INTRODUCTION

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Receptor recognition by murine leukemia viruses (MLVs) and feline leukemia viruses (FeLVs) is determined by the amino-terminal domain of their surface envelope glycoproteins (2, 4, 5, 7, 22, 28, 35). This domain consists of several conserved sequences that are interrupted by variable regions termed VRA and VRB. VRA and VRB are highly divergent between MLVs, FeLVs, and other type C retroviruses, both in length and in sequence, and they have been implicated in receptor specificity (4, 5, 7, 35). Most of the sequence diversities in VRA and VRB occur in regions that are enclosed by cysteine residues that are conserved in all mammalian type C envelope glycoproteins. As confirmed by a recent X-ray crystallographic study of an ecotropic MLV envelope glycoprotein (10), these conserved cysteines form disulfide-bonded loops, with two loops within VRA and one loop in VRB. Consistent with these ideas, specific residues critical for receptor recognition have been identified within the first disulfide-bonded loop of VRA (VRA1), in the envelope glycoproteins of ecotropic MLV (2), amphotropic MLV (A-MLV) (3, 35), and feline leukemia virus subgroups B (FeLV-B) (35) and C (8, 32). Residues outside VRA1 have also been implicated in receptor recognition. Two amino acids located between VRA1 and VRA2 in the envelope glycoprotein of PVC-211 MLV, an ecotropic MLV variant, are responsible for its enhanced ability to infect Chinese hamster ovary cells (21). Residues adjacent to VRA1 in A-MLV and 10A1 MLV envelope glycoproteins have also been implicated in receptor recognition (12, 13).

Surprisingly, the amino-terminal domains of FeLV-B and A-MLV gp70s share substantial sequence identity in VRA and VRB, despite their use of different cell surface receptors. FeLV-B uses the Na⁺-dependent phosphate symporter Pit1 (15, 26, 27, 37), which was originally identified as a receptor for gibbon ape leukemia virus (26), whereas A-MLV uses a related Na⁺-dependent phosphate symporter, Pit2 (15, 24, 38). Several studies have suggested that the presumptive extracellular loops 2 and 4 (ECL2 and -4) of Pit1 and Pit2 are essential for receptor function (14, 17-19, 30, 35, 36). Additional studies have implicated Pit1 ECL5 in FeLV-B infections (29, 35), and we have previously reported that Pit2 ECL5 may also be involved in A-MLV infections (35).

A major problem in previous efforts to identify critical sites involved in functional interactions of retroviral envelope glycoproteins and receptors has derived from the possible existence of multiple contact sites in both components. Consequently, identification of an important amino acid in either gp70 or its receptor does not provide evidence concerning its contact site in other component. Moreover, traditional mutagenesis approaches have been complicated because loss-of-function mutations in gp70 or the receptor can be caused by diverse mechanisms including global abnormalities of folding or posttranslational processing. Because of this problem, effects of mutations are often considered to be significant only if they reduce function by several orders of magnitude.

Recently, we initiated studies that can potentially overcome some of these problems. We found that viruses pseudotyped with several chimeric FeLV-B/A-MLV envelope glycoproteins were unable to utilize native Pit1 or Pit2 as a receptor but could efficiently use specific Pit1/Pit2 chimeras (35). This gain-of-function evidence enabled us to identify specific functional interactions of VRA and VRB with particular correspondent sites in the receptors. Specifically, we obtained evidence that FeLV-B and A-MLV VRA functionally interact with the receptor's ECL4/ECL5 region, whereas VRB interact with ECL2 (35). By using an FeLV-B/A-MLV chimeric envelope that was specifically deficient in its VRA interactions with a Pit1/Pit2 chimeric receptor and by substituting specific residues within this chimeric envelope so that it could gain receptor recognition, we determined that residues FeLV-B F60 and P61 and residues A-MLV Y60 and V61 were important for specific recognition of the receptor's ECL4/ECL5 region. As more thoroughly discussed below, subsequent mutagenesis studies of the A-MLV envelope glycoprotein appeared to contradict several of our results. Battini et al. reported that A-MLV VRB is not essential for Pit2 utilization (3). Furthermore, Han et al. suggested that A-MLV residues Y60 and V61 were not individually necessary for interaction with Pit2, whereas a sequence between VRA1 and VRA2 was essential (13).

