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

Date Published: June 12, 2018

Publisher: Public Library of Science

Author(s): Sampath Gamage, Marquez Howard, Hiroki Makita, Brendan Cross, Gary Hastings, Ming Luo, Yohannes Abate, Eve-Isabelle PECHEUR.


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.

Partial Text

Many enveloped viruses continue to be a persistent health threat to human populations. To enter a cell, viruses attach to host-cell receptors. Detailed understanding of how viral entry proteins interact with their host-cell receptors and how the viral membrane envelope undergoes changes that lead to entry offer opportunities for the development of novel therapeutics and vaccines. For example, influenza virus (IFV) has been used as a prototype enveloped virus to study virus entry into the host cell. Hemagglutinin (HA) is a major surface glycoprotein embedded in the IFV membrane envelope. HA is responsible for IFV attachment to the host cell receptor and is involved in mediating membrane fusion during virus entry. Previous studies have led to a generally accepted model for the fusion mechanism between the target and viral membranes [1]. In this model, the postulated role of HA is primarily to bring the two membranes close to each other, so that they fuse forming a fusion pore. This process was proposed to be initiated by the conformational change of HA induced by low pH. The HA fusion peptide is released and then inserted in the target membrane as the result of this conformation change. Afterward, the trimeric coiled coil of HA refolds to bring the target and viral membranes together for fusion. The fusion process may consist of steps of hemifusion, pore formation and expansion to allow the release of the viral genome. The key point here is that the pore will only form when membrane fusion occurs. However, other reports have observed “rupture” of the target and viral membranes independently before fusion occurs [2, 3]. When a liposome was incubated with a vesicle that has HA on its surface, the low pH induced structural change of HA actually caused rupture in the liposomal membrane (forming gaps) prior to HA refolding [2]. Similar disruption of the cellular membrane was also observed when an adenovirus protein was incubated with the host cell [4]. These results clearly demonstrated that the target host membrane may be disrupted for virus entry without membrane fusion. On the other hand, the viral membrane and the target host membrane do not have the same chemical composition or structure. The requirements for pore formation in each respective membrane are different as shown previously [5–7]. When HA is inserted in the viral envelope, it increases the lipid order [5]. Acylation of HA increased the curvature of the viral membrane [6]. These suggest that the requirement for disrupting the viral membrane may be different from that for the cellular membrane. In addition, it is also unclear if disruption of the viral membrane requires membrane fusion. pH-dependent rupture of the vesicle membrane was observed at low pH when the inside of the vesicle was lined with influenza virus protein M1 that is the viral matrix protein bound with the cytoplasmic tail of HA [7]. These data imply that the disruption of the target or the viral membrane may be induced independently.

As shown in Fig 1A s-SNOM functions as a nanolocalized probe via scattering of optical fields from a probe tip either at single frequency or using broadband light sources covering a wide IR frequency range. Fig 1B shows a topography image of viruses with height in the range ~20–30 nm (Fig 1F and 1G) and diameter ~70–100 nm. Near-field phase (φ3) images are shown at 1088 (Fig 1C), 1400 (Fig 1D) and 1659 cm-1 (Fig 1E). It is well established that the near-field phase, being directly proportional to the imaginary part of the permittivity, represents infrared vibrational absorption of a sample [8–10]. The contrast at 1088 and 1659 cm-1 indicate strong infrared absorption bands of the virus at these frequencies. The weak contrast at 1400 cm-1 demonstrates weak absorption at this frequency. From these near-field spectral images chemical specific structural properties of single viral particles can be assessed [11]. Remarkably, the spectral image at 1659 cm-1 (Fig 1E) and the corresponding line profile (Fig 1G) show HA aggregates protruding out from the surface of the virus envelope. These aggregates are not well resolved in the topography map (Fig 1B). We believe these protrusions are HA protein aggregates because they are missing in the 1088 cm-1spectral image, which occurs because protein absorbs at 1659 cm-1 but not at 1088 cm-1. In this way these spectral images provide insight into not only the spatial structure, but also the chemical composition of the surface of the virus. The near-field amplitude images also can distinctly identify the HA protein from the inner structure of the virus (S1 Fig). However, detailed assignment of vibrational modes requires detailed spectra over a broad range, and it is to this issue that we now turn.




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