Fusion activation through attachment protein stalk domains indicates a conserved core mechanism of paramyxovirus entry into cells

Abstract

Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes, resulting in viral nucleocapsid entry into the cytoplasm. However, the sequence of events that closely links the timing of receptor recognition by HN, H, or G and the "triggering" interaction of the attachment protein with F is unclear. F activation results in F undergoing a series of irreversible conformational rearrangements to bring about membrane merger and virus entry. By extensive study of properties of multiple paramyxovirus HN proteins, we show that key features of F activation, including the F-activating regions of HN proteins, flexibility within this F-activating region, and changes in globular head-stalk interactions are highly conserved. These results, together with functionally active "headless" mumps and Newcastle disease virus HN proteins, provide insights into the F-triggering process. Based on these data and very recently published data for morbillivirus H and henipavirus G proteins, we extend our recently proposed "stalk exposure model" to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.

Importance: Paramyxoviruses are a large family of membrane-enveloped negative-stranded RNA viruses causing important diseases in humans and animals. Two viral integral membrane glycoproteins (fusion [F] and attachment [HN, H, or G]) mediate a concerted process of host receptor recognition, followed by the fusion of viral and cellular membranes. We describe here the molecular mechanism by which HN activates the F protein such that virus-cell fusion is controlled and occurs at the right time and the right place. We extend our recently proposed "stalk exposure model" first proposed for parainfluenza virus 5 to other paramyxoviruses and propose an "induced fit" hypothesis for F-HN/H/G interactions as conserved core mechanisms of paramyxovirus-mediated membrane fusion.

FIG 1

Expression and surface transport of PIV5 HN stalk point mutant proteins. (A) Radioimmunoprecipitation of Tran³⁵S-labeled 293T cell lysates, transfected with PIV5 HN single point mutants in the HN stalk domain. Polypeptides were immunoprecipitated with a PIV5 HN polyclonal antibody (R471) and analyzed on a 10% reducing SDS-PAGE gel. (B) Detection of PIV5 HN point mutant proteins at the surface of 293T cells at 18 h posttransfection. Proteins were detected using the PIV5 HN PAb R471 and a goat α-rabbit fluorescein isothiocyanate (FITC)-conjugated secondary antibody by flow cytometry. The mean fluorescence intensity (MFI) is shown as a percentage of wt PIV5 HN protein expression levels (n = 3).

FIG 2

Hydrophobic residues of the PIV5 HN stalk domain form the basis of F activation. (A) Luciferase reporter assay for cell-cell fusion in Vero cells cotransfected with wt PIV5 F and wt PIV5 HN or PIV5 HN stalk point mutants. The extent of cell-cell fusion was measured as relative luciferase units (RLU), expressed as a percentage of wt PIV5 F and PIV5 HN fusion, with data from three independent experiments. (B) Atomic structure of PIV5 HN in the “2 heads up, 2 heads down” form (41) showing where the globular head protomer (surface representation) makes contacts with a single helix from the 4HB stalk domain (diagram representation). Side chains of residues that were mutated are shown as sticks on the stalk helix, and the mutated residues are colored according to their ability to activate PIV5 F, as observed in panel A and as previously described (41). Colors: red, complete block of F activation; blue, no effect on F activation; yellow, core hydrophobic residue within the 4HB that requires a hydrophobic substitution to be functionally active. (C) Atomic structure of NDV HN in the “4 heads down” form (23) showing one globular head protomer (surface representation) forming contacts with one helix of the stalk (diagram representation). NDV HN residues functionally characterized in previous studies (33, 36) are highlighted with their side chains shown as sticks. Color coding is the same as in panel B. (D) Gapless alignment of PIV5 HN (W3A strain) and NDV HN (Australia-Victoria strain) protein sequences showing the part of the HN stalk domains harboring putative F-activating domains. Color coding of residues is the same as in panels B and C, with all of the residues present within the hydrophobic core of the PIV5 HN and NDV HN stalk 4HBs highlighted with a yellow box. (E) Receptor-binding activity of PIV5 HN stalk point mutants, as measured by the ability of these mutant proteins to bind chicken RBCs. Specifically bound RBCs were lysed, and the hemoglobin absorbance (Abs) at 410 nm was expressed as a percentage of the wt PIV5 HN protein hemadsorption activity. The results are from three independent experiments. Asterisks indicate a significant variation in hemadsorption activity compared to that of wt PIV5 HN (P < 0.05). (F) Neuraminidase (receptor-destroying) activity of PIV5 HN stalk point mutants was measured using a fluorogenic substrate (MU-NANA). Emission of the cleaved fluorescent product was measured at 450 nm and is expressed as a percentage of wt PIV5 HN neuraminidase activity (n = 3).

