction with CXCR4 have not been identified.

The purpose of this study was to define the domains of SU that are crucial for the interaction with CXCR4 in order to better understand the mechanism of FIV entry and infection. Analysis of genetic variations in SU proteins of FIV, simian immunodeficiency virus, and HIV-1 has revealed analogies in the locations and distribution of the variable domains V3, V4, and V5 (35). A structural model of FIV SU identified these variable domains as potential exposed loop segments (36). A segment, denoted loop II (L2), in the N-terminal part of FIV SU has also been predicted to fold as an exposed loop (36). As a step toward a better understanding of the molecular interaction between FIV and its target cells, we created SU mutants with deletions of L2, V3, V4, or V5. These mutants were used together with a panel of SU-specific monoclonal antibodies (MAbs) in SU-CXCR4 binding studies. Using nested V3 peptides and site-directed mutagenesis, the CXCR4 binding region of SU was further localized to a discrete stretch of nine amino acids at the predicted tip of the V3 loop, with critical involvement of serine 393 and tryptophan 394 to facilitate binding and virus entry into the target cell.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell lines and virus. The interleukin-2 (IL-2)-independent feline lymphoma cell line 3201 was obtained from W. Hardy (Sloan-Kettering Memorial Hospital, NY). 3201 cells are CXCR4^hi and have a very low or negative phenotype for heparan sulfate proteoglycans and CD134 (16). CHO-K1 cells were obtained from the American Type Culture Collection (Rockville, MD). GFox cells are Crandell Feline Kidney (CrFk) cells engineered to express CD134 (12). Cat peripheral blood mononuclear cells (PBMCs) were prepared by Ficoll-Hypaque Plus (Amersham Biosciences, Sweden) density gradient purification from heparinized whole feline blood as previously described (28). The primary IL-2-dependent T-cell line 104-C1 is a clone isolated from feline PBMCs by limiting dilution (27). Propagation of the different cell lines was performed as previously described (13, 44).

FIV-PPR is a molecularly cloned clade A primary isolate (37).

Recombinant SU proteins. Deletion mutants of FIV-PPR SU were generated using PCR-ligation-PCR mutagenesis (1). Briefly, fragments of FIV-PPR SU were joined after amplification in separate PCRs. The PCR primers used for these reactions were designed to contain a blunt-ended restriction enzyme site, and products from the initial PCRs were digested with the corresponding restriction enzymes, gel purified, and ligated. A fraction of each ligation reaction mixture was then used to amplify the ligated DNA by a second PCR, using the 5' primer for the upstream fragment and the 3' primer for the downstream fragment. The PCR products were then subcloned into pRSC-GS-SU-Fc (13), followed by sequencing to confirm the presence of the desired deletion and the absence of any other mutation. The pRSC-GS-SU-Fc delMut plasmids were then used for production of stable CHO-K1 cell lines, as previously described (13), expressing SU-Fc fusion proteins (adhesins) with the desired deletions. Fc (13) or SU-Fc proteins from stable CHO cell supernatants were purified by affinity chromatography over protein A-Sepharose and quantitated as described previously (13). The following amino acids were removed in the SU deletion mutants: delL2, amino acids 249 to 353; delV3, amino acids 378 to 416; delV4, amino acids 463 to 499; and delV5, amino acids 535 to 560). All deletions were constructed with the amino acids serine-glycine-glycine as a linker.

Western blot analysis. Purified Fc and Fc-tagged fusion proteins were loaded on an 8 to 16% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and the presence of SU-Fc proteins was revealed by Western blotting with a peroxidase-conjugated goat antibody specific for human immunoglobulin G (IgG) Fc (Pierce, Rockford, IL).

Flow cytometry analysis. Binding of SU-Fc fusion proteins or Fc (negative control) to the surfaces of 3201 cells, 104-C1 cells, or PBMCs was analyzed by flow cytometry, using a phycoerythrin-conjugated goat anti-human IgG1 Fc antibody (MP Biomedicals, Aurora, OH) and FLOWJO software (Tree Star, San Carlos, CA). CXCR4-specific binding was confirmed by preincubation of cells with the CXCR4 inhibitor AMD3100 (obtained through the AIDS Research and Reference Reagent Program) (10) at 1 µg/ml (1.3 µM) for 0.5 h at 25°C. For inhibition studies, antibodies or peptides were preincubated with the SU adhesin or cells, respectively, at the indicated concentration for 0.5 h at 25°C. Percent inhibition was calculated as previously described (11) and expressed as follows: +++++, 90 to 100% inhibition; ++++, 70 to 90% inhibition; +++, 50 to 70% inhibition; ++, 30 to 50% inhibition; +, 10 to 30% inhibition; and –, 0 to 10% inhibition.

Preparation of pseudovirions and virus entry assays. Production of pseudovirions that were pseudotyped with either wild-type (wt) SU or SUs in which specific deletions or point mutations had been introduced was performed as previously described, using a beta-galactosidase (β-Gal)-expressing packaging vector termed pCFIV (11). Point mutations were performed using the QuikChange site-directed mutagenesis strategy (Stratagene, La Jolla, CA) as recommended by the manufacturer. Each pseudovirion was assessed for the level of reverse transcriptase (RT) in the supernatants of transfected 293T cells. RT values were then normalized before assessing infection of Gfox (CXCR4⁺ CD134⁺) target cells in single-round infections as described previously (11).

