Glycoprotein interactions in paramyxovirus fusion

Abstract

The Paramyxoviridae are enveloped, negative-stranded RNA viruses, some of which recognize sialic acid-containing receptors, while others recognize specific proteinaceous receptors. The major cytopathic effect of paramyxovirus infection is membrane fusion-induced syncytium formation. Paramyxoviruses are unusual in that the receptor-binding and fusion-promoting activities reside on two different spike structures, the attachment and fusion glycoproteins, respectively. For most paramyxoviruses, this distribution of functions requires a mechanism by which the two processes can be linked for the promotion of fusion. This is accomplished by a virus-specific interaction between the two proteins. An increasing body of evidence supports the notion that members of this family of viruses utilize this glycoprotein interaction in different ways in order to mediate the regulation of the fusion protein activation, depending on the type of receptor utilized by the virus.

Figure 1. A linear correlation between the level of fusion promotion and the extent of complex formation at the cell surface between Newcastle disease virus HN and fusion proteins

Several individual point mutations were introduced for residues A89, L90, P93 and L94 in a short domain in the stalk of the Newcastle disease virus HN protein. The mutated proteins were coexpressed with fusion (F) protein in baby hamster kidney cells using the vaccinia-T7 expression system. The abilities of the mutated HN proteins to complement the homologous F protein in the promotion of fusion were determined in a content-mixing assay. The amounts of the mutated HN proteins associated with a cleavage-site mutant form of the F protein (csmF) at the cell surface were determined using a co-IP assay involving radiolabeling and biotinylation of the two proteins and co-IP of HN with a monoclonal antibody to the F protein. The rationale for using csmF in these studies was to make it possible to compare complex formation in two nonfusing monolayers, thus separating complex formation from the possible dissolution of the complex following F protein activation and fusion. The percentage of mutated HN that co-IPs with the F protein relative to that of the wt protein is graphed versus the percentage of wt fusion. Each data point is labeled with the amino acid substitution of the mutated HN protein and represents the mean of at least four independent determinations of fusion by the content-mixing assay and at least two independent determinations of the amount of HN immunoprecipitated by an anti-F protein monoclonal antibody in the co-IP assay. The graph establishes a linear correlation between the extent of Newcastle disease virus HN–F protein complex formation at the cell surface and the extent of fusion. The failure to detect co-IP of HN at fusion levels less than 20% of wt indicates the detection limit of the co-IP assay. co-IP: Coimmunoprecipitation; HN: Hemagglutinin–neuraminidase; wt: Wild-type. Data taken from [67].

Figure 2. Two models for the promotion of paramyxovirus fusion

(A) The association model shows the attachment protein homotetramer and the metastable prefusion form of the F homotrimer existing separately at the surface prior to receptor binding (left panel). The attachment protein spike is depicted as being significantly taller than that of the prefusion F spike based on modeling studies [Lamb, Jardetzky, Pers. Comm.]. This aligns the head of F with the stalk of the attachment protein. The attachment protein is labeled as an HN protein in this figure based on the available data, suggesting that this mode of fusion promotion is specific to viruses that recognize sialic acid-containing receptors. Attachment of HN to receptors results in a conformational change in the protein that induces an interaction with the F protein, which, in turn, triggers the latter into its fusion-active form (right panel). This results in the insertion of the previously sequestered fusion peptide into the target membrane. In this model, the interaction between the two proteins may be quite transient, although this has not been proven. (B) The dissociation model proposes that the attachment protein and the metastable prefusion form of F are associated in a complex on the surface prior to receptor binding (left panel). The association with the attachment protein presumably serves to maintain F in its metastable, prefusion conformation. The attachment protein in this figure is labeled as H/G because the available data suggest that this mode of fusion promotion is specific to viruses whose attachment proteins, specifically, measles virus H and Nipah/Hendra G, recognize specific protein receptors. Attachment of H/G to protein receptors results in a conformational change in the protein that releases F to assume its fusion-active form (right panel). F: Fusion; G: Glycoprotein; H: Hemagglutinin; HN: Hemagglutinin–neuraminidase. Adapted from [40].

Figure 3. Nipah virus fusion is inversely correlated with the avidity of the F–G protein interaction

Reciprocal coimmunoprecipitations were performed on cell lysates of 293T cells transfected with wild-type Nipah virus (NiV) G protein and either wild-type NiV F or mutated forms of F protein lacking one or more N-glycans. The deletion of the N-glycans increases the extent of fusion, yet decreases the avidity of the complex between the F and G proteins. (A) The G protein was immunoprecipitated with anti-NiV-G-specific antisera and the samples were western blotted for NiV F protein. (B) The F protein was immunoprecipitated and the samples were western blotted for the G protein. The F–G protein interaction avidities were calculated and are graphed here against the fusion indices. A Pearson correlation analysis is included for each graph. The graph shows that the fusogenicity of the F protein mutants inversely correlates with the avidity of the complex formed with the wild-type G protein. F: Fusion; G: Glycoprotein. Reproduced with permission from [63].