Progressive ordering with decreasing temperature of the phospholipids of influenza virus

Abstract

Using linewidth and spinning sideband intensities of lipid hydrocarbon chain resonances in proton magic angle spinning NMR spectra, we detected the temperature-dependent phase state of naturally occurring lipids of intact influenza virus without exogenous probes. Increasingly, below 41 degrees C ordered and disordered lipid domains coexisted for the viral envelope and extracts thereof. At 22 degrees C much lipid was in a gel phase, the fraction of which reversibly increased with cholesterol depletion. Diffusion measurements and fluorescence microscopy independently confirmed the existence of gel-phase domains. Thus the existence of ordered regions of lipids in biological membranes is now demonstrated. Above the physiological temperatures of influenza infection, the physical properties of viral envelope lipids, regardless of protein content, were indistinguishable from those of the disordered fraction. Viral fusion appears to be uncorrelated to ordered lipid content. Lipid ordering may contribute to viral stability at lower temperatures, which has recently been found to be critical for airborne transmission.

Figure 1

¹H MAS NMR spectra of model and biomembranes. (a) Typical ¹H MAS NMR spectra of lipids in l_d, l_o and s_o phases. The inset shows the first-order spinning sidebands at 20-fold magnification. The l_d spectrum is shown ten-fold attenuated. The spectra in the l_d and s_o phases are from DPPC, and those in l_o are from DPPC/cholesterol, 7:3 mol/mol mixture . (b) ¹H MAS NMR spectra of lipids extracted from influenza virus (top), intact influenza virus (middle) and allantoic membranes of embryonated eggs used for virus growth (bottom). Spectra were recorded at a MAS frequency of 10 kHz at 40 °C. The inset shows first-order spinning sidebands at 20-fold magnification. The sharp peak at 4.8 ppm is from water. (c) Example of spectral deconvolution (see Supplementary Methods); influenza virus ¹H MAS NMR spectrum from b (black line). The methylene resonance of hydrocarbon chains at 1.3 ppm (blue line) is fitted as a superposition of narrow (75 Hz–wide, green line) and broad (1,000 Hz–wide, red line) components. The lower panel shows the two superimposed resonances and the ¹H NMR spectrum after subtracting them (gray line). (d) ¹H MAS NMR spectra of intact influenza virus (top) and RBC membranes (erythrocyte ghosts membranes, bottom). Spectra were recorded at a MAS frequency of 10 kHz at 25 °C. The inset shows first-order spinning sidebands at ten-fold magnification.

Figure 2

¹H MAS NMR spectra as a function of temperature. (a) ¹H MAS NMR spectra of influenza virus recorded at 10 kHz at temperatures of 40 °C, 25 °C, 13 °C and −5 °C. (b) ¹H MAS NMR spectra of influenza virus total extracted lipids recorded at 10 kHz at temperatures of 40 °C, 25 °C, 13 °C and −5 °C. The inset shows first-order spinning sidebands at 20-fold magnification.

Figure 3

Fraction of ordered lipids as a function of temperature. (a) Temperature dependence of the 1.3 ppm resonance of influenza virus, total extracted viral lipids, allantoic membranes and RBC ghost membranes. (b) Temperature dependence of the fraction of membranes in ordered domains in influenza virus, viral lipids, allantoic membranes and RBC ghost membranes calculated from the 1.3 ppm resonance height, as explained in the Results. (c) Temperature dependence of the ratio of sideband to centerband height of the 1.3 ppm resonance of influenza virus, viral lipids, allantoic membranes and RBC ghost membranes.

Figure 4

¹H MAS NMR diffusion measurements. (a) Time dependence of the average diffusion displacement in viral lipid extract at 37 °C (◊) and at 17 °C (♦). The solid line is a fit with a diffusion rate of 30 μm² s⁻¹ and diffusion confinement of 4.3 μm. The dashed line is a ft with a diffusion rate of 7 μm² s⁻¹ and diffusion confinement of 2.5 μm. (b) Temperature dependence of diffusion confinement in viral lipid extract. For comparison, the fraction of disordered lipid in viral lipid extract is plotted as well (○, axis on the right).

Figure 5

Fluorescence microscopy detection of ordered lipid domains. (a) Solid-supported multibilayers of viral lipids containing 1% of Texas Red–DPPE, hydrated at room temperature. (b) Ordered and disordered lipid domains coexist on the surface of giant unilamellar vesicles of viral lipids containing 1% of Texas Red–DPPE formed by electroformation on the ITO surface. Vesicle is attached to the surface, forming a hemisphere. Bright area is in the liquid disordered state, while circular dark domains are liquid ordered and dendritic dark domains are solid ordered.

Figure 6

Effect of temperature on lipid mixing assay of influenza virus fusion with RBC membranes. Virus labeled with R18 at self-quenching concentration was mixed with RBC membranes and incubated for 5 min to equilibrate temperature. Fusion was triggered by adjusting the pH to 5 and monitored by the increase of fluorescence intensity due to probe dequenching. Membrane solubilization with 1% Triton X-100 gave complete dequenching, defined as 100% fusion. Decrease in temperature results in a decrease of both the rate and extent of fusion, but fusion is robust at temperatures above 40 °C, where the amount of ordered lipids is negligible.