ELISA. All enzyme-linked immunosorbent assay (ELISA) operations were carried out at room temperature. Immulon II HB plates (Thermo, Milford, MA) were coated overnight with 100 ng purified SU deletion construct proteins in phosphate-buffered saline, pH 7.2. Plates were then washed twice with saline and tap dried. ELISA buffer (100 µl of 0.15 M NaCl, 0.05 M Tris-HCl, 1 mM EDTA, 3% bovine serum albumin fraction V, 3.5% fetal calf serum, and 0.05% Tween 20, pH 7.4) was added to all wells. Staphylococcal protein A purified MAb (100 µl in 20 µg/ml ELISA buffer) was added to the first well of each row (total volume, 200 µl). The contents of the first well were then twofold serially diluted over eight wells, resulting in a dilution series from 1,000 ng/100 µl in the first well to 8 ng/100 µl in the last well. Plates were incubated for 45 min, washed three times, and tap dried. Goat anti-mouse IgG-horseradish peroxidase (Bio-Rad, Hercules, CA) was diluted 1:1,000 in ELISA buffer, and 100 µl/well was added; the plates were incubated for 45 min, washed three times, and tap dried. The substrate (15 ml/plate, for 150 µl per well) used was o-phenylenediamine (Sigma), as follows: 20 mg was dissolved in 15 ml of 50 mM citric acid and 100 mM dibasic sodium phosphate, pH 5.0, and activated with 80 µl per plate hydrogen peroxide (30% solution; Fisher Scientific). The reaction proceeded for 8 min and was then stopped by the addition of 50 µl/well 2.0 M sulfuric acid. The reaction color was read at 493 nm.

Peptide synthesis. Peptides were synthesized on a PTI Symphony multiple peptide synthesizer utilizing standard 9-fluorenylmethoxy carbonyl chemistry. Crude peptide material was purified by high-pressure liquid chromatography, and purity was determined to exceed 90% for all material. The masses of peptides were confirmed by mass spectrometry.

Infections. Cells were seeded at 3 x 10⁴ cells per well in a 96-well plate and infected for 2 h at room temperature with 20 µl virus stock. Cells were then washed once and incubated in cell medium until day 7, when supernatants were analyzed for virus production by using a micro-RT assay as described below. For analysis of V3 peptide inhibition of infection, cells were preincubated for 30 min with peptides before infection and throughout the incubation time.

Micro-RT activity assay. The micro-RT activity assay was performed as previously described (13, 15). Briefly, 50 µl of cell-free supernatant was incubated for 10 min at room temperature with 10 µl of lysis buffer (0.75 M KCl, 20 mM dithiothreitol, 0.5% Triton X-100). Forty microliters of a mixture containing 125 mM Tris-HCl (pH 8.1), 12.5 mM MgCl₂, 1.25 µg poly(rA)-poly(dT)_12-18 (Amersham Biosciences, Piscataway, NJ), and 1.25 µCi of [³H]dTTP (DuPont, Boston, MA) was added to the sample, followed by 2 h of incubation at 37°C. RT activity was measured as previously described (15).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Construction and expression of FIV SU deletion mutants. We previously reported an experimental approach using a FIV SU-Fc immunoadhesin (soluble FIV surface glycoprotein fused in frame with the Fc domain of human IgG1) (2) to screen for cell surface receptors involved in FIV binding and infection (11-14, 16, 17). The envelope glycoproteins of FIV, simian immunodeficiency virus, and HIV-1 share only minor sequence similarity in SU. However, there are analogies in the locations and distribution of the SU variable domains V3 to V5 (30, 35-37, 42). To characterize the interaction of FIV with its entry receptor, CXCR4, four FIV SU-Fc constructs with deletions of variable domain V3, V4, or V5 or the predicted loop II were generated and used to produce soluble proteins by establishment of stable CHO cell lines (Fig. 1). Purified wt and deletion mutants of SU were subjected to SDS-polyacrylamide gel electrophoresis and confirmed to have the expected size by Western blotting with an anti-human IgG1 Fc antibody (Fig. 1b).

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FIG. 1. Construction (a) and expression (b) of FIV SU-immunoadhesins with deletions of variable domains. Recombinant deletion mutants of FIV SU-Fc were generated by PCR-ligation-PCR mutagenesis, expressed by stable transfection of CHO-K1 cells, and batch purified from cell supernatants by affinity chromatography over protein A. (b) Samples (500 ng) of SU-Fc (wt and deletion mutants) and Fc protein were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions and immunoblotted with an antibody specific for human IgG1 Fc (anti-Fc).

Binding of SU to 3201 cells is CXCR4 specific and dependent on V3. 3201 is a feline lymphoma cell line with relatively high expression levels of CXCR4 but lacking CD134 (13). To analyze the binding of SU to CXCR4, we incubated 3201 cells together with the full-length wt SU-Fc immunoadhesin, followed by flow cytometric analysis using a phycoerythrin-conjugated anti-IgG1 Fc antibody. As previously reported (16), SU-Fc bound 3201 cells, and the CXCR4 specificity of this binding was demonstrated by a complete inhibition of binding upon preincubation with the CXCR4 antagonist AMD3100 (Fig. 2a). The lack of binding of Fc to 3201 cells confirmed that cellular binding of the immunoadhesin is entirely attributed to the SU part of the SU-Fc fusion protein (Fig. 2a).

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FIG. 2. Binding of FIV SU-Fc deletion mutants to cell surface CXCR4 was detected by FACS, using a phycoerythrin-conjugated anti-Fc antibody. (a) FACS-detected binding of wt and deletion mutants of SU-Fc to CXCR4 on 3201 cells in the presence and absence of the CXCR4 antagonist AMD3100. CXCR4-specific binding is evident after deletion of L2, V4, and V5, but deletion of V3 abolishes SU binding to CXCR4. Cells were incubated with Fc protein (solid gray areas) or wt or deletion mutants of SU (gray lines) or preincubated with AMD3100 (black lines) before the addition of wt or deletion mutants of SU. Results are representative of three independent determinations. (b) Production and infection analysis of β-Gal-expressing pseudovirions with deletions in SU. Supernatants from transfected 293 cells were analyzed for RT activity 48 h after transfection. RT values were normalized for wt and deletion mutants, and supernatants were then used in single-round infections of CD134- and CXCR4-expressing CrFK cells (Gfox cells). β-Gal activity was then analyzed 48 h after infection. None of the deletion mutants were able to facilitate virus entry, consistent with the failure to bind CD134.

In order to localize the SU-CXCR4 interaction, we performed similar binding studies using the L2, V3, V4, and V5 deletion mutants in the presence or absence of a saturating concentration (1.3 µM) of AMD3100 (Fig. 2a). SU-Fc adhesins with deletion of L2, V4, or V5 were functional and retained the capacity to bind 3201 cells, albeit more weakly than full-length wt SU-Fc (Fig. 2a). Binding was totally abolished in the presence of AMD3100, demonstrating CXCR4 specificity. In contrast to the other deletion mutants, the V3-deleted SU failed to bind 3201 cells, indicating that the V3 region is specifically required for interaction of SU with cell surface CXCR4 (Fig. 2a).

Virtually all SU binding observed by fluorescence-activated cell sorting (FACS) analysis of 104-C1 T cells (CD134^hi CXCR4^lo) is via binding to CD134, and binding is not significantly inhibited by AMD3100 (ref 13, 14, 16). Thus, this cell line serves as a venue for measuring CD134 binding by SU, with a negligible contribution from the entry receptor, CXCR4. Attempts were made to identify the CD134 binding site, using the four deletion mutants described above. In contrast to results with CXCR4^hi 3201 cells, the CD134^hi 104-C1 cells failed to bind any of the deletion mutants (not shown). In addition to binding studies, vectors expressing wt SU and the four deletion mutants were cotransfected with a β-Gal-expressing pCFIV vector in 293T cells in order to generate pseudotyped virus particles expressing each envelope protein. After standardizing the virus supernatants for RT activity, the pseudovirions were used in single-round infections of Gfox cells (CD134⁺ CXCR4⁺). Only wt SU facilitated virus entry, consistent with failure of the mutants to bind to CD134 (Fig. 2b). Given the diverse group of deletion mutants, the results indicate that the binding of CD134 is heavily dependent on the overall conformation of SU rather than on a short contiguous epitope as with CXCR4.

Structural mapping of a panel of MAbs to SU. To further characterize the FIV-CXCR4 interaction, we employed the SU deletion proteins and synthetic peptides encompassing the whole or parts of the V3 loop to map the binding locations of SU-specific MAbs generated by immunizing mice with SU-Fc (14). By performing ELISAs using the MAb panel versus full-length or deletion proteins, we were able to assign four MAbs to SU-L2, seven to SU-V3, and one to SU-V4 (Table 1). No reactivity was noted to V5, suggesting that this region may either be nonimmunogenic in mice or reside in an immunologically privileged environment.

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TABLE 1. Mapping of binding locations of SU-specific MAbs by ELISA, using full-length or deletion mutant SU proteins

Antibodies specific for the central parts of V3 can block SU-CXCR4 interaction. Next, we analyzed the capacity of the characterized MAbs to interfere with the SU-CXCR4 interaction. SU-Fc was preincubated with the various anti-SU MAbs before addition to 3201 cells, followed by analysis of binding by flow cytometry (Fig. 3). Eleven of the 12 characterized SU MAbs inhibited SU binding to CXCR4. Among these, MAbs with binding reactivity specific for V3 were more efficient in interfering with binding of SU to CXCR4 than MAbs recognizing L2 or V4 (Fig. 3a and b). Four of the V3-specific MAbs (SU1-30, SU2-4, SU2-5, and SU2-10) are specific to the E60 region in the center of V3 and are CD134-dependent neutralizing MAbs (14). The V3 E60 region is highly conserved between FIV clade A, B, and C isolates (14) and is the FIV equivalent of a region previously identified as critical for HIV entry (31). Interestingly, MAbs recognizing E60 gave the strongest inhibition of SU binding to CXCR4 (Fig. 3). MAbs SU1-5, SU1-7, and SU1-10 also recognized the V3 peptide and failed to bind SU-3 but had a specificity outside E60 (Table 1 and data not shown). Of these MAbs, SU1-5 and SU1-7 exhibited less influence on SU binding than the MAbs specific for the E60 region (Fig. 3).

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FIG. 3. (a) Inhibition of SU-Fc binding to 3201 cells by SU MAbs. SU-Fc was preincubated with the indicated anti-SU Abs for 30 min before being added to cells and incubated for 1 hour at room temperature. SU-Fc binding was analyzed by FACS with anti-Fc antibody and is expressed as percent inhibition of SU binding. Results are representative of three independent determinations. (b) Summary of SU MAb specificity versus blockage of SU binding to CXCR4 on 3201 cells. Inhibition scale: black, strong; dark gray, moderate; light gray, weak; white, none or upregulation of binding.