We have attempted to address these issues and to develop a more comprehensive map of the functional gp70-receptor interactions. For this purpose, we generated pseudotype virions bearing mutant FeLV-B envelopes in which specific FeLV-B VRA sequences were substituted with corresponding A-MLV sequences, and we assessed the infectivity of the virions on mouse cells that expressed specific chimeric Pit1/Pit2 receptors. These Pit1/Pit2 chimeras were all active in phosphate transport, confirming their proper folding and processing to the cell surfaces. Moreover, all of the FeLV-B/A-MLV chimeric or mutant envelope glycoproteins used in this investigation were processed and incorporated into virion particles (data not shown). Our results strongly suggest that VRA1 residues 60 and 61 are important for receptor recognition and that they functionally interact in a specific manner with a receptor sequence in the presumptive ECL5 region. In addition, a gp70 sequence at positions 66 to 78, located between VRA1 and VRA2, specifically interacts with ECL4 of the receptor. Finally, we identified a region of A-MLV VRB that functionally interacts with ECL2 of the receptor. These results provide a map of the functionally important virus-receptor interactions and indicate that the virus specifically recognizes multiple sites in the receptor protein.

	MATERIALS AND METHODS

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Cell lines. Mus dunni tail fibroblast (MDTF) cells and TELCeB6 packaging cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum. TELCeB6 cells (provided by Y. Takeuchi and F. L. Cosset) (9) produce noninfectious virions which contain the nlslacZ retroviral vector (11). MDTF cells expressing chimeric Pit1/Pit2 proteins were generated by transduction with pseudotyped A-MLV containing chimeric Pit1/Pit2 genes. The pseudotype virions were generated by transfecting the Pit1/Pit2 cDNA expression vectors pLGGRSN, pLGRRSN, and pLGGrGSN (provided by A. Dusty Miller) into PA12 amphotropic packaging cells (23). Transfected cells were incubated with G418 selection medium (1.2 mg of G418/ml); resistant cells were pooled, and the viral supernatant was harvested to transduce MDTF cells. Transduced MDTF cells were selected with 1.5 mg of G418 per ml, and individual resistant clones were analyzed for phosphate uptake. Clones showing the best uptake, and therefore representing high Pit1/Pit2 expression levels, were used for infection studies. MDTF cells expressing Pit1 (GGG) were generated as described before (35).

Mutagenesis of FeLV-B and A-MLV envelopes. The baBB, baBB1, baBB2, and baBB3 mutant FeLV-B envelopes and the ABA envelope (see Fig. 1 and 3) were generated in our previous study (35). Specific FeLV-B VRA residues were mutated by PCR mutagenesis as described before (35). The mutant envelopes were subsequently cloned into the FBsalf retroviral expression vector (9). The ABA1 mutant envelope was generated by mutagenesis of specific A-MLV VRB sequence to the corresponding FeLV-B VRB sequence (see Fig. 4). In addition, the A-MLV VRB disulfide-loop sequence was mutated to a foreign sequence that encoded a 16-amino-acid peptide containing the RGD-integrin recognition motif. This was done by PCR mutagenesis using the specific primer 5'-TGCCAAGCCGGTACCTTTGCGCTCCGCGGCGATAATCCCCAAGGATGC-3'. A BamHI-EcoRI A-MLV PCR fragment which includes the VRB mutations was ligated with an EcoRI-ClaI A-MLV envelope cDNA fragment into a BamHI-ClaI-digested FBsalf expression vector. All envelope cDNAs were sequenced to confirm the mutations by the Microbiology and Molecular Immunology Core Facility on a PE/ABD 377 sequencer by using dye terminator cycle-sequencing chemistry (Applied Biosystems, Foster City, Calif.).

