Fabian Käsermann

Photodynamic inactivation of enveloped viruses using buckminsterfullerene

In a process for the inactivation of enveloped viruses in a biological fluid buckminsterfullerene (C60) is used as a photosensitizer. In a photodynamic processes singlet oxygen is generated which is the active agent for the inactivation of viruses present in the fluid. The process comprises the steps of (a) contacting of a solution or dispersion of material derived from the human body or from the bodies of animals with buckminsterfullerene as a photosensitizer, of (b) saturating the solution or dispersion with oxygen; and of (c) irradiating the solution or dispersion with visible or invisible light for activating the oxygen into the singlet state until viruses contained in the solution or dispersion are inactivated. This virus inactivation is specially suitable for protein solutions, e.g. bovine serum albumin, BSA or plasma products of human origin.

Buckminsterfullerene and photodynamic inactivation of viruses

The development of new virus inactivation procedures has become an area of growing interest mainly due to increased demands concerning the safety of biological products. Photochemical processes represent the most promising methods for the future to inactivate viruses. In these methods, dyes are the most widely used photosensitising reagents. The current article covers a new interesting alternative, namely the use of buckminsterfullerene (C60). The unique properties of this molecule make it a valid candidate for future applications in the inactivation of viruses in biological fluids.

Identification of the pore forming element of Semliki Forest virus spikes

Pore formation at mildly acidic pH by SFV spike proteins was investigated using isolated and modified virions. Modification of the virions was performed by limited proteolysis in presence of octylglucoside and resulted in the formation of E1 particles and spikeless particles, respectively. Pore formation was detected by measuring the influx of propidium iodide into the viral particles. The results obtained clearly showed that the presence of E1 alone is sufficient to promote pore formation at mildly acidic pH. Thus E1 represents the pore forming element of the viral spike proteins.

Virus inactivation and protein modifications by ethyleneimines

Virus inactivation by ethyleneimines was first introduced more than 30 years ago. Selective targeting of nucleic acids was reported for oligomeric ethyleneimines. In this study, trimeric ethyleneimine (TEI) was used to inactivate minute virus of mice (MVM; Parvoviridae) and Semliki forest virus (SFV; Togaviridae). The pH-dependency of the inactivation kinetics observed with MVM was different compared to the kinetics reported for other viruses. The higher inactivation rate at higher pH favoured the idea of a mechanism involving protein modifications. Alteration of the isoelectric point and changes in mass could be observed after treatment of soluble proteins with TEI. The uptake of MVM by host cells was reduced or completely blocked by TEI treatment, as shown by monitoring viral internalisation of DNA into target cells. The observed loss of virus infectivity coincided with the inhibition of virus uptake. Thus, virus inactivation by TEI is most likely also a result of chemical modifications of viral surface proteins.

Low pH-induced pore formation by spike proteins of enveloped viruses.

Exposure of Aedes albopictus cells infected with Semliki Forest virus (SFV; Togaviridae) to mildly acidic pH (5.6) results in a dramatic increase in the host cell membrane permeability due to pore formation by the virus spike proteins. Identical results were obtained when the cells were infected with two other viruses, Sindbis virus (SIN, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae). This permeability change could also be observed on isolated virions of SFV, SIN and VSV by measuring the influx of propidium iodide, a nucleic acid-specific fluorescent marker, into the virions. This influx was dependent on the presence of the ectodomains of the viral spikes and could be hampered by zinc ions. Furthermore, haemagglutinin, a membrane protein of influenza A virus (Orthomyxoviridae), expressed in Aedes cells induced a change in membrane permeability identical to that induced by the spike proteins of SFV, SIN and VSV when exposed to low pH. Thus acid-induced membrane permeability changes produced by spike proteins of three different virus families could be demonstrated in infected cells as well as in virions. Therefore, the low pH-induced pore formation by viral spike proteins seems to be more than an event specific for togaviruses and might well be an inherent property of enveloped viruses that use the endocytotic pathway to infect a cell.

Inactivation of enveloped viruses by singlet oxygen thermally generated from a polymeric naphthalene derivative.

Inactivation of viruses can be induced by singlet oxygen generating agents. The water-insoluble polymeric compound PVNE (poly (1,4-dimethyl-6-vinylnaphthalene-1,4-endoperoxide)) is used as a storage for reactive oxygen and is able to produce thermally generated 1O2 in a dark-reaction. Enveloped viruses from two different families, Semliki Forest virus (SFV, Togaviridae) and vesicular stomatitis virus (VSV, Rhabdoviridae) showed a loss of infectivity of up to 8 log10/ml (TCID50) when incubated at 37 degrees C with PVNE in buffered solutions. PVNE produces singlet oxygen by thermal decomposition without irradiation. Such chemically generated oxygen excludes reactions involving radicals (type I photoreactions), a problem often encountered in photodynamic processes utilizing dyes as sensitizers. In addition, the water insolubility of the oxygen-carrier allows an easy removal and recycling from aqueous solutions. Therefore, it may prove useful in the inactivation of viruses in biological systems and may be a helpful tool in studies concerning the inactivation mechanism by 1O2.

Virus membrane proteins and proteinaceous pores

Enveloped viruses penetrate the host cells by fusion of the viral envelope with a cellular target membrane. One of the best studied viruses with respect to its penetration and uncoating is the alphavirus Semliki Forest virus that is taken up by endocytosis. The alphavirus membrane glycoprotein E1 harbors a so-called fusion peptide, which is responsible for interaction with the endosomal membrane, leading to fusion. Besides this fusion process, cell infection by alphaviruses is accompanied by membrane permeability changes, thus implying some form of pore across the membrane. However, the ability of E1 protein to form ion pores has not been widely accepted. This review provides an overview of studies that confirm earlier results predicting the formation of a proteinaceous pore by the alphavirus spike proteins. Furthermore, different models to explain this pore formation during virus entry are discussed. Viruses are genetic elements that are able to reproduce in a host cell-dependent way. The virus genome, RNA or DNA, is packaged into a protective protein coat; in enveloped viruses this nucleocapsid is surrounded by a lipid bilayer, derived from the host cell membrane during virus budding, which contains viral spike glycoproteins. This packaging protects the virus during the extracellular journey and presents recognition signals for adsorption to the cellular membrane. It provides the means for viral structural conversions upon interaction with the host cell. This leads to successful penetration of the cellular membrane and to the uncovering and release of the nucleic acid for replication (uncoating of the nucleocapsid).