FIG 3

The PIV5 HN F activation region is able to substitute for the NDV HN F activation domain. (A) Gapless protein sequence alignment of portions of HN, H, or G stalk domains from various paramyxoviruses. Predicted transmembrane domains (TM), identified as a stretch of hydrophobic residues flanked by charged residues, are underlined in red. The sequence of seven residues of the putative F-activating domain of PIV5 HN and those replaced in the NDV HN stalk domain to create the NDV-PIV5-Fact-HN chimera, are shown enclosed within black boxes. Color coding of boxes highlighting individual residues correspond to that described in Fig. 2B. Negatively charged aspartic acid or glutamic acid residues of rubulaviruses, including D88 of PIV5 HN and E105 of MuV HN, are colored red. (B) Schematic representation of the NDV HN wt and the NDV-PIV5-Fact-HN chimera proteins. The PIV5 HN region (residues 81 to 87) that replaced the corresponding NDV HN region (residues 90 to 96) is highlighted in black on the NDV-PIV5-Fact-HN stalk. CT, cytoplasmic tail. (C) NDV HN wt and NDV-PIV5-Fact-HN expression in 293T cells labeled with Tran³⁵S-label. The proteins were immunoprecipitated from transfected cell lysates using a NDV HN polyclonal antibody (R4722). The proteins were analyzed on a 10% reducing gel. Numbers on the right are molecular masses in kilodaltons. (D) Surface expression of the NDV-PIV5-Fact-HN chimera protein compared to wt NDV HN surface expression determined by flow cytometry. Proteins were detected on the surfaces of transfected cells using the NDV HN R4722 antibody and labeled with a goat α-rabbit FITC-conjugated secondary antibody. The results from three independent experiments. (E) Cell-cell fusion mediated by the NDV-PIV5-Fact-HN chimeric protein when cotransfected with PIV5 F or NDV F, as measured using a luciferase reporter assay for fusion. Fusion activity was measured as RLU and is expressed as a percentage of wt NDV F and wt NDV HN fusion activity. Results from three independent experiments are shown. (F) Representative micrographs showing cell-cell fusion in BHK-21 cells transfected with NDV F or PIV5 F alone or in combination with wt NDV HN, PIV5 HN, or the NDV-PIV5-Fact-HN chimera. Cells were fixed, stained, and photographed at 18 h posttransfection. (G) Receptor-binding ability of the NDV-PIV5-Fact-HN chimera protein as measured using a hemadsorption assay. Absorbance of hemoglobin from RBCs specifically bound by the expressed proteins on 293T cells is measured at 410 nm and shown as a percentage of wt NDV HN hemadsorption activity. Asterisk indicates a P value of <0.05 from three independent experiments. (H) Receptor-destroying activity of the NDV-PIV5-Fact-HN chimera protein as measured by a neuraminidase assay using a fluorogenic substrate. The fluorescent emission is measured at 450 nm and expressed as a percentage of wt NDV HN neuraminidase activity (n = 3).