Viruses and infection. The envelope cDNA expression vectors were transfected into TELCeB6 cell lines by calcium phosphate coprecipitation (Stratagene). Transfectants were selected with phleomycin (50 µg/ml), and resistant colonies were pooled 2 weeks after addition of selection. Viral supernatants were harvested and infection assays were carried out as previously described (35). Viral titers were expressed as the number of CFU per milliliter of viral supernatant.

	RESULTS

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Characteristics of chimeric FeLV-B/A-MLV envelopes and Pit1/Pit2 receptors. Figure 1 shows the chimeric FeLV-B/A-MLV envelopes (Fig. 1A) and Pit1/Pit2 receptors (Fig. 1B) used in this investigation. The gp70 envelope glycoproteins were separated into three distinct domains, an N-terminal domain containing VRA, a mid-domain containing VRB, and a C-terminal domain. Expression vectors for the chimeric or wild-type envelope genes were transfected into TELCeB6 retroviral packaging cells to produce pseudotype virions that encode -galactosidase (LacZ), and the viral infectivities were assayed in MDTF cells that expressed the specific Pit1/Pit2 chimeras. In our previous study, we found that the BBB virus (FeLV-B) could use GGG (Pit1) but not GGR as a receptor, whereas the ABB virus used GGR but not GGG (35); we have expanded on that observation in this investigation (see below). In addition, we used the GGrG receptor chimera, which was generated by replacing the critical nine-amino-acid ECL4 region A of Pit1 (14, 36) with the corresponding sequence from Pit2 (25).

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Structures of chimeric FeLV-B/A-MLV envelopes and chimeric Pit1/Pit2 proteins. (A) The chimeric FeLV-B/A-MLV envelopes were generated in our previous study (35). The variable regions VRA and VRB, the proline-rich region (PRR), and the restriction sites used to generate the chimeric envelope cDNAs are indicated. The two potential disulfide-bonded loops in VRA and one disulfide-bonded loop in VRB are indicated by the loop structures. BBB and AAA represent wild-type FeLV-B and A-MLV envelopes, respectively. (B) Topological model of wild-type and chimeric Pit1 and Pit2 receptors showing the five presumptive ECLs and the large cytoplasmic loop. The chimeric Pit1/Pit2 cDNAs were generated by Miller and Miller (25).

Amino acids 66 to 78 of FeLV-B and A-MLV gp70s functionally interact with ECL4 and/or ECL5 of their receptors. Recent mutagenesis studies by Han et al. suggested that residues Y60 and V61 of A-MLV gp70 are not individually essential for Pit2 recognition (13). Similarly, in our previous study we used mutant FeLV-B viruses in which specific VRA sequences were replaced with corresponding sequences from A-MLV, and we found that Y60 and V61 of A-MLV were individually insufficient to switch the receptor usage from GGG to GGR (35). However, in the context of other A-MLV VRA residues, our results suggested that the presence of the Y60-V61 dipeptide sequence was essential for utilization of the GGR receptor. To more thoroughly investigate this issue, we constructed the FeLV-B VRA substitution mutations shown in Fig. 2 and analyzed viral infectivities in MDTF cells that expressed GGG (Pit1) or GGR receptors. Mutation of either FeLV-B F60 to Y (BBB1 virus) or P61 to V (BBB2 virus) reduced titers of infection on GGG expressing cells only two- to fivefold compared to the BBB virus (Fig. 2), whereas no infection was observed on GGR cells. This confirms that these individual substitutions were insufficient for recognition of Pit2 sequences in the GGR receptor. However, the BBB3 virus that contains both F60Y and P61V mutations only weakly infected GGG cells, suggesting that FeLV-B residues F60 and P61 are important for efficient recognition of the Pit1 ECL4/ECL5 region. In addition, other FeLV-B VRA residues must also contribute to specific recognition of Pit1 ECL4/ECL5 because BBB3 virus weakly utilizes the GGG receptor. Moreover, the BBB3 virus was unable to utilize the GGR receptor, indicating that A-MLV residues Y60 and V61 alone were insufficient for functional recognition of Pit2 ECL4/ECL5. In contrast, the ABB virus that contains the complete A-MLV VRA sequence efficiently utilizes the GGR receptor (Fig. 2), suggesting that additional A-MLV VRA residues are involved in recognition of Pit2 ECL4/ECL5. Evidence described below strongly suggests that A-MLV residues Y60 and V61 are important for Pit2 recognition.

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Mutagenesis of FeLV-B VRA. (A) Sequences of BBB (FeLV-B) and AAA (A-MLV) VRAs. The VRA residues of FeLV-B were mutated to the corresponding A-MLV VRA residues by PCR mutagenesis. Unchanged amino acids are indicated by dots, and divergent amino acids are underlined in the AAA VRA sequence. The cysteine residues that form the first disulfide-bonded loop in VRA and which are conserved in all mammalian type C retroviruses are in boldface. (B) Infection of MDTF cells expressing GGG (Pit1) or GGR receptor with lacZ pseudotype virus bearing mutant BBB (FeLV-B) envelopes. Topological diagrams of the chimeric receptors expressed by MDTF cells are shown above the histograms. Titers are mean values of three infection studies; error bars are indicated.

Residues 60 and 61 specifically recognize the ECL5 region, whereas residues 66 to 78 recognize ECL4. The identification of two subregions within VRA that may functionally interact with the receptor's ECL4/ECL5 region raised the possibility that the gp70 subregions recognize distinct receptor sequences. To test this and to further investigate the above evidence, we used the GGG, GGR, and GGrG receptors (Fig. 1) to analyze virions containing the mutant FeLV-B envelopes, baBB1, baBB2, and baBB3 (Fig. 3A). The baBB and baBB1 viruses have the same host range as the BBB virus (Fig. 3B and reference 35), suggesting that the substituted A-MLV sequences including the VRA sequence EEWD at positions 50 to 53 are unimportant for specific receptor recognition. The baBB virus was not used in this study.

View larger version (47K): [in this window] [in a new window]   FIG. 3.   Susceptibility of GGG, GGR, and GGrG cells to pseudotype virus bearing mutant FeLV-B envelopes. (A) The chimeric baBB and mutant baBB envelopes were generated in our previous study (35). Divergent residues are underlined in the AAA sequence, and unchanged amino acids are indicated by dots. The conserved cysteines are highlighted in boldface. (B) Infection of MDTF cells expressing the GGG (Pit1), GGR, or GGrG receptor with lacZ pseudotype virus bearing mutant baBB envelopes. Topological diagrams of the chimeric receptors are shown above the histograms. Titers are mean values of three infection studies; error bars are indicated.

A-MLV and FeLV-B VRB residues 129 to 139 interact with ECL2. Previously we reported that FeLV-B and A-MLV VRB functionally interact with ECL2 of their receptors (35). This conclusion was based on studies of a chimeric A-MLV envelope glycoprotein (ABA in Fig. 1), in which the A-MLV VRB sequence had been substituted with the corresponding VRB sequence of FeLV-B. As shown in Fig. 4, the ABA virus only weakly infects normal MDTF cells which contain an endogenous Pit2 receptor (RRR) but strongly infects GRR cells. Because Pit1 and Pit2 contain identical ECL1 sequences, the presumptive extracellular surfaces of GRR and RRR (Pit2) differ only in ECL2.

View larger version (43K): [in this window] [in a new window]   FIG. 4.   Mutagenesis of A-MLV VRB. (A) A-MLV VRB residues were mutated to the corresponding FeLV-B VRB residues by PCR mutagenesis. The ABA envelope contains the full FeLV-B VRB sequence. Unchanged residues are represented by dots; divergent amino acids are underlined in the ABA sequence. The cysteine residues that form the disulfide-bonded loop in VRB are in boldface. (B) Infection of parental MDTF cells, which naturally express RRR, and MDTF cells expressing GRR receptor with pseudotype virions bearing mutant AAA (A-MLV) envelopes. Titers are mean values of three infection studies; error bars are indicated.

