Analysis and function of prototype foamy virus envelope N glycosylation

Abstract

The prototype foamy virus (PFV) glycoprotein, which is essential for PFV particle release, displays a highly unusual biosynthesis, resulting in posttranslational cleavage of the precursor protein into three particle-associated subunits, i.e., leader peptide (LP), surface (SU), and transmembrane (TM). Glycosidase digestion of metabolically labeled PFV particles revealed the presence of N-linked carbohydrates on all subunits. The differential sensitivity to specific glycosidases indicated that all oligosaccharides on LP and TM are of the high-mannose or hybrid type, whereas most of those attached to SU, which contribute to about 50% of its molecular weight, are of the complex type. Individual inactivation of all 15 potential N-glycosylation sites in PFV Env demonstrated that 14 are used, i.e., 1 out of 2 in LP, 10 in SU, and 3 in TM. Analysis of the individual altered glycoproteins revealed defects in intracellular processing, support of particle release, and infectivity for three mutants, having the evolutionarily conserved glycosylation sites N8 in SU or N13 and N15 in the cysteine-rich central "sheets-and-loops" region of TM inactivated. Examination of alternative mutants with mutations affecting glycosylation or surrounding sequences at these sites indicated that inhibition of glycosylation at N8 and N13 most likely is responsible for the observed replication defects, whereas for N15 surrounding sequences seem to contribute to a temperature-sensitive phenotype. Taken together these data demonstrate that PFV Env and in particular the SU subunit are heavily N glycosylated and suggest that although most carbohydrates are dispensable individually, some evolutionarily conserved sites are important for normal Env function of FV isolates from different species.

FIG. 1.

Schematic outline of the PFV glycoprotein domain organization. A) Schematic outline of the PFV Env domain organization. B) Alignment of wild-type and mutant protein sequences around potential glycosylation sites 8, 13, and 15. Amino acids altered in the mutant constructs are shown, and the corresponding construct names are given on the left. FP, fusion peptide; MSD, membrane-spanning domain; h, hydrophobic domain of the leader peptide. Potential N-linked carbohydrate chain attachment sites are indicated by Y and numbered 1 to 15. Asterisks mark evolutionarily conserved sites.

FIG. 2.

Glycosidase digestion of particle-associated FV proteins. FV particles were generated by cotransfection of 293T cells with pMH118 and the indicated Env expression constructs. Thirty-two hours posttransfection, cells were metabolically labeled with [³⁵S]methionine and [³⁵S]cysteine for 16 h. Subsequently, viral particles were purified by ultracentrifugation through 20% sucrose and digested with endo H or PNGase F as indicated or mock incubated before separation of viral proteins by SDS-PAGE and exposure to X-ray film. The individual PFV proteins are indicated on the right, and the assignment of the specific bands to individual subunits is indicated by different symbols: SU (*), TM (#), and SU-TM (†). Cells were cotransfected with pMH118 and the following: lanes 1, 4, and 7, pcDNA3.1+zeo (pcDNA); lanes 2, 5, and 8, pczHFVenvEM002 (wt); lanes 3, 6, and 9, pczHFVenvEM020 (ΔSU/TM).

FIG. 3.

Biochemical analysis of PFV Env N-glycosylation mutants. Western blot analysis of 293T cell (cell) and purified PFV particle (virus) lysates using (A) anti-PFV Gag, (B) anti-PFV Env LP (aa 1 to 86)-specific polyclonal rabbit antiserum, or (C) anti-PFV-SU (P3E10) monoclonal hybridoma supernatant is shown. The individual PFV proteins are indicated. 293T cells were cotransfected with the PFV Gag/Pol-expressing, replication-defective retroviral vector pMH118 and the respective PFV Env expression construct as indicated: lane 1, pczHFVenvEM058 (ΔN1); lane 2, pczHFVenvEM077 (ΔN2); lane 3, pczHFVenvEM078 (ΔN3); lane 4, pczHFVenvEM105 (ΔN4); lane 5, pczHFVenvEM106 (ΔN5); lane 6, pczHFVenvEM107 (ΔN6); lane 7, pczHFVenvEM108 (ΔN7); lane 8, pczHFVenvEM109 (ΔN8); lane 9, pczHFVenvEM110 (ΔN9); lane 10, pczHFVenvEM111 (ΔN10); lane 11, pczHFVenvEM112 (ΔN11); lane 12, pczHFVenvEM113 (ΔN12); lane 13, pczHFVenvEM114 (ΔN13); lane 14, pczHFVenvEM115 (ΔN14); lane 15, pczHFVenvEM116 (ΔN15); lane 16, pczHFVenvEM002 (wt); lane 17, pcDNA3.1+zeo (pcDNA). For lane 18, transfection was with only empty expression vector.