FIG 4

A single charged amino acid in the HN stalk domain determines F activation promiscuity of two rubulaviruses. (A) Sequence alignment of the PIV5 HN F activation domain and the corresponding region of MuV HN. PIV5 HN residues critical for F activation are shown in red. Arrowheads mark the MuV HN residues that were mutagenized. (B) Protein expression levels of MuV HN single point mutants, as observed after radioimmunoprecipitation of Tran³⁵S-labeled proteins from transfected 293T cell lysates using whole MuV polyclonal antisera. Samples were analyzed on a SDS–10% PAGE gel. Numbers on the left indicate molecular masses markers in kilodaltons. (C) Protein expression and transport to the surface of 293T cells were quantified in cells transfected with MuV HN point mutants. MuV HN proteins were detected on the surfaces of cells using the MuV antisera and fluorescently labeled with a goat α-rabbit FITC-conjugated secondary antibody for detection by flow cytometry. MFI values were expressed as a percentage of wt MuV HN surface expression, and the results are representative of three independent experiments (n = 3). (D) Hemadsorption assay of MuV HN stalk point mutants. Absorbance of hemoglobin from lysed RBCs that were specifically bound to HN proteins expressed on 293T cells was measured at 410 nm and expressed as a percentage of wt MuV HN hemadsorption. (n = 3). (E) Neuraminidase assay of MuV HN stalk point mutants. Emission at 450 nm on cleavage of MU-NANA was expressed as a percentage of the wt MuV HN neuraminidase activity (n = 3). (F) Luciferase reporter assay for fusion showing quantitative cell-cell fusion for MuV HN point mutant proteins coexpressed in Vero cells with MuV F (gray bars) or PIV5 F (white bars). The results are expressed in RLU as a percentage of the wt MuV F and MuV HN fusion activity (n = 3). (G) Representative micrographs showing cell-cell fusion observed when MuV HN point mutants are coexpressed with either MuV F or PIV5 F. Cells were fixed, stained, and photographed at 18 h posttransfection.

FIG 5

Disulfide bonds introduced within the NDV HN four-helix bundle stalk F activation region abrogate fusion promotion. (A) Top and side views of the NDV HN “4 heads down” structure mapping the positions of surface-exposed residues that are critical for F activation (red) and the S92 residue (green) in the core of the 4HB. (B) Immunoprecipitation of NDV HN point mutants from transfected 293T cell lysates with the NDV HN R4722 polyclonal antibody after labeling with Tran³⁵S-label. The proteins were analyzed on a 10% reducing SDS-PAGE gel. M, monomer. (C) Same as in panel B, but proteins were analyzed on a 10% nonreducing gel. The numbers on the right indicate molecular mass markers in kilodaltons. D, dimers. (D) Surface expression of NDV HN mutants as detected using the above antibody and analyzed by flow cytometry. Mean fluorescent intensities (MFI) of the NDV HN mutant proteins expressed on the surface of 293T cells are shown as a percentage of wt NDV HN (n = 3). (E) Syncytium fusion assay showing cell-cell fusion of the NDV HN mutants cotransfected with NDV F or the NDV F L289A mutant in BHK-21 cells. Samples were fixed, stained, and imaged 18 h posttransfection. (F) Luciferase reporter assay for fusion showing cell-cell fusion activity for NDV HN point mutants cotransfected with the NDV F L289A mutant. The luciferase reporter activity generated in fused cells (RLU) is expressed as a percentage of NDV F L289A and wt NDV HN fusion (n = 3). (G) Hemadsorption assay of NDV HN mutants. RBCs bound specifically to HN proteins on transfected cells were lysed, and the hemoglobin absorbance was measured at 410 nm. The absorbance values are represented as a percentage of the wt NDV HN hemadsorption activity (n = 3). (H) Neuraminidase activity of NDV HN mutant proteins. Emission at 450 nm upon cleavage of MU-NANA by the NDV HN mutant proteins is represented as a percentage of the wt NDV HN neuraminidase activity (n = 3).

