Activity of the Mason-Pfizer monkey virus fusion protein is modulated by single amino acids in the cytoplasmic tail

Abstract

Mason-Pfizer monkey virus (M-PMV) encodes a transmembrane glycoprotein with a 38-amino-acid-long cytoplasmic tail. After the release of the immature virus, a viral protease-mediated cleavage of the cytoplasmic tail (CT) results in the loss of 17 amino acids from the carboxy terminus and renders the envelope protein fusion competent. To investigate the role of individual amino acid residues in the CT in fusion, a series of mutations was introduced, and the effects of these mutations on glycoprotein biosynthesis and fusion were examined. Most of the alanine-scanning mutations in the CT had little effect on fusion activity. However, four amino acid substitutions (threonine 4, lysine 7, glutamine 9, and isoleucine 10) resulted in substantially increased fusogenicity, while six (leucine 2, phenylalanine 5, isoleucine 13, lysine 16, proline 17, and glycine 31) resulted in much-reduced fusion. Interestingly, the bulk of these mutations are located upstream of the CT cleavage site in a region that has the potential to form a coiled-coil in the Env trimer. Substitutions at glutamine 9 and isoleucine 10 with alanine had the most dramatic positive effect and resulted in the formation of large syncytia. Taken together, these data demonstrate that individual residues within the cytoplasmic domain of M-PMV Env can modulate, in both a positive and negative manner, biological functions that are associated with the extracellular domains of the glycoprotein complex.

FIG. 1.

Schematic diagram of M-PMV TM protein organization. The amino acid sequence of the cytoplasmic tail is shown. Amino acid residues that were substituted with alanines in this study (position 2 to position 33 of the cytoplasmic domain of the TM protein) are shown in bold. The designation of the truncation mutants is given on the left. The * indicates a stop codon introduced on that site to generate a truncation mutation.

FIG. 2.

Synthesis and processing of mutant and wild-type glycoproteins. (A) COS-1 cells were transfected with the pTMT expression vector containing either the wild-type or mutant env genes; at 48 h posttransfection, the cells were labeled with [³H]leucine for 30 min and immunoprecipitated with goat anti-M-PMV serum as described in the text. The mutant designation is shown above each lane, and the position of the precursor glycoprotein, Pr86, is indicated in the center. (B) Processing of the Env polyprotein precursor protein. Following a 4-h chase in unlabeled medium, Env proteins were immunoprecipitated with goat anti-M-PMV serum and analyzed by 12% SDS-PAGE. The positions of the precursor glycoprotein, Pr86, and the cleavage products, gp70 and gp22, are shown. (C) Secretion of SU into culture media. Following pulse-chase, culture media were collected, immunoprecipitated with goat anti-M-PMV serum, and analyzed for the released SU proteins. (D) Surface biotinylation of envelope glycoproteins. Transfected cells expressing radiolabeled glycoprotein were biotinylated and immunoprecipitated with goat anti-M-PMV serum. The immunoprecipitated samples were boiled in SDS, and streptavidin-agarose beads were added to isolate biotinylated Env proteins. The biotinylated Env proteins were then analyzed by SDS-PAGE. Mutations are designated by the standard one-letter code of the wild-type amino acid followed by its numerical position within the cytoplasmic domain and then the resulting mutant amino acids. The mutant designation is shown above each lane, and the positions of the viral bands are indicated in the middle.

FIG. 3.

Cell-cell fusion assay of the alanine-scanning mutants. COS-1 cells were transfected with the pTMT expression vector encoding either the mutant or wild-type env genes. COS-1 cells expressing glycoproteins were mixed at a 1:2 ratio with HOS-CD4/LTR-hGFP cells and replated. The cells were analyzed for expression of GFP and quantitated by FACS 36 h later as described in Materials and Methods. The * indicates syncytium formation in the context of full-length glycoprotein. The mean percentage (± the standard deviation) of GFP-expressing cells relative to wild type from three independent experiments is shown for each of the mutants.

FIG. 4.

Fusion of COS-1 cells with GHOST cells by Q9 mutant envelope proteins. COS-1 cells were transfected as described for protein expression. At 24 h posttransfection, they were mixed with GHOST cells. Cells were fixed and then stained with Giemsa stain. Cells were photographed 24 h later.

FIG. 5.

Cell-cell fusion assay of the truncation mutants. COS-1 cells were transfected with the pTMT expression vector encoding either the truncation mutant or wild-type env genes. COS-1 cells expressing glycoproteins were mixed at a 1:2 ratio with HOS-CD4/LTR-hGFP cells and replated. The cells were analyzed for expression of GFP and quantitated by FACS 24 h later as described in Materials and Methods. The * indicates syncytium formation in the context of glycoprotein.

FIG. 6.

Expression of Q9 mutant envelope proteins. Proteins were expressed and labeled with ³H as described in Materials and Methods. (A) Pulse; (B) pulse-chase; (C) supernatants; (D) surface biotinylation. The mutant designation is shown above each lane, and the positions of the viral bands are indicated on the left.

FIG. 7.

Effects of mutations on the fusion kinetics of the glycoprotein in time course experiments. (A) Fusion activities of Q9 envelope proteins monitored by fluorescent dye transfer. COS-1 cells expressing both GFP and the M-PMV Env constructs were used as effector cells, and target JC-53BL cells were labeled with the cytoplasmic dye CellTracker Blue and the lipid dye Fast DiI. Doubly labeled JC-53BL cells were overlaid on the COS-1 cells, incubated for 2 h at 37°C, and then fixed in 4% paraformaldehyde. After fixation, cells were analyzed by flow cytometry for the percentage of Fast DiI- and CellTracker Blue-positive cells. Cells that were positive for GFP, Fast DiI, and CellTracker Blue were determined to be fused cells. (B and C) COS-1 cells were transfected with the pTMT expression vector encoding either the mutant or wild-type env genes. COS-1 cells expressing Q9 mutants were mixed with GHOST cells and replated. The cells were analyzed for expression of GFP and quantitated by FACS 24 h later (B) or 48 h later (C) as described in Materials and Methods. The * indicates syncytium formation in the context of the Q9 mutants. The mean percentage (± the standard deviation) of GFP-expressing cells relative to wild type from three independent experiments is shown for each of the mutants.

FIG. 8.

Predicted probability for coiled-coil formation in the cytoplasmic domain of M-PMV Env. The amino acid sequences of the membrane-spanning domain and cytoplasmic domain (residues 520 to 586 of Env) for the wild-type and mutant proteins were used for analysis by COILS (40). The output for the region spanning the first 30 amino acids of the cytoplasmic domain is shown.