Differentially-Charged Phospholipids Interact with Alphaherpesviruses and Interfere with Virus Entry

Oleksandr Kolyvushko*, Juliane Latzke*, Ismail Dahmani, Nikolaus Osterrieder, Salvatore Chiantia, Walid Azab 1 Institut für Virologie, Robert von Ostertag-Haus, Zentrum für Infektionsmedizin, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany 2 University of Potsdam, Institute of Biochemistry and Biology, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany * These authors contributed equally † Correspondence to: Dr. Walid Azab, walid.azab@fu-berlin.de Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 14 April 2020 doi:10.20944/preprints202004.0220.v1


Introduction
Phospholipids have been shown to be necessary for enveloped viruses to promote infection.
Phosphatidylserine (PS) is used as a receptor by different viruses [1,2]. Recently, it was shown that PS exposure on cell surface occurs shortly after equine herpesvirus type 1 (EHV-1) contacts the cell [3]. Similar findings for another alphaherpesvirus, herpes simplex virus type 1 (HSV-1), were reported [4]. Exogenous PS and the anionic lipid phosphatidylglycerol facilitate cell-to-cell fusion of cells expressing human immunodeficiency virus 1 (HIV-1) proteins. This led to the conclusion that specific interactions can occur between virus particles and cellular phospholipids [5]. Here, we investigated the role of PS as well as other phospholipids during virus infection. Furthermore, we assessed the specificity of the interaction between EHV-1 and differentially charged phospholipids. To this end, we to infection with EHV-1. ED cells infected without previous SUV treatment were used as a control.
Positively and negatively charged DOTAP and PS lipids significantly inhibited EHV-1 infection ( Figure   1A). In contrast, neutral PC SUVs had no effect on virus infection. At a concentration of 300 μM of all lipid preparations, none of the SUVs significantly affected cell viability after a 24 hour incubation period ( Figure 1C).
In another experiment, ED cells were incubated with 300 μM PS SUVs for different times (0, 1, Longer incubation of cells with PS rescued EHV-1 infectivity, although at significantly lower levels when compared with that in non-treated cells ( Figure 1B). It is worth mentioning that EHV-4, a close relative to EHV-1, was also inhibited in a similar fashion (data not shown).

Discussion
We report here that PS and DOTAP inhibit the infection of ED cells by EHV-1. The inhibition is immediate, reversible, and dose-dependent. Previously, we showed that EHV-1 facilitates scramblase-dependent exposure of PS on the outer leaflet of the plasma membrane, suggesting that there is a specific, yet unknown, role for PS in fusion with the plasma membrane [3]. Thus, we surmised that integration of external PS would promote fusion and enhance the infection process [5].
Contrary to our expectations, we found that EHV-1 infection was reduced in the presence of PS.
The hypothesis that PS liposomes are blocking the infection after integration into the plasma membrane of the target cell, as was described for HIV before [5], was not supported by our data,  [3,6,7]. Addition of exogenous PS can interact with herpesviral glycoproteins, particularly gH/gL and gB, and block virus entry as was described for other viruses [6]. On the other hand, it is possible that PS is redistributing the charges on the cell surface in a fashion that makes the initial contact between virus particles and cell surface proteoglycans containing heparan sulfate less likely, thus reducing the probability of virus entry.
However, the exact mechanism of how PS is able to reduce infection needs to be studied further.
Our confocal microscopy and SPR data both confirmed that PS interact with EHV-1. Although the microscopy data did not show strong interaction between PS and virus particles, the SPR data indicated a stronger interaction as compared to neutral liposomes (PC). It became clear in our study that SPR analysis is more sensitive than microscopic examination. It can detect binding of small

Materials and Methods
Viruses and Cells. Equine herpesvirus type 1 strain RacL11 EHV-1-RFP [9] with a red fluorescent protein (RFP) fused to the small capsid protein VP26 [3] was used in this study. The virus further expresses the enhanced green fluorescent protein (eGFP) for efficient identification of infected cells. The virus was grown on primary equine dermal (ED) cells (CCLV-RIE 1222, Federal Research Institute for Animal health, Germany) as described before [3]. The cells were propagated in Isocove's Liquid Medium with stable glutamine (Pan -Biotech GmbH) supplemented with 20% fetal calf serum (Pan -Biotech GmbH), 0.5% penicillin (Roth), 0.5% streptomycin (Alfa Aesar), 1% sodium pyruvate 100 mM (Pan -Biotech GmbH) and 1% nonessential amino acids (Merck KGaA). For microscopy experiments, virus was purified by ultracentrifugation over a 30% sucrose solution followed by sucrose step gradient ultracentrifugation exactly as described before [10]. During the preparation process, a final concentration of 0.01 to 0.05 mol% was used for both fluorescent lipids. All lipid preparations were tested for their possible cytotoxic effects on cells using WST-1 kit (Cayman chemical, USA) [11,12].
EHV-1-RFP virus was added to allow the interaction with the GUVs. The mixture was incubated at room temperature for at least 20 min prior to imaging.
Statistical analysis. Experiments were performed in triplicate, unless specified otherwise. Image analysis was performed in a blinded fashion. One-way ANOVA was performed using GraphPad Prism