**4. Discussion**

In this study, we identified the CPXV012 peptide as a broