A comprehensive approach to mapping the interacting surfaces of murine amphotropic and feline subgroup B leukemia viruses with their cell surface receptors

Abstract

Because mutations in envelope glycoproteins of retroviruses or in their cell surface receptors can eliminate function by multiple mechanisms, it has been difficult to unambiguously identify sites for their interactions by site-directed mutagenesis. Recently, we developed a gain-of-function approach to overcome this problem. Our strategy relies on the fact that feline leukemia virus subgroup B (FeLV-B) and amphotropic murine leukemia virus (A-MLV) have closely related gp70 surface envelope glycoproteins and use related Na(+)-dependent phosphate symporters, Pit1 and Pit2, respectively, as their receptors. We previously observed that FeLV-B/A-MLV envelope glycoprotein chimeras spliced between the variable regions VRA and VRB were unable to use Pit1 or Pit2 as a receptor but could efficiently use specific Pit1/Pit2 chimeras. The latter study suggested that the VRA of A-MLV and FeLV-B functionally interact with the presumptive extracellular loops 4 and 5 (ECL4 and -5) of their respective receptors, whereas VRB interacts with ECL2. We also found that FeLV-B gp70 residues F60 and P61 and A-MLV residues Y60 and V61 in the first disulfide-bonded loop of VRA were important for functional interaction with the receptor's ECL4 or -5. We have now extended this approach to identify additional VRA and VRB residues that are involved in receptor recognition. Our studies imply that FeLV-B VRA residues F60 and P61 interact with the Pit1 ECL5 region, whereas VRA residues 66 to 78 interact with Pit1 ECL4. Correspondingly, A-MLV VRA residues Y60 and V61 interact with the Pit2 ECL5 region, whereas residues 66 to 78 interact with Pit2 ECL4. Similar studies that focused on the gp70 VRB implicated residues 129 to 139 as contributing to specific interactions with the receptor ECL2. These results identify three regions of gp70 that interact in a specific manner with distinct portions of their receptors, thereby providing a map of the functionally interacting surfaces.

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).

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.

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.

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.