N-glycans on other paramyxovirus fusion proteins are known to be required for proper expression, conformational integrity, and efficient fusogenicity of the protein (13
). Surprisingly, we show in this report that many of the N-glycans on NiV-F are not required for conformational integrity and actually reduce fusion efficiency but protect the fusion protein from neutralizing antibodies. Indeed, removal of multiple N-glycans on NiV-F resulted in hyperfusogenic phenotypes, and the presence of N-glycans on NiV-F likely reduced the rate of six-helix bundle formation, resulting in slower fusion kinetics. The hyperfusogenic phenotypes of the NiV-F N-glycan mutants were most marked in 293T cells, slightly less apparent in Vero cells, and least apparent in MDCK cells. This is consistent with the slight increase in fusogenicity noted for the F3 and F5 N-glycan mutants in MDCK cells by Moll et al. (21
). Cell-type-specific glycosylation (the type and quantity of glycans that are added on) and expression levels likely account for these differences. Indeed, Western blotting of NiV-F produced in 293T cells versus that produced in MDCK cells indicate that they migrate with slightly different mobilities, suggestive of glycosylation differences (data not shown).
N-glycans on viral envelope glycoproteins can “shield” viruses from NAbs. To our knowledge, the role of N-glycans in viral escape from NAb has not been documented for paramyxoviruses but is well documented for viruses such as HIV (42
), SIV (32
), EIAV (33
), HepB (16
), and influenza virus (37
) (reviewed in reference 27
). Here, we uncovered a role for N-glycans on the NiV fusion protein as a “glycan shield” against NAb (Fig. ). The “unshielded” NiV-F N-glycan mutants were more neutralizable by polyclonal anti-NiV-F than was fully glycosylated NiV-F. Future testing of these N-glycan mutans against convalescent-phase sera from NiV-infected patients will help determine the veracity of this “glycan shield” hypothesis. Our results may have implications for NiV vaccine development, as this increased neutralization sensitivity may be a result of increased peptide epitope exposure. Selective deglycosylation and the resultant increased epitope exposure may result in more potent and/or broader neutralizing antibody responses, as has been observed for SIV (32
), EIAV (33
), and HepB (16
). Thus, we speculate that these neutralization-sensitive N-glycan mutants may elicit improved NAb responses.
We found that N-glycans on the NiV fusion protein (NiV-F) “protected” NiV-F against neutralization by NAb (Fig. ). However, removal of these N-glycans, in general, also greatly increased its fusogenicity (Fig. and ). These varied roles for N-glycans on NiV-F contrast sharply with the role of N-glycans on other paramyxoviral glycoproteins, as removal of specific N-glycans from the Newcastle disease, measles, and Sendai virus F proteins results in severe defects in fusion (13
). Nevertheless, our findings that the hyperfusogenic NiV-F N-glycan mutants also displayed increased fusion kinetics and increased resistance to a novel NiV-F heptad repeat fusion inhibitory protein (NiV-HR2-Fc) (Fig. ) are consistent with the mechanisms underlying the hyperfusogenic phenotypes in other viruses with class I fusion proteins. For example, the hyperfusogenic V3 loop and cytoplasmic tail of HIV-1 envelope glycoprotein mutants also show faster fusion kinetics and display increased resistance to heptad repeat peptide inhibition (1
). We note that while hyperfusogenic phenotypes in viruses with class I fusion proteins are commonly identified, it is less common, if not novel, to find a whole class of mutations, such as removal of N-glycans, that result additively or synergistically in hyperfusogenicity. We cannot formally exclude the possibility that the resistance of our N-glycan mutants to heptad repeat inhibition is due to conformational differences that result in the heptad repeat region binding sites being less accessible. Therefore, it remains to be determined how N-glycans on NiV-F actually modulate the kinetics of fusion. Do the N-glycans stabilize the metastable prefusogenic conformation of NiV-F, or do they physically impede six-helix bundle formation due to their bulky hydrophilic nature?
To obtain further insights into the relatively novel roles for N-glycans in NiV-F, we compared the primary and tertiary structures of 12 paramyxovirus fusion proteins. The three-dimensional (3-D) structure of each paramyxovirus F protein was modeled based on the solved crystal structure of the HPIV-3 F protein (see Materials and Methods), and the established N-linked glycosylation sites were mapped onto the predicted structure (Fig. ). Our modeling of the NiV-F N-glycan sites showed that while the F2, F3, and F4 N-glycan sites do not align with the primary sequence of other paramyxoviruses (except for HeV) (Fig. ), the F2 and F4 N-glycan sites clustered in the similar globular head and neck regions with N-glycans from many other paramyxoviruses (Fig. ) (40
). The F5 N-glycan site aligned only with SV5 and NDV (in addition to HeV) in primary sequence and also mapped to the HR-B region, a relatively uncommon site for N-glycans. Despite this similarity, the N-glycans in the HR-B region of the SV5 and NDV F proteins seem to be important for efficient cleavage and cell surface expression (3
) or fusogenicity (19
). This is clearly different from the NiV/HeV F5 N-glycan mutation, which enhances fusogenicity, as seen from our data for NiV and those of Carter et al. for HeV (7
). The modulating parameter(s) of the relatively unique nature of the F5 N-glycan remains to be determined.
FIG. 7. Comparison of the primary and tertiary structures of the NiV, HeV, MeV, RPV, PPRV, CDV, PDV, SeV, HPIV-1, HPIV-3, SV5, and NDV paramyxovirus F proteins. The 3-D structure of each paramyxovirus F protein was modeled based on the solved crystal structure (more ...)
However, we note that the NiV/HeV F3 N-glycan mapped to a unique position in the HR-C region. It is interesting that the F3 NiV-F protein had the highest level of fusogenicity of all the single N-glycan mutant proteins. To our knowledge, the HR-C region has not been previously implicated in the fusion process, but our data suggest that a bulky hydrophilic structure in this area clearly inhibits fusion, perhaps by impeding the conformational changes that NiV-F undergoes between the pre- and postfusion states.
Although some level of association is generally required between the fusion and attachment proteins of paramyxoviruses in order for productive fusion to occur (15
), current models suggest that dissociation of the attachment protein from the fusion protein after receptor engagement is required for efficient fusion peptide exposure (38
). These models predict that a greater propensity for attachment protein/fusion protein dissociation might lead to greater fusion peptide exposure and thus increased fusogenicity. Indeed, our results provide direct experimental evidence for this model. The fusogenicity of the N-glycan mutants was negatively correlated with the relative avidity of F/G interactions as quantified by coimmunoprecipitation (Fig. ). Higher levels of shedding of the attachment subunit from the fusion glycoprotein subunit have been linked to higher fusogenicity in viruses such as HIV and Moloney murine leukemia virus, where similar models of attachment protein dissociation from the fusion protein prior to fusion peptide exposure have been suggested (2
It remains to be determined how N-glycans on NiV-F actually stabilize F/G interactions and why N-glycans on NiV-F appear to play a different role than N-glycans on the fusion proteins of other paramyxoviruses. Do the unique roles of the NiV-F N-glycans in NiV entry contribute to the unusual pathogenicity of the virus? Moreover, is there an advantage of down regulating the fusogenic capacity of the NiV-F protein by N-glycan addition so that the virus does not kill the host before it can successfully spread? Can the infectivity of NiV differ in vivo when produced in cell types with differential glycosylation machinery? Answers to these questions will enhance our understanding of the pathobiology of this deadly emerging virus.