Probing structural changes in single enveloped virus particles using nano-infrared spectroscopic imaging

<div><p>Enveloped viruses, such as HIV, Ebola and Influenza, are among the most deadly known viruses. Cellular membrane penetration of enveloped viruses is a critical step in the cascade of events that lead to entry into the host cell. Conventional ensemble fusion assays rely on collective responses to membrane fusion events, and do not allow direct and quantitative studies of the subtle and intricate fusion details. Such details are accessible via single particle investigation techniques, however. Here, we implement nano-infrared spectroscopic imaging to investigate the chemical and structural modifications that occur prior to membrane fusion in the single archetypal enveloped virus, influenza X31. We traced in real-space structural and spectroscopic alterations that occur during environmental pH variations in single virus particles. In addition, using nanospectroscopic imaging we quantified the effectiveness of an antiviral compound in stopping viral membrane disruption (a novel mechanism for inhibiting viral entry into cells) during environmental pH variations.</p></div

Spatial evolution of virus particles when acidity changed from pH 7 to pH 5.

<p>Topography (black & white), and near-field amplitude (A₃) and phase (φ₃) images of four virus particles at pH 7 (a) and pH 5 (b) taken at 1225 and 1665 cm^-1. Scale bar 100 nm.</p

Probing structural changes in single enveloped virus particles using nano-infrared spectroscopic imaging - Fig 5

<p>(A) Nano-FTIR spectra of influenza virus particles at neutral pH (green) and at pH 5 (red). Topography and hyperspectral images of a virus particle at pH 7. Topography, and spectra obtained at two different points on the particle (A and B) (a). Near-field amplitude, A₂ (b and d) and phase φ₂ (c and e) images at frequencies 1400 and 1225 cm^-1 sliced from hyperspectral data. Scale bar 100 nm.</p

Schematic of s-SNOM nanoimaging and nano-FTIR setup.

<p>Schematic of s-SNOM nanoimaging and nano-FTIR setup.</p

Probing structural changes in single enveloped virus particles using nano-infrared spectroscopic imaging - Fig 4

<p>Nano-FTIR spectra of influenza virus particles from three different samples at neutral pH (a) and spectra from virus particles taken on day 1 and day 7 (b). Topography, near-field amplitude (A₃) and phase (φ₃) images of two influenza virus particles on day 1 and day 7 (c). Scale bar 100 nm.</p

Probing structural changes in single enveloped virus particles using nano-infrared spectroscopic imaging - Fig 3

<p>Topography, near-field amplitude (A₃) and phase (φ₃) images of several influenza virus particles i-(a), after compound 136 interaction (15 min) i-(b), followed by acid exposure i-(c). ii-(a-b) shows a different set of virus particles at neutral pH ii-(a) and ii-(b) shows the particles after acid exposure following compound 136 incubation (1 hr.). Scale bar 100 nm.</p

s-SNOM experimental setup and near-field infrared spectral images of influenza virus at pH 7.4.

<p>(a) schematic of the s-SNOM experiment, (b) topography of four viruses (scale bar is 100 nm), (c-e) near-field phase (φ₃) spectral images at three different frequencies. Line profiles of topography (f) and phase (g) representing the red broken lines shown in the topography (b) and phase (c, e) images.</p