Hydrostatic Pressure Induces the Fusion-active State of Enveloped Viruses

Abstract

Enveloped animal viruses must undergo membrane fusion to deliver their genome into the host cell. We demonstrate that high pressure inactivates two membrane-enveloped viruses, influenza and Sindbis, by trapping the particles in a fusion-intermediate state. The pressure-induced conformational changes in Sindbis and influenza viruses were followed using intrinsic and extrinsic fluorescence spectroscopy, circular dichroism, and fusion, plaque, and hemagglutination assays. Influenza virus subjected to pressure exposes hydrophobic domains as determined by tryptophan fluorescence and by the binding of bis-8-anilino-1-naphthalenesulfonate, a well established marker of the fusogenic state in influenza virus. Pressure also produced an increase in the fusion activity at neutral pH as monitored by fluorescence resonance energy transfer using lipid vesicles labeled with fluorescence probes. Sindbis virus also underwent conformational changes induced by pressure similar to those in influenza virus, and the increase in fusion activity was followed by pyrene excimer fluorescence of the metabolically labeled virus particles. Overall we show that pressure elicits subtle changes in the whole structure of the enveloped viruses triggering a conformational change that is similar to the change triggered by low pH. Our data strengthen the hypothesis that the native conformation of fusion proteins is metastable, and a cycle of pressure leads to a final state, the fusion-active state, of smaller volume.Enveloped viruses utilize regulated membrane fusion to introduce their genomes in the cytoplasm of the host cell. The fusion is mediated by surface envelope proteins of the virus in response to a trigger (1, 2). Once triggered, the fusion process leads to a conformational change that promotes the interaction of a specific sequence (fusion peptide) with the target membrane and initiates membrane fusion. Membrane fusion is crucial in other biological functions such as myotube formation, fertilization, and trafficking of endocytic and exocytic vesicles within eukaryotic cells (1, 3). Many enveloped animal viruses have been studied as models for understanding the mechanism of membrane fusion. While the fusion proteins of many viruses reveal significant similarity in their putative fusogenic conformation, such as those of influenza virus (hemagglutinin HA2), 1 human immunodeficiency virus (gp41 protein), Moloney murine leukemia virus (TM protein), and Ebola virus (GP2 protein) (4), the events of membrane fusion for other virus families (e.g. Flaviviridae and Togaviridae) are beginning to be understood. Alphavirus and the flavivirus fusion proteins appear to have evolved from a common ancestor (5) and possess a similar new class of membrane fusion proteins that do not form coiledcoils (6 -8).Sindbis and influenza are enveloped viruses that first enter a cell by endocytosis and then fuse with the cellular membrane in response to acidic conditions. Sindbis virus is the prototype of the Alphavirus genus, Togaviridae family. The Alphavirus spike is...

Lack of Correlation between Virus Barosensitivity and the Presence of a Viral Envelope during Inactivation of Human Rotavirus, Vesicular Stomatitis Virus, and Avian Metapneumovirus by High-Pressure Processing

“…Since HPP has no adverse effect on covalent bonds, it is generally accepted that HPP alters quaternary and tertiary structures, but not primary and secondary structures, of proteins (3,5,20,22,26). Consistent with this, the abundance of all VSV structural proteins was not significantly altered despite the fact that virion structure was completely disrupted.…”

“…Although most enveloped viruses are not food borne, a better understanding of the inactivation of enveloped viruses would help to identify the optimal processing parameters, which can facilitate many other important applications of HPP such as preparation of inactivated vaccines. To date, there are only a few studies on high-pressure inactivation of enveloped viruses (20,22,27,41,49). For example, the titers of herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) were reduced by more than 7 and 4 logs, respectively, after treatment at 300 MPa at 25°C for 10 min (41).…”

Recent Advances in the Use of High Pressure as an Effective Processing Technique in the Food Industry

“…Pressure can also cause the dissociation of virus particles; depending on the virus and the treatment conditions, pressure-induced dissociation may be fully reversible or irreversible (Da Poian et al 1994). High pressure can also induce minor changes in viral structures without disassembling the whole particle (Gaspar et al 2002). The formation of non-infectious particles after HP treatment has been observed for many viruses, including rotavirus (Pontes et al 2001), HIV (Nakagami et al 1996), lambda phage (Bradley et al 2000) and picornaviruses (Oliveira et al 1999).…”

Strategy for Nonenveloped Virus Entry: a Hydrophobic Conformer of the Reovirus Membrane Penetration Protein μ1 Mediates Membrane Disruption

“…HA can also be induced to change conformation by nonphysiological stimuli such as elevated temperature and pressure (14,28). For both HA and E, the conformationally rearranged form of the fusion protein is more thermostable than its precursor (18,65).…”