	DISCUSSION

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Mapping the functionally interacting surfaces of the A-MLV and FeLV-B glycoproteins with their cell surface receptors. In this investigation, we have further developed a novel strategy for simultaneously mapping the functionally interacting surfaces of a retroviral envelope glycoprotein and its cell surface receptor. Our approach has relied on the close sequence similarities between the gp70 glycoproteins of FeLV-B and A-MLV and also between their Pit1 and Pit2 cell surface receptors. Initially, we anticipated that chimeras of these viral glycoproteins and of these receptors would very likely fold into potentially functional structures that would be properly processed to cell surfaces or into virion particles; these expectations have been, with one exception (35), confirmed by our results. Thus, the Pit1/Pit2 chimeras were all processed to cell surfaces where they functioned as Na⁺-dependent phosphate symporters, and the envelope glycoprotein chimeras and site-directed mutants used in this study were also all properly processed and incorporated into virion particles (results not shown). Interestingly, many of the chimeric or mutated gp70s that we have analyzed cannot functionally recognize the native Pit1 or Pit2 receptors, although they can interact functionally with specific Pit1/Pit2 chimeras (Fig. 2 to 4). By using this approach, we have obtained evidence that A-MLV VRA1 residues Y60 and V61 specifically interact with the Pit2 presumptive ECL5 region and that the nearby sequence between amino acids 72 and 78 functionally interacts with ECL4. In addition, the VRB sequence appears to functionally interact with ECL2, and at least part of this interaction appears to involve gp70 residues 129 to 139 (Fig. 4). Interestingly, our results suggest that the corresponding sequences of FeLV-B gp70 also participate in functional interactions with the same regions of its Pit1 receptor. Thus, the maps of the functionally interacting surfaces of A-MLV with Pit2 and of FeLV-B with Pit1 are highly homologous and strikingly similar. These gp70-receptor interactions appear to involve multiple functionally important contacts that occur at discrete positions on their surfaces.

A possible alternative topology for Pit1 and Pit2. Recently, we have learned that the presumptive topology of Pit1 and Pit2 that has been assumed by workers in this field, including ourselves, may be incorrect (33; P. Rodrigues, C. Salaun, and J. M. Heard, personal communication). From the perspective of our results, it is relevant that the regions we have termed ECL2 and ECL4 are exposed to the extracellular milieu in both models. However, in the new model the region that we have termed ECL5 is intracellular, whereas the carboxyl-terminal sequence is extracellular. Consequently, the evidence we have presented with reference to ECL5 may, alternatively, pertain to this extreme carboxyl-terminal region of the receptors. If this alternative model is correct, our evidence would suggest that VRA residues 60 and 61 of FeLV-B and A-MLV functionally interact with this carboxyl-terminal region. Interestingly, the carboxyl termini of Pit1 and Pit2 are highly divergent in sequence. Resolution of these issues will require more detailed evaluations of these topological models. Until such resolution is available, studies designed to evaluate our conclusions regarding ECL5 must also investigate the extreme carboxyl-terminal region of the receptors.

Relationship to previous results. We believe that our results are generally in close agreement with previous evidence. For example, several groups have determined that the presumptive ECL2 and ECL4 of Pit1 and Pit2 are required for receptor function (14, 17-19, 29, 30, 35, 36). In addition, a region encompassing ECL5 and the extreme carboxyl-terminal region of Pit1 has also been implicated in FeLV-B reception (29, 35). Furthermore, the amino-terminal region of gp70 that includes VRA and VRB has been strongly implicated in specific associations with cell surface receptors for diverse type C retroviruses (4, 5, 7, 22, 35). As described above, specific amino acids essential for receptor utilization have been identified within the first disulfide-bonded VRA loop (VRA1) of several retroviruses (2, 3, 8, 32, 35) and between the two disulfide-bonded loops of VRA (12, 13, 21) in the region corresponding to residues 66 to 78 of FeLV-B and A-MLV that we have identified as important for interactions with Pit1 and Pit2 ECL4.