**1. Introduction**

The continuous threat of conventional viruses and emergence of new virus species requires an ongoing search for new antiviral compounds. Broad-spectrum antiviral inhibitors have become promising therapeutic candidates [1]. These compounds act on common elements shared by many groups of viruses, including the synthesis of viral RNA and DNA, viral proteases, and glycosylation of viral proteins (reviewed by [2]). An important class of broad-spectrum antivirals targets the lipid envelope of viruses [3–7]. These include not only antibodies and chemical compounds, but also cationic peptides present as defense peptides in the human host and other organisms. In essence, the applicability of such peptides as antiviral compounds targeting the envelope of viruses will depend on their ability to discriminate viral from human membranes.

Enveloped viruses, in contrast to non-enveloped ones, have their nucleocapsid surrounded by a lipid membrane. Depending on the virus, this lipid bilayer is derived from the plasma membrane or intracellular organelles. Although the membrane is host cell-derived, it may di ffer functionally and structurally from the membrane of origin in several aspects, including the lipid composition. Compared to host cell membranes, the envelope of many viruses is enriched for the phospholipid phosphatidylserine (PS). PS is the most abundant anionic lipid in eukaryotic cells. In resting cells, PS is mainly contained within the cell, with only very limited exposure to the extracellular environment. PS is enriched in some intracellular organelles, but the majority of PS is asymmetrically distributed on the inner leaflet of the plasma membrane [8]. This organization results from constantly flipping PS from the outer membrane leaflet to the inner membrane leaflet by ATP-dependent flippases. As a result, the outer exposed membrane leaflet of the cellular plasma membrane is devoid of PS. In apoptotic cells, this organization is lost due to the inactivation of flippases and the activation of scramblases. The resulting PS exposure on apoptotic cells and cell debris induces uptake by surrounding cells through PS receptors. Many viruses take advantage of this uptake mechanism by exposing PS on their envelope, thereby facilitating virus entry. The increase in PS on the virus membrane may be due to the lack of an active flippase. Alternatively, some viruses actively accumulate PS at sites of virus budding, or bud from subcellular microdomains enriched in PS [9].

Another important aspect of the host membrane is its property of self-renewal upon injury. This fast-acting mechanism involves several events, including the detection of the damaged site, exocytosis of nearby cytosolic vesicles, cytoskeletal remodeling, endocytosis of the damaged membrane, and reconstitution of membrane homeostasis [10,11]. The lipid membrane loses its self-renewal capacity once surrounding the viral nucleocapsid, thereby becoming particularly prone to membrane damage [5]. The structural and functional di fferences between virus and host cell membranes make viral membranes ideal targets for antiviral therapy.

Here, we identify a peptide with broad-spectrum antiviral activity, whose sequence is derived from the cowpox virus protein CPXV012. Within virus-infected cells, this protein helps to evade the immune system by inhibiting the transporter associated with antigen processing (TAP), thereby interfering with MHC I-dependent antigen presentation [12–16]. We pinpointed the segmen<sup>t</sup> of the CPXV012 protein responsible for blocking TAP: a peptide comprising the C-terminal domain of CPXV012. It should be noted that we have no indication that this peptide is produced upon CPXV infection. Interestingly, when doing functional assays with this peptide, upon infection we noticed that the percentage of infected cells decreased significantly. This finding was the basis for the current study. Here, we show that this peptide inhibits infection of many enveloped viruses by interacting with virus particles. Variants of the CPXV012 peptide revealed that basic residues within the peptide are important for this inhibitory e ffect. Furthermore, CPXV012 strongly interacts with lipid monolayers and membranes enriched for the anionic phospholipid PS. These results sugges<sup>t</sup> that this CPXV012 peptide disturbs the integrity of viral membranes enriched for PS, and thus may be explored as an antiviral agen<sup>t</sup> against a broad range of enveloped viruses.

### **2. Materials and Methods**

### *2.1. Cells and Viruses*

MelJuSo (MJS) and BHK21 cells were cultivated with RPMI 1640 containing 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine (complete medium). HEK293T, HeLa, Huh7.5, and Vero cells were grown in DMEM supplemented with 10% FCS (5% for Vero cells), 100 U/mL penicillin, 100 μg/mL streptomycin, 1% nonessential amino acids, 2 mM L-glutamine, and 1% sodium pyruvate (complete DMEM). HepRG cells were differentiated in Williams E medium supplemented with 10% FCS (Fetalclone II from Hyclone, Thermo Scientific; Breda, The Netherlands), 100 U/mL penicillin, 100 μg/mL streptomycin, glutaMax, 0.023 IE/mL human insulin (Sanofi Aventis, Amsterdam, Netherlands), 4.7 μg/mL hydrocortisone (Pfizer, Capelle a/d IJssel, The Netherlands), 80 μg/mL Gentamicin, and 1.8% DMSO. HepG2.2.15 cells were cultivated in Williams E medium with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1% nonessential amino acids. LC5-RIC cells (EASY-HIT assay) were maintained as described previously [17].

Recombinant modified vaccinia virus Ankara (MVA) expressing eGFP under the natural early/late promoter p7.5 was used in this study (MVA-eGFP) [18]. MVA-eGFP was propagated and titrated in chicken embryonic fibroblasts (CEFs) according to standard methodology [19]. Vaccinia virus expressing eGFP (VACV-eGFP) was a generous gift from Dr. Jon Yewdell (NIH, Bethesda, USA). VACV-eGFP was propagated and titrated on CV-1 cells. Cowpox virus strain Brighton Red (CPXV-BR) expressing RFP/eGFP virus was originally obtained from Dr. Karsten Tischer (FU Berlin, Germany). HSV-1 expressing the VP16-eGFP was kindly provided by Dr. Peter O'Hare (Imperial College London, UK). Stocks were prepared and titrated on Vero cells.

HBV particles were concentrated from the supernatant of HepG2.2.15 cells as described previously [20]. Infectious HIV-1 stocks were prepared as described before [17]. Measles virus expressing eGFP (MV-eGFP) was generated as previously described [21]. Vesicular stomatitis virus expressing Luciferase (VSV-deltaG (Luc)) was a kind gift from Dr. Gert Zimmer (Institute of Virology and Immunology, Mittelhäusern, Switzerland). Recombinant adenovirus expressing GFP and ovalbumin (AdGOva) was kindly provided by Dr. Percy Knolle (Institutes of Molecular Medicine and Experimental Immunology, Bonn, Germany) and virus stocks were prepared as previously described [22]. Non-spreading Rift Valley fever virus (RVFV) replicon particles were produced and titrated as described previously [23]. Coxsackie B3 virus expressing Renilla Luciferase (RLuc-CVB3) was produced and titrated as described previously [24].