FIG 6

A destabilized NDV F protein causes significant cell-cell fusion, when triggered by an NDV HN “headless” stalk. (A) Schematic representation of NDV HN full-length and “headless” stalk mutant proteins NDV HN 1-123 and NDV 1-123 S92C. CT, cytoplasmic tail; TM, transmembrane domain. (B) NDV HN “headless” stalk mutants were expressed in 293T cells. The cells were labeled with Tran³⁵S-label and lysed, and proteins were immunoprecipitated with the NDV HN polyclonal antibody R9722. Polypeptides were analyzed by SDS-PAGE on a 15% gel. Numbers indicate molecular masses in kilodaltons; M, HN monomer. (C) 293T cells expressing the NDV HN 1-123 “headless” stalk protein grown in the presence of 5 μg of tunicamycin/ml. Radiolabeled polypeptides were immunoprecipitated and analyzed as in panel B; M, HN monomer; D, HN dimer. (D) Surface expression of NDV HN mutants as detected by the above NDV HN polyclonal antibody on the surface of transfected 293T cells. MFI of the NDV HN mutants are represented as a percentage of wt NDV HN surface expression (n = 3). (E) Receptor binding of the NDV HN full-length and “headless” stalk mutants analyzed using a hemadsorption assay. The ability of the mutant proteins to bind chicken RBCs was quantified and is expressed as a percentage of the wt NDV HN absorbance at 410 nm. (F) Cell-cell fusion as observed in a syncytium assay of NDV F cotransfected with the NDV HN 1-123 headless stalk. Samples were fixed, stained, and photographed at various times posttransfection as indicated. (G) Syncytium assay showing the ability of the NDV HN headless stalk constructs, NDV HN 1-123 and NDV HN 1-123 S92C, to trigger NDV F or the hyperfusogenic NDV F L289A mutant. Cells were fixed, stained, and photographed 20 h posttransfection.

FIG 7

A mumps virus HN “headless” stalk is able to trigger both cognate and noncognate F proteins. (A) Schematic representation of MuV wt HN protein and MuV HN “headless” stalks of various lengths. CT, cytoplasmic tail; TM, transmembrane domain. (B) Migration pattern of MuV HN “headless” stalk proteins on a SDS–15% PAGE gel. Polypeptides were immunoprecipitated from radiolabeled lysates of transfected 293T cells using the MuV polyclonal sera. (C) Expression of MuV HN “headless” stalk domains detected on the surface of transfected 293T cells using the MuV polyclonal sera. A goat α-rabbit FITC-conjugated secondary antibody was used for flow cytometry. The mean fluorescence intensities of the MuV HN “headless” stalks were expressed as a percentage of wt MuV HN surface expression (n = 3). (D) Luciferase reporter assay for fusion showing fusion promotion by the MuV HN “headless” stalk mutants cotransfected with MuV-F, expressed as a percentage of wt MuV-F and wt MuV HN fusion (n = 3). (E) In the first row, representative micrographs of syncytia show cell-cell fusion in BHK-21 cells transfected with MuV F and MuV HN 1-132 “headless” stalk at 18, 20, 22, 24, and 26 h posttransfection. For the second row, a syncytium assay was performed to show the ability of the MuV HN 1-132 “headless” stalk to cause cell-cell fusion when cotransfected with PIV5 F. Cells were fixed, stained, and photographed 18 to 26 h posttransfection. For the third row, a syncytium assay was performed to assess the expression of PIV5 F alone, so that spontaneous cell-cell fusion could be compared to fusion (26 h posttransfection) caused by cotransfection of MuV HN 1-132 and PIV5 F. (F) Receptor-binding ability of the MuV HN 1-132 mutant, as measured with a hemadsorption assay. The hemadsorption activity of the MuV HN 1-132 mutant was expressed as a percentage of the wt MuV HN activity (n = 3).