Fine mapping of the V3 region required for interaction with CXCR4. Our analyses using SU-Fc deletion mutants and SU-specific MAbs strongly support the involvement of SU V3 in the FIV-CXCR4 interaction. Therefore, the 41-amino-acid V3 loop peptide was assessed for the ability to block SU binding to CXCR4. As analyzed by flow cytometry, preincubation of 3201 cells with the V3 peptide abrogated binding of SU to CXCR4 (Fig. 4a). We next sought to determine the minimal domain within the V3 region needed for SU-CXCR4 interaction by evaluating a panel of nested peptides overlapping the E60 region of FIV SU. The peptides spanning this region were truncated at either the N or carboxyl (C) terminus and exhibited various capacities to interact with SU-CXCR4 binding (Fig. 4b; also see Fig. 6a). All V3 peptides tested displayed a clear dose-dependent inhibition (data not shown), and peptides with an intact N terminus, starting with two conserved (>90%) serines, and with a truncated C terminus gave a much stronger inhibition of SU binding than did peptides with truncations in the N terminus (Fig. 4b; see Fig. 6a). Remarkably, a peptide as short as nine amino acids (SSWKQRNRW), designated N44, was equally potent at blocking SU binding to CXCR4 as the full-length V3 peptide. The C-terminal end of N44 consists of an arginine and a tryptophan that are more than 99% conserved among 227 different FIV isolates (see Fig. 6b and data not shown). Interestingly, truncation of these two amino acids in peptides P26 and P27 abolished most of the inhibitory activity for binding of SU to CXCR4 (Fig. 4b). A peptide corresponding to the C-terminal half of V3 (P28) exhibited no inhibition of SU binding. In summary, these findings verify the significance of the central portion of V3, and in particular the nine-amino-acid long "N44" region, for SU-CXCR4 interaction.

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FIG. 4. Peptides corresponding to the V3 loop interfere with binding of SU to CXCR4. (a) Preincubation of 3201 cells with a peptide corresponding to V3 before addition of SU-Fc efficiently reduces binding of SU to CXCR4. The solid grey area corresponds to cells incubated with Fc protein, the grey line corresponds to cells incubated with SU-Fc, and the black line corresponds to cells preincubated with V3 peptide before the addition of SU-Fc. (b) Analysis of inhibition of SU-CXCR4 binding by a panel of short peptides overlapping the central region of V3. (c) The V3 peptide can inhibit FIV infection. PBMCs were pretreated with the indicated peptides before addition of FIV-PPR. RT activity was measured 7 days after infection. A peptide corresponding to a region of the feline leukemia virus (FELV) transmembrane region was used as a negative control. Results are representative of three independent determinations.

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FIG. 6. (a) Sequences of a panel of V3 peptides and summary of the ability of the peptides to interfere with SU binding to CXCR4. (b) Consensus sequence and amino acid percent frequency of the "N44 domain." (c) JPRED prediction of the secondary structure of V3. E, beta strand; -, indicates coil. Amino acids corresponding to the "N44 domain" are underlined.

Attempts were also made to utilize both the full-length V3 peptide and N44 peptide to block virus infection ex vivo. Relative RT activity was measured in culture supernatants 7 days after infection with FIV-PPR as a function of peptide addition. A 50% inhibition of infection was observed with the full-length V3 peptide, but the shorter N44 peptide did not block infection in these experiments (Fig. 4c). We surmise that the peptide may have been degraded ex vivo or lacked the avidity required to continuously block virus entry.

The importance of the V3 region of SU for receptor binding is not type specific but is limited to CXCR4. We assessed the blocking activities of the V3 and N44 peptides by analyzing their effects on Fc-tagged SU proteins from genetically divergent FIV subtypes. SU proteins derived from FIV 34TF10 Petaluma (an FIV subgroup A clone) (16, 45) and FIV C36 (19) both bound CXCR4 on 3201 cells (Fig. 5a). The binding of SU-34TF10-Fc or SU-C36-Fc was potently blocked by N44 and V3 peptides in a manner similar to that with FIV-PPR SU (Fig. 5a), indicating that the involvement of the N44 domain for CXCR4 interaction is a general feature among different strains of FIV.

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FIG. 5. (a) Binding of SU from FIV strains 34TF10 Petaluma and C36 to CXCR4 is blocked by preincubation with N44 and V3 peptides. (b) Analysis of the effects of peptides on SU binding to PBMCs and 104-C1 cells. A peptide corresponding to a region of the feline leukemia virus (FELV) transmembrane region was used as a negative control. Results are representative of three independent determinations.

As indicated above, FIV SU also binds cell surface CD134 (12, 16, 40). Therefore, the N44 and V3 peptides were tested for the ability to block SU-Fc binding to CD134 expressed on PBMCs (CXCR4⁺ CD134⁺) and 104-C1 cells (CD134^hi CXCR4^lo). Only a limited inhibition of SU binding by N44, V3, or AMD3100 was observed for PBMCs (Fig. 5b). Furthermore, there was no effect of either of these peptides or AMD3100 on SU binding to 104-C1 cells (Fig. 5b), again consistent with an independent binding mode for CD134, i.e., V3, in particular its "N44 region," is required for interaction of SU with the entry receptor CXCR4 but not with the binding receptor CD134.

No three-dimensional analyses have been performed with FIV SU. However, JPRED analysis (8) of FIV SU indicates that FIV V3 has a similar secondary structure to that of HIV V3 (Fig. 6c) (23). Notably, the SSWKQRNRW amino acids of N44 are located at the predicted tip of V3, between two beta sheets (Fig. 6c). A similar motif has been identified in HIV V3 in the crystal structure of HIV-1 SU (23).

To further define residues critical for CXCR4 binding, site-directed mutagenesis was performed on the N-terminal portion of the N44 peptide region, with particular emphasis on the relatively conserved Ser392-Ser393-Trp394 sequence highlighted as important from the above studies. These three residues were mutated, individually or as a group, to alanine in the context of SU, and pseudovirions were then prepared as described above in order to test the effects of these mutations on virus entry. As shown in Fig. 7, Ser392 tolerated the change, but the Ser393Ala or Trp394Ala substitution or substitution of all three residues abrogated the ability of the mutant SU to facilitate virus entry into Gfox cells.

