Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity

Abstract

Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering.

Importance: Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus (PIV5), a region of the HN stalk that has not had its structure determined by X-ray crystallography. Our data suggest that the MPSR influences receptor binding and neuraminidase activity via an indirect mechanism. Moreover, the receptor binding head group stabilizes the 4HB stalk as part of the general mechanism to fine-tune F-activation.

FIG 1

Expression of MPSR triple alanine mutants. The 3× Ala mutants were made by mutations at the first 20 residues of the ectodomain of PIV5 headless HN1-117 to determine its functional significance. (A) Atomic model of PIV5 headless HN1-117 showing unresolved membrane-proximal stalk region (MPSR). F-interacting region (FAR) at the upper portion of the HN stalk is indicated. (B) Schematic diagram illustrating the PIV5 HN protein and showing the different domains: F-interacting region (blue), membrane-proximal stalk region (green), and transmembrane (TM) domain (red). The scheme of the 3× Ala mutants is shown. (C) Protein expression was determined using 30 min of ³⁵S labeling followed by immunoprecipitation. Glycosylated species were digested by peptide-N-glycosidase F ([PNGase F]) treatment. Polypeptides were analyzed by 17.5% SDS-PAGE under reducing conditions. (D) Cell surface expression of HN1-117 and 3× Ala mutants was determined by flow cytometry. M.F.I., mean fluorescent intensity. Values are expressed as a percentage of HN1-117. Error bars represent standard deviations from three experiments. P values were calculated using Student's t test. *, P < 0.05; **, P < 0.01; otherwise, P > 0.05.

FIG 2

Fusogenic properties of HN1-117 MPSR 3× Ala mutants. (A) BHK cells were transfected with PIV5 F and HN1-117 or the respective triple mutants. Formation of syncytia was observed at 16 h p.t., and the cells were stained. (B) Quantitative luciferase activity (fusion) of both HN1-117 and 3× Ala mutants. Fusion was performed at 33°C (black bars) or 37°C (checkered bars). Luciferase activity was expressed as relative light units (RLU) of HN1-117. Error bars represent standard deviations from three independent experiments. Statistical significance of difference between each mutant and HN1-117 was assessed using Student's t test. *, P < 0.05; ***, P < 0.001; otherwise, P > 0.05.

FIG 3

Effect of single point mutations of HN1-117 MPSR on fusion promotion. (A) Schematic diagram of HN1-117 showing positions of single Ala point mutants. (B) Protein expression was analyzed by ³⁵S metabolic labeling and radioimmunoprecipitation. Proteins were treated with or without PNGase F to assess susceptibility to digestion. Polypeptides were analyzed by 17.5% SDS-PAGE. (C) Cell surface expression of HN1-117 and single point Ala mutants was quantified by flow cytometry. (D) Fusion promotion of HN1-117 mutants were assessed using the luciferase fusion assay. Error bars represent standard deviations from three experiments. P values were calculated using paired Student's t test to determine statistical significance of difference between each mutant and HN1-117. **, P < 0.01; ***, P < 0.001; otherwise, P > 0.05.

FIG 4

Point mutations in the MPSR of full-length HN affect receptor binding and NA activity but not fusion promotion. The point mutations shown in Fig. 3 were made in full-length WT HN to assess its biological function. (A) 293T cells were transfected with pCAGGS vector encoding full-length WT HN or the MPSR point mutants. HN was metabolically ³⁵S labeled and immunoprecipitated, and polypeptides were analyzed by 10% SDS-PAGE. (B) Cell surface expression of WT HN and the point Ala mutants. (C and D) NA activity and HAd activity of both WT HN and HN point mutants, respectively. Error bars are standard deviations from three independent experiments. (E) Fusion promotion by WT HN and mutants was quantified using the luciferase fusion assay. Student's t test was used to determine statistical significance. *, P < 0.05; otherwise, P > 0.05.

FIG 5

Disulfide-linked stabilization of HN MPSR. (A) 293T cells expressing HN with cysteine mutations at residues 40 to 45 were ³⁵S metabolically labeled and HN immunoprecipitated, and polypeptides were analyzed by reducing (top) and nonreducing (middle) 10% SDS-PAGE. The different mobilities of WT HN and the mutants are indicated by the monomer (M), dimer (D), and tetramer (T). ImageJ was used to quantify band intensity, and the ratio of tetramer to dimer was estimated (bottom). (B) Cell surface expression of WT HN and cysteine mutants and (C) the effect of disulfide linked tetramerization of the MPSR on receptor binding (HAd) were measured under reducing (10 mM TCEP) and nonreducing conditions. Asterisks indicate statistical significance between TCEP treated and untreated. (D) NA activity of Cys mutants under reducing (TCEP) and nonreducing conditions. (E) Quantitative luciferase fusion assay of WT HN and HN cysteine mutants. *, P < 0.05; **, P < 0.01; ***, P < 0.001; otherwise, P > 0.05.

FIG 6

The stalk of HN1-117 forms limited disulfide-linked tetramers. MPSR residues 37 to 56 in HN1-117were substituted for with cysteine to determine if the stalk could be trapped via covalent disulfide bonds. (A) 293T cells expressing HN1-117 and Cys mutants were ³⁵S labeled, HN was immunoprecipitated, and polypeptides were analyzed by 17.5% SDS-PAGE under reducing (top) and nonreducing (bottom) conditions. Arrows indicate HN1-117 monomer (M), dimer (D), and tetramer (T). (B) Cell surface expression was quantified by flow cytometry and expressed as mean fluorescent intensity (M.F.I.) of HN1-117. The horizontal dashed line represents the HN1-117 expression level. (C) ³⁵S metabolically labeled 293T cells were treated with 3 μM (final concentration) copper(II) phenanthroline (CuP) prior to lysis and immunoprecipitation. Both HN1-117and Cys mutants were resolved by nonreducing 17.5% SDS-PAGE after PNGase F digestion to remove carbohydrate chains. (D) The ratio of tetramer to dimer was estimated by quantifying band intensities with ImageJ. Error bars are standard deviations from two experiments. ‡, tetramer/dimer ratios were not estimated for C41 and C48 due to lack of detectable signal on the gel. *, P < 0.05; ***, P < 0.001; otherwise, P > 0.05.

FIG 7

The stalk of full-length HN can be trapped as disulfide-linked tetramers at the MPSR. (A) ³⁵S metabolically labeled Cys mutants of full-length HN were resolved on a 10% SDS-PAGE under reducing (top) or nonreducing (bottom) conditions. Arrows indicate monomers (M), dimers (D), and tetramers (T) were ³⁵S metabolically labeled. HN was immunoprecipitated, and polypeptides were analyzed by 10% SDS-PAGE. (B) Cell surface expression was quantified by flow cytometry and expressed as percentage of WT mean fluorescent intensity (M.F.I.). The horizontal dashed line represents the WT HN expression level. (C) WT HN and MPSR Cys mutants treated with CuP were resolved by nonreducing 10% SDS-PAGE. (D) Intensity bands of CuP-labeled proteins (C37 to C56) were quantified using ImageJ software, and the ratio of tetramer to dimer was estimated. *, P < 0.05; otherwise, P > 0.05.