Regulation of paramyxovirus fusion activation: the hemagglutinin-neuraminidase protein stabilizes the fusion protein in a pretriggered state

Abstract

The hemagglutinin (HA)-neuraminidase protein (HN) of paramyxoviruses carries out three discrete activities, each of which affects the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. Binding of HN to its sialic acid receptor on a target cell triggers its activation of the fusion protein (F), which then inserts into the target cell and mediates the membrane fusion that initiates infection. We provide new evidence for a fourth function of HN: stabilization of the F protein in its pretriggered state before activation. Influenza virus hemagglutinin protein (uncleaved HA) was used as a nonspecific binding protein to tether F-expressing cells to target cells, and heat was used to activate F, indicating that the prefusion state of F can be triggered to initiate structural rearrangement and fusion by temperature. HN expression along with uncleaved HA and F enhances the F activation if HN is permitted to engage the receptor. However, if HN is prevented from engaging the receptor by the use of a small compound, temperature-induced F activation is curtailed. The results indicate that HN helps stabilize the prefusion state of F, and analysis of a stalk domain mutant HN reveals that the stalk domain of HN mediates the F-stabilization effect.

Fig 1

HPIV3 fusion protein mediates cell fusion at 37°C with uncleaved influenza virus hemagglutinin (HA) as a nonspecific binding protein. Cells expressing HPIV3 HN-wt and HPIV3 F (A), HPIV3 HN-D216R and HPIV3 F (B), uncleaved influenza virus HA and HPIV3 F (C), HPIV3 F (D), uncleaved influenza virus HA (E), or pCAGGS empty vector (F) were overlaid with receptor-bearing cells expressing red fluorescent protein (RFP) and incubated for 18 h at 37°C. Fusion under each condition was quantitated as described in Materials and Methods (G). Cell-cell fusion is observed on the left by syncytium formation (visible microscopy) and on the right by RFP redistribution (fluorescence microscopy). RLU, relative light units.

Fig 2

HN, before receptor engagement, prevents F activation. (A) Cell-to-cell fusion at 37°C mediated by HPIV3 F coexpressed with uncleaved influenza virus HA and HN wt, uncleaved influenza virus HA and HN P111S/D216N, uncleaved influenza virus HA and HN-D216R, uncleaved influenza virus HA, or alone at 1, 4, 8, and 12 h in the presence of treatments as indicated on the x axis. Fusion, quantitated as described in Materials and Methods, is shown as a percentage of the fusion mediated by the HA, HN wt, and HPIV3 F in the no-treatment group (± standard deviation). (B) Fusion was visualized at 18 h by visible and fluorescence microscopy for the no-treatment group and the zanamivir-treated group. The data reflect experiments repeated at least three times.

Fig 3

Temperature dependence of fusion mediated by F in the presence of HA alone or with D216R HN, N551D HN, or P111S/D216N HN. For the F-activation assay, monolayers of 293T cells coexpressing wt F and uncleaved influenza virus HA, alone or in the presence of wt HN, D216R HN, N551D HN, or P111S/D216N HN were allowed to bind to receptor-bearing RBCs at 4°C and then transferred to the indicated temperatures (x axis). After 60 min of incubation, fused RBCs were quantified, and results are expressed as a percentage of the total bound RBCs (y axis). The ordinate values are means of results (± standard deviations) of three experiments.

Fig 4

The HN stalk domain is critical for preventing F from undergoing thermal activation. Monolayers of 293T cells coexpressing wt F and uncleaved influenza virus HA, either alone (no HN) or in the presence of the indicated HN molecules, were allowed to bind to receptor-bearing RBCs at 4°C and then transferred to 37°C for 60 min (A and E) or to 45°C for the indicated times (B, C, D, F, G, and H). Zanamivir to prevent HN′s receptor engagement was either present or absent, as indicated. After incubation, fused RBCs were quantified, and results are expressed as a percentage of the total bound RBCs (y axis). The ordinate values are means of results (± standard deviations) of at least three experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5

Structural rearrangements in F in are detected by altered protease sensitivity. (A) Monolayers of cells coexpressing F and uncleaved HA with the indicated HN or F and HA alone (bottom row) were incubated for 60 min at 37°C in medium either without zanamivir (receptor-engaged; R+) or with zanamivir to disengage HN from its receptor (R−). After the cells were lysed, the envelope glycoproteins were immunoprecipitated and then incubated in the presence or absence of protease K. The representative Western blot shows F proteolysis, detected by polyclonal anti-F HRC antibodies. F0 (precursor) protein and F1 are indicated. When each HN is present, but not engaging receptor (R−), F does not become protease sensitive. The stalk domain mutant HN (P111S HN) prevents protease digestion of F even when HN is receptor engaged (R+). (B) Flow cytometric analysis of cell surface F expression using rabbit polyclonal anti-F antibodies. The mean fluorescent intensity (MFI) of the cells expressing F with the indicated HN/HA pair is presented as a percentage of the mean fluorescent intensity of cells expressing F with HA alone.

Fig 6

Negative-stain electron microscope tomography of HPIV3 virus particles. (A) A subpopulation of small particles (V, virus interior) bear predominantly F proteins in postfusion conformation (white arc and carat), with some F in prefusion form (black carat). (B and C) The majority of typical particles in our preparations show a double-layered surface (white box), with little postfusion F observed. (D) Close-up of the upper region of the particle shown in panel A. (E) Close-up of the lower region of the particle in panel A. (F) Average radial density plots show clear differences between regions of primarily F in postfusion conformation (red) and the more typical double-layer coat on most particles (black). (G) Models and dimensions for coupled HN/F and postfusion F based upon reported crystal structures (, , –69) (yellow, HN; cyan, prefusion F; red, postfusion F) and a cryo-negative stain electron microscopy reconstruction for postfusion F (red density) (32). au, arbitrary units.