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FIG. 7. Site-directed mutagenesis of highly conserved amino acids at the predicted tip of the V3 loop of FIV SU. Residue Ser392, Ser393, or Trp394 or all three amino acids were replaced with alanine, and β-Gal-expressing pseudovirions were prepared to display each mutant SU, as described in Materials and Methods. Supernatants from transfected 293T cells containing each pseudovirion were assessed for RT activity, normalized, and then used to infect Gfox cells as detailed in the legend to Fig. 2. An alanine at Ser392 was tolerated, but substitution of alanine for either Ser393 or Trp394 abrogated the ability of SU to facilitate CXCR4 binding and virus entry.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
To characterize the interplay between FIV and its entry receptor, CXCR4, we generated deletion mutants of the FIV SU protein and analyzed their binding characteristics. These studies suggested that the V3 loop of SU contains the main determinants for FIV SU binding to CXCR4. Given the lower level of total binding of mutants relative to that of wt SU, the L2 and V4 loops may also contribute, directly or indirectly, to the interaction with CXCR4. However, these regions appear to be much less critical than V3. Next, a panel of SU MAbs was mapped based on their binding locations. These characterized MAbs were then employed to further study the SU-CXCR4 interaction. Binding studies demonstrated that MAbs specific for the V3 region of SU were more efficient than L2- or V4-specific MAbs in blocking the SU binding to CXCR4. Furthermore, the most efficient inhibition of SU binding was primarily displayed by MAbs recognizing the central part of the V3 loop. A set of peptides overlapping this region was analyzed for the ability to block SU binding to CXCR4. By this approach, the chemokine receptor binding region of SU was narrowed down to nine amino acids corresponding to the predicted tip of the V3 loop. Further mutagenesis studies demonstrated particular involvement of residues Ser393 and Trp394 in the SU-CXCR4 interaction.

Although the consensus sequences between the conserved cysteine residues of the FIV and T-cell-tropic HIV-1 V3 domains display a low degree of homology, V3 domains from both viruses are positively charged (13, 21). Furthermore, a JPRED analysis predicted the secondary structure of FIV V3 to display a high degree of similarity to that of the HIV V3 loop. Both V3 regions are conserved in length (HIV V3 is 35 residues long, and FIV V3 is 41 residues long), and they exhibit a relatively conserved centrally located tip flanked by two beta-sheets (23, 26) (Fig. 6b and c). The consensus sequence of the HIV V3 tip is comprised of the amino acid sequence GPGR or GPGQ. Similarly, the amino acids in the "N44 region" at the tip of FIV V3 are also fairly conserved, with six of the amino acids having a frequency of 90% or higher in over 200 FIV isolates (Fig. 6b and data not shown). The V3 loop of HIV gp120 is the major determinant for coreceptor usage and hence cell tropism (4, 6, 22, 24, 25, 32, 43). In particular, the amino acid sequence at the tip/crown of HIV V3 is important for coreceptor usage (6, 32). In this region, basic residues and conserved hydrophobic residues have been shown to contribute to CXCR4 binding (4). Interestingly, the amino acid sequence in the FIV V3 region corresponding to N44 is also rich in hydrophobic and basic residues, including two tryptophans (98 and 100% conserved) and two arginines that are 73% and 99% conserved.

The "N44 domain" in FIV V3 is located outside the lysine and glutamic acid residues at positions 407 and 409 that have been implicated in the affinity of SU for heparan sulfate proteoglycans and in CrFK cell tropism for tissue culture-adapted FIV (16, 41, 46). Furthermore, the amino acids of the "N44 domain" correspond to the central section of peptides that have been reported to both absorb neutralizing activity in pooled cat sera and induce neutralizing Abs in cats (29, 38). Together with our data, these findings suggest that FIV neutralizing Abs recognize the CXCR4 binding part of SU or epitopes in close proximity to this domain.

As noted previously (12), and in contrast to SU binding to CXCR4^hi 3201 cells, the majority of SU binding to either PBMCs or 104-C1 T cells occurs via CD134. Barely detectable contributions from direct binding to CXCR4 are evident by FACS analysis, as shown by the relative insensitivity of binding to the latter cells in the presence of the CXCR4 antagonist AMD3100 (Fig. 5). The present studies show that the V3 peptides did not block SU binding to either PBMCs or 104-C1 cells, which indicates that binding of SU to CD134 either occurs via regions other than V3 or is more conformation dependent, such that short peptides fail to block the CD134-SU interaction. In keeping with this observation, studies using HIV SU deletion mutants have demonstrated that V3 is not directly involved in CD4-SU interactions (51). Furthermore, none of the diverse deletion mutants employed here were able to facilitate virus entry when expressed on pseudovirions, consistent with the interpretation that there is a conformation-dependent binding of CD134 to noncontiguous regions of SU.

CXCR4-independent binding of the virus to CD134 on total PBMCs and 104-C1 cells may explain the low success of the V3 peptides in competing for virus infection ex vivo. FIV will bind to these cells via CD134 in the presence of peptides capable of blocking CXCR4 binding. Therefore, when peptide exchange/uptake occurs and/or when new CXCR4 entry receptors are generated, the virus is able to bind CXCR4 and enter the cell. However, it has been reported previously for HIV V3 peptides that circularized peptides can compete successfully for virus entry, whereas linear forms cannot (39). Consistent with this observation, the FIV full-length V3 peptide that demonstrated inhibitory effects on virus replication ex vivo had N- and C-terminal cysteine residues and thus could cyclize, whereas the shorter N44 peptide which failed to inhibit virus replication did not.