FIG. 4.

Cell surface expression of selected PFV Env N-glycosylation mutants. Transfected 293T cells were metabolically labeled, and subsequently cell surface proteins were specifically biotinylated. (A) Following immunoprecipitation with an FV-positive monkey serum, SDS-polyacrylamide gel electrophoresis, and blotting to nitrocellulose membranes, biotinylated PFV Env proteins were chemiluminescently detected by incubation with streptavidin-horseradish peroxidase, ECL-Plus. (B) Subsequently, total cellular Env expression was visualized by PhophorImager analysis after the chemiluminescence signal was allowed to fade overnight. 293T cells were cotransfected with the PFV Gag/Pol-expressing, replication-defective retroviral vector pMH118 and the respective PFV Env expression construct as indicated: lane 1, pczHFVenvEM109 (ΔN8); lane 2, pczHFVenvEM114 (ΔN13); lane 3, pczHFVenvEM116 (ΔN15); lane 4, pczHFVenvEM112 (ΔN11); lane 5, pczHFVenvEM002 (wt); lane 6, pcDNA3.1+zeo (pcDNA).

FIG. 5.

Infectivity of viral supernatants. The infectivities of supernatants of 293T cells cotransfected with the indicated PFV Env expression constructs and A) the EGFP-expression FV vector pMH118 or B) the β-galactosidase-expressing FV vector pMH120 are shown. For calculation of the relative infectivities by the EGFP transfer assay, the values for EGFP-positive target cells obtained by using the wild-type PFV Env expression plasmid were arbitrarily set to 100%. The mean values and standard deviations for two independent experiments with a total of 8 to 12 values for each individual Env protein are shown.

FIG. 6.

Analysis of selected PFV Env mutants. A to D) Western blot analysis of 293T cell (cell) and purified PFV particle (virus) lysates using anti-PFV Gag and anti-PFV Env LP (aa 1 to 86)-specific polyclonal rabbit antisera. The individual PFV proteins are indicated. E) Infection analysis of supernatants of 293T cells cotransfected with pMH118 and the individual PFV Env expression constructs as indicated. For calculation of the relative infectivities by the EGFP transfer assay, the values of EGFP-positive target cells obtained by using the wild-type PFV Env expression plasmid were arbitrarily set to 100%. The mean values and standard deviations for two independent exper-iments with a total of 4 to 12 values for each individual Env protein are shown. Cotransfection was with pMH118 and the following: lane 1, pczHFVenvEM002 (wt); lane 2, pczHFVenvEM109 (ΔN8); lane 3, pczHFVenvEM131 (ΔN8.1); lane 4, pczHFVenvEM151 (ΔN8.2); lane 5, pczHFVenvEM114 (ΔN13); lane 6, pczHFVenvEM132 (ΔN13.1); lane 7, pczHFVenvEM152 (ΔN13.2); lane 8, pczHFVenvEM116 (ΔN15); lane 9, pczHFVenvEM133 (ΔN15.1); lane 10, pczHFVenvEM153 (ΔN15.2); lane 11, pczHFVenvEM112 (ΔN11); lane 12, pczHFVenvEM002 (wt); lane 13, pcDNA3.1+zeo (pcDNA).

FIG. 7.

Temperature sensitivity analysis of selected PFV Env mutants. The infectivities of supernatants of 293T cells cotransfected with the indicated PFV Env expression constructs generated at 37°C (solid bars) or 30°C (open bars) was determined on HT1080 cells by using the EGFP transfer assay. For calculation of the relative infectivities, the values of EGFP-positive target cells obtained by using the wild-type PFV Env expression plasmid were arbitrarily set to 100%. The mean values and standard deviations for two independent experiments with a total of 4 to 8 values for each individual Env protein are shown.