The PFV envelope glycoprotein displays a highly unusual biosynthesis. Similar to other retroviral glycoprotein precursor proteins, it is proteolytically processed into mature subunits during its transport to the cell surface. However, unlike as reported for other retroviral glycoproteins, two cleavage events, which result in the generation of LP, SU, and TM subunits, take place only late during intracellular transport, most likely by furin-like proteases (4
). In this study we analyzed the extent of N glycosylation of the different PFV Env subunits and the function of individual N-glycosylation sites for viral replication. We demonstrate that all potential N-glycosylation sites except one that are present in the PFV Env protein sequence are indeed modified. The LP subunit contains only one carbohydrate chain, whereas the TM and SU subunits have oligosaccharides attached at 3 and 10 sites, respectively. Interestingly, the pattern of digestion by different glycosidases suggests that the LP and TM subunits contain only carbohydrate chains of the high-mannose or hybrid type, whereas the majority of the oligosaccharides attached to the SU subunit are of the complex type. Recent results on the binding of HIV and Ebola virus GPs to the C-type lectins DC-SIGN and DC-SIGNR suggest a role for high-mannose-type carbohydrate structures in determining the specificity of the interaction (16
). Therefore, it might be possible that the specific carbohydrate composition of individual PFV Env subunits, in particular that of SU, may influence binding to target cells and interaction with cellular receptors.
Sequence comparison with glycoproteins from other FV species revealed the conservation of 2 out of 10 N-glycosylation sites in SU and all three sites in TM (data not shown). Mutation analysis of the individual glycosylation sites demonstrated that all the nonconserved sites are dispensable for PFV envelope function in vitro when inactivated. Interestingly, only mutation of some of the conserved carbohydrate attachment sites resulted in detectable phenotypes in our analysis, suggesting that these sites might be important for glycoprotein function of all FV isolates. Mutation of the first (N13) and third (N15) glycosylation site in the TM subunit, which are located in the unique cysteine-rich, prolonged central “sheets-and-loops” region of unknown function (30
), resulted in greatly diminished particle release. In addition, both mutants showed a reduced cell-associated LP processing and cell surface expression, indicating that the intracellular transport of these proteins is less efficient than wild type. Since the extracellular domain of retroviral TM subunits normally harbors the oligomerization domains and correct oligomerization of glycoproteins is essential for proper intracellular transport, the results might indicate that these two mutations affect oligomerization, although this was not addressed directly in our analysis. The results with alternative mutants affected in both glycosylation sites (ΔN13.1 and ΔN15.1) or neighboring sequences (ΔN13.2 and ΔN15.2) suggest that in case of N13 the glycosylation at this position itself is important for Env function. For N15, alternative inhibition of glycosylation (ΔN15.1) led to a significant improvement of particle release and infectivity, and an amino acid change not affecting glycosylation (ΔN15.2) resulted in a slight reduction in the infectivity of the released particles. Furthermore, analysis of the temperature dependence of the phenotypes observed for individual mutants suggests that only the ΔN15 mutant displays a significant temperature-sensitive phenotype. Therefore, in this case the sequence surrounding N15 rather than the attached carbohydrate chain, or a perhaps a mixture of both, seems to be important for normal glycoprotein function. This resembles the case for Friend ecotropic murine leukemia virus Env protein, where only one of eight signals for N-linked glycan attachment is critical for envelope function (12
). Subsequently, a more detailed mutational analysis of this MuLV N-linked glycosylation site and surrounding sequences revealed that N glycosylation per se is not required for MuLV Env function but that the region surrounding this glycosylation site mediates envelope folding and the stability of the interaction between SU and TM (15
In the PFV Env SU subunit, only inactivation of the conserved carbohydrate attachment site N8 impaired Env function. Similar to what was observed for the ΔN13 and ΔN15 mutants, the ΔN8 mutant protein was not properly transported intracellularly, as indicated by the lack of LP cleavage of the mutant precursor protein and cell surface expression. However, unlike the two mutants with mutations in TM, neither ΔN8 nor the alternative glycosylation site mutant ΔN8.1 showed any detectable infectivity or particle release. In contrast, the ΔN8.2 mutant, retaining glycosylation but having the variable second amino acid of the N-X-S/T glycosylation site signal sequence mutated, displayed a wild-type phenotype. This strongly suggests that the carbohydrate chain itself serves an important role in PFV Env function, most likely in folding, as both glycosylation mutants showed no proteolytic processing of the precursor protein. However, since this glycosylation site is conserved in the SU subunits of FV isolates from different species, it is possible that it may be involved in other Env function, such as interaction with the cellular receptor, although this apparently cannot be addressed in the context of the full-length PFV Env protein due to the transport defect of these mutants. However, preliminary data obtained using PFV Env immunoadhesins indicate that glycosylation site N8 or surrounding sequences might be important for receptor interaction (A. Duda and D. Lindemann, unpublished observations). Since the digestion pattern of the SU subunit with different glycosidases suggests that the majority of the carbohydrate chains in SU are of the complex type, it would be interesting to know which sites are of the high-mannose or hybrid type and in particular what type of carbohydrate chain is added to N8.