FIV and primate lentiviruses share the same model of cell entry, with a sequential interaction of SU with a binding receptor followed by binding and entry via a chemokine entry receptor. These binding steps are critical for viral entry and therefore offer potential targets for therapeutic or prophylactic intervention. The studies reported here have defined the residues critical for CXCR4 binding, which will aid in the use of the FIV-cat system as a small-animal model for development of inhibitors relevant to the treatment of HIV/AIDS.

 
We thank Erika Assarsson and Ying-Chuan Lin for valuable comments and Elizabeth Fink for excellent technical assistance.

This work was supported by grants AI025825 (J.H.E.) and AI069984 (A.D.P.) from the National Institutes of Health (NIH). Peptide synthesis was supported by grants AI0426969 and AI065402 (K.J.S.). The peptide facility at M. D. Anderson Cancer Center was supported with funds from NIH grant CA 16672. Magnus Sundstrom was supported by a fellowship from the Sweden-America Foundation.

 
* Corresponding author. Mailing address: Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB-14, La Jolla, CA 92037. Phone: (858) 784-8270. Fax: (858) 784-2750. E-mail: jelder{at}scripps.edu

Published ahead of print on 2 July 2008.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1
1. Ali, S. A., and A. Steinkasserer. 1995. PCR-ligation-PCR mutagenesis: a protocol for creating gene fusions and mutations. BioTechniques 18:746-750.[Medline]
2
2. Aruffo, A., I. Stamenkovic, M. Melnick, C. B. Underhill, and B. Seed. 1990. CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303-1313.[CrossRef][Medline]
3
3. Avrameas, A., J. G. Guillet, L. Chouchane, A. Moraillon, P. Sonigo, and A. D. Strosberg. 1992. Localisation of three epitopes of the env protein of feline immunodeficiency virus. Mol. Immunol. 29:565-572.[CrossRef][Medline]
4
4. Basmaciogullari, S., G. J. Babcock, D. Van Ryk, W. Wojtowicz, and J. Sodroski. 2002. Identification of conserved and variable structures in the human immunodeficiency virus gp120 glycoprotein of importance for CXCR4 binding. J. Virol. 76:10791-10800.[Abstract/Free Full Text]
5
5. Brelot, A., N. Heveker, K. Adema, M. J. Hosie, B. J. Willett, and M. Alizon. 1999. Effect of mutations in the second extracellular loop of CXCR4 on its utilization by human and feline immunodeficiency viruses. J. Virol. 73:2576-2586.[Abstract/Free Full Text]
6
6. Cormier, E. G., and T. Dragic. 2002. The crown and stem of the V3 loop play distinct roles in human immunodeficiency virus type 1 envelope glycoprotein interactions with the CCR5 coreceptor. J. Virol. 76:8953-8957.[Abstract/Free Full Text]
7
7. Croft, M. 2003. Costimulation of T cells by OX40, 4-1BB, and CD27. Cytokine Growth Factor Rev. 14:265-273.[CrossRef][Medline]
8
8. Cuff, J. A., M. E. Clamp, A. S. Siddiqui, M. Finlay, and G. J. Barton. 1998. JPred: a consensus secondary structure prediction server. Bioinformatics 14:892-893.[Abstract/Free Full Text]
9
9. Dean, G. A., S. Himathongkham, and E. E. Sparger. 1999. Differential cell tropism of feline immunodeficiency virus molecular clones in vivo. J. Virol. 73:2596-2603.[Abstract/Free Full Text]
10
10. De Clercq, E., N. Yamamoto, R. Pauwels, J. Balzarini, M. Witvrouw, K. De Vreese, Z. Debyser, B. Rosenwirth, P. Peichl, R. Datema, et. al. 1994. Highly potent and selective inhibition of human immunodeficiency virus by the bicyclam derivative JM3100. Antimicrob. Agents Chemother. 38:668-674.[Abstract/Free Full Text]
11
11. de Parseval, A., U. Chatterji, G. Morris, P. Sun, A. J. Olson, and J. H. Elder. 2005. Structural mapping of CD134 residues critical for interaction with feline immunodeficiency virus. Nat. Struct. Mol. Biol. 12:60-66.[CrossRef][Medline]
12
12. de Parseval, A., U. Chatterji, P. Sun, and J. H. Elder. 2004. Feline immunodeficiency virus targets activated CD4+ T cells by using CD134 as a binding receptor. Proc. Natl. Acad. Sci. USA 101:13044-13049.[Abstract/Free Full Text]
13
13. de Parseval, A., and J. H. Elder. 2001. Binding of recombinant feline immunodeficiency virus surface glycoprotein to feline cells: role of CXCR4, cell surface heparans, and an unidentified non-CXCR4 receptor. J. Virol. 75:4528-4539.[Abstract/Free Full Text]
14
14. de Parseval, A., C. K. Grant, K. J. Sastry, and J. H. Elder. 2006. Sequential CD134-CXCR4 interactions in feline immunodeficiency virus (FIV): soluble CD134 activates FIV Env for CXCR4-dependent entry and reveals a cryptic neutralization epitope. J. Virol. 80:3088-3091.[Abstract/Free Full Text]
15
15. de Parseval, A., D. L. Lerner, P. Borrow, B. J. Willett, and J. H. Elder. 1997. Blocking of feline immunodeficiency virus infection by a monoclonal antibody to CD9 is via inhibition of virus release rather than interference with receptor binding. J. Virol. 71:5742-5749.[Abstract]
16
16. de Parseval, A., S. Ngo, and J. H. Elder. 2004. Factors that increase the effective concentration of CXCR4 dictate feline immunodeficiency virus tropism and kinetics of replication. J. Virol. 78:9132-9143.[Abstract/Free Full Text]
17
17. de Parseval, A., S. V. Su, J. H. Elder, and B. Lee. 2004. Specific interaction of feline immunodeficiency virus surface glycoprotein with human DC-SIGN. J. Virol. 78:2597-2600.[Abstract/Free Full Text]
18
18. de Ronde, A., J. G. Stam, P. Boers, H. Langedijk, R. Meloen, W. Hesselink, L. C. Keldermans, A. van Vliet, E. J. Verschoor, M. C. Horzinek, et al. 1994. Antibody response in cats to the envelope proteins of feline immunodeficiency virus: identification of an immunodominant neutralization domain. Virology 198:257-264.[CrossRef][Medline]
19
19. de Rozières, S., C. K. Mathiason, M. R. Rolston, U. Chatterji, E. A. Hoover, and J. H. Elder. 2004. Characterization of a highly pathogenic molecular clone of feline immunodeficiency virus clade C. J. Virol. 78:8971-8982.[Abstract/Free Full Text]
20
20. English, R. V., C. M. Johnson, D. H. Gebhard, and M. B. Tompkins. 1993. In vivo lymphocyte tropism of feline immunodeficiency virus. J. Virol. 67:5175-5186.[Abstract/Free Full Text]
21
21. Fouchier, R. A., M. Groenink, N. A. Kootstra, M. Tersmette, H. G. Huisman, F. Miedema, and H. Schuitemaker. 1992. Phenotype-associated sequence variation in the third variable domain of the human immunodeficiency virus type 1 gp120 molecule. J. Virol. 66:3183-3187.[Abstract/Free Full Text]
22
22. Henkel, T., P. Westervelt, and L. Ratner. 1995. HIV-1 V3 envelope sequences required for macrophage infection. AIDS 9:399-401.[Medline]
23
23. Huang, C. C., M. Tang, M. Y. Zhang, S. Majeed, E. Montabana, R. L. Stanfield, D. S. Dimitrov, B. Korber, J. Sodroski, I. A. Wilson, R. Wyatt, and P. D. Kwong. 2005. Structure of a V3-containing HIV-1 gp120 core. Science 310:1025-1028.[Abstract/Free Full Text]
24
24. Hung, C. S., N. Vander Heyden, and L. Ratner. 1999. Analysis of the critical domain in the V3 loop of human immunodeficiency virus type 1 gp120 involved in CCR5 utilization. J. Virol. 73:8216-8226.[Abstract/Free Full Text]
25
25. Hwang, S. S., T. J. Boyle, H. K. Lyerly, and B. R. Cullen. 1991. Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1. Science 253:71-74.[Abstract/Free Full Text]
26
26. LaRosa, G. J., J. P. Davide, K. Weinhold, J. A. Waterbury, A. T. Profy, J. A. Lewis, A. J. Langlois, G. R. Dreesman, R. N. Boswell, P. Shadduck, et al. 1990. Conserved sequence and structural elements in the HIV-1 principal neutralizing determinant. Science 249:932-935.[Abstract/Free Full Text]
27
27. Lerner, D. L., C. K. Grant, A. de Parseval, and J. H. Elder. 1998. FIV infection of IL-2-dependent and -independent feline lymphocyte lines: host cells range distinctions and specific cytokine upregulation. Vet. Immunol. Immunopathol. 65:277-297.[CrossRef][Medline]
28
28. Lerner, D. L., P. C. Wagaman, T. R. Phillips, O. Prospero-Garcia, S. J. Henriksen, H. S. Fox, F. E. Bloom, and J. H. Elder. 1995. Increased mutation frequency of feline immunodeficiency virus lacking functional deoxyuridine-triphosphatase. Proc. Natl. Acad. Sci. USA 92:7480-7484.[Abstract/Free Full Text]
29
29. Lombardi, S., C. Garzelli, C. La Rosa, L. Zaccaro, S. Specter, G. Malvaldi, F. Tozzini, F. Esposito, and M. Bendinelli. 1993. Identification of a linear neutralization site within the third variable region of the feline immunodeficiency virus envelope. J. Virol. 67:4742-4749.[Abstract/Free Full Text]
30
30. Morikawa, S., H. Lutz, A. Aubert, and D. H. Bishop. 1991. Identification of conserved and variable regions in the envelope glycoprotein sequences of two feline immunodeficiency viruses isolated in Zurich, Switzerland. Virus Res. 21:53-63.[CrossRef][Medline]
31
31. Nehete, P. N., E. M. Vela, M. M. Hossain, A. K. Sarkar, N. Yahi, J. Fantini, and K. J. Sastry. 2002. A post-CD4-binding step involving interaction of the V3 region of viral gp120 with host cell surface glycosphingolipids is common to entry and infection by diverse HIV-1 strains. Antivir. Res. 56:233-251.[CrossRef][Medline]
32
32. Nolan, K. M., A. P. Jordan, and J. A. Hoxie. 2008. Effects of partial deletions within the human immunodeficiency virus type 1 V3 loop on coreceptor tropism and sensitivity to entry inhibitors. J. Virol. 82:664-673.[Abstract/Free Full Text]
33
33. Olmsted, R. A., V. M. Hirsch, R. H. Purcell, and P. R. Johnson. 1989. Nucleotide sequence analysis of feline immunodeficiency virus: genome organization and relationship to other lentiviruses. Proc. Natl. Acad. Sci. USA 86:8088-8092.[Abstract/Free Full Text]
34
34. Pancino, G., C. Chappey, W. Saurin, and P. Sonigo. 1993. B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins. J. Virol. 67:664-672.[Abstract/Free Full Text]
35
35. Pancino, G., H. Ellerbrok, M. Sitbon, and P. Sonigo. 1994. Conserved framework of envelope glycoproteins among lentiviruses. Curr. Top. Microbiol. Immunol. 188:77-105.[Medline]
36
36. Pancino, G., I. Fossati, C. Chappey, S. Castelot, B. Hurtrel, A. Moraillon, D. Klatzmann, and P. Sonigo. 1993. Structure and variations of feline immunodeficiency virus envelope glycoproteins. Virology 192:659-662.[CrossRef][Medline]
37
37. Phillips, T. R., R. L. Talbott, C. Lamont, S. Muir, K. Lovelace, and J. H. Elder. 1990. Comparison of two host cell range variants of feline immunodeficiency virus. J. Virol. 64:4605-4613.[Abstract/Free Full Text]
38
38. Rigby, M. A., N. Mackay, G. Reid, R. Osborne, J. C. Neil, and O. Jarrett. 1996. Immunogenicity of a peptide from a major neutralising determinant of the feline immunodeficiency virus surface glycoprotein. Vaccine 14:1095-1102.[CrossRef][Medline]
39
39. Sakaida, H., T. Hori, A. Yonezawa, A. Sato, Y. Isaka, O. Yoshie, T. Hattori, and T. Uchiyama. 1998. T-tropic human immunodeficiency virus type 1 (HIV-1)-derived V3 loop peptides directly bind to CXCR-4 and inhibit T-tropic HIV-1 infection. J. Virol. 72:9763-9770.[Abstract/Free Full Text]
40
40. Shimojima, M., T. Miyazawa, Y. Ikeda, E. L. McMonagle, H. Haining, H. Akashi, Y. Takeuchi, M. J. Hosie, and B. J. Willett. 2004. Use of CD134 as a primary receptor by the feline immunodeficiency virus. Science 303:1192-1195.[Abstract/Free Full Text]
41
41. Siebelink, K. H., M. L. Bosch, G. F. Rimmelzwaan, R. H. Meloen, and A. D. Osterhaus. 1995. Two different mutations in the envelope protein of feline immunodeficiency virus allow the virus to escape from neutralization by feline serum antibodies. Vet. Immunol. Immunopathol. 46:51-59.[CrossRef][Medline]
42
42. Siebelink, K. H., I. H. Chu, G. F. Rimmelzwaan, K. Weijer, A. D. Osterhaus, and M. L. Bosch. 1992. Isolation and partial characterization of infectious molecular clones of feline immunodeficiency virus obtained directly from bone marrow DNA of a naturally infected cat. J. Virol. 66:1091-1097.[Abstract/Free Full Text]
43
43. Speck, R. F., K. Wehrly, E. J. Platt, R. E. Atchison, I. F. Charo, D. Kabat, B. Chesebro, and M. A. Goldsmith. 1997. Selective employment of chemokine receptors as human immunodeficiency virus type 1 coreceptors determined by individual amino acids within the envelope V3 loop. J. Virol. 71:7136-7139.[Abstract]
44
44. Sundstrom, M., U. Chatterji, L. Schaffer, S. de Rozieres, and J. H. Elder. 2008. Feline immunodeficiency virus OrfA alters gene expression of splicing factors and proteasome-ubiquitination proteins. Virology 371:394-404.[Medline]
45
45. Talbott, R. L., E. E. Sparger, K. M. Lovelace, W. M. Fitch, N. C. Pedersen, P. A. Luciw, and J. H. Elder. 1989. Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proc. Natl. Acad. Sci. USA 86:5743-5747.[Abstract/Free Full Text]
46
46. Verschoor, E. J., L. A. Boven, H. Blaak, A. van Vliet, M. C. Horzinek, and A. de Ronde. 1995. A single mutation within the V3 envelope neutralization domain of feline immunodeficiency virus determines its tropism for CRFK cells. J. Virol. 69:4752-4757.[Abstract]
47
47. Willett, B. J., K. Adema, N. Heveker, A. Brelot, L. Picard, M. Alizon, J. D. Turner, J. A. Hoxie, S. Peiper, J. C. Neil, and M. J. Hosie. 1998. The second extracellular loop of CXCR4 determines its function as a receptor for feline immunodeficiency virus. J. Virol. 72:6475-6481.[Abstract/Free Full Text]
48
48. Willett, B. J., M. J. Hosie, J. C. Neil, J. D. Turner, and J. A. Hoxie. 1997. Common mechanism of infection by lentiviruses. Nature 385:587.[Medline]
49
49. Willett, B. J., E. L. McMonagle, F. Bonci, M. Pistello, and M. J. Hosie. 2006. Mapping the domains of CD134 as a functional receptor for feline immunodeficiency virus. J. Virol. 80:7744-7747.[Abstract/Free Full Text]
50
50. Willett, B. J., L. Picard, M. J. Hosie, J. D. Turner, K. Adema, and P. R. Clapham. 1997. Shared usage of the chemokine receptor CXCR4 by the feline and human immunodeficiency viruses. J. Virol. 71:6407-6415.[Abstract]
51
51. Wyatt, R., N. Sullivan, M. Thali, H. Repke, D. Ho, J. Robinson, M. Posner, and J. Sodroski. 1993. Functional and immunologic characterization of human immunodeficiency virus type 1 envelope glycoproteins containing deletions of the major variable regions. J. Virol. 67:4557-4565.[Abstract/Free Full Text]

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Journal of Virology, September 2008, p. 9134-9142, Vol. 82, No. 18
0022-538X/08/$08.00+0     doi:10.1128/JVI.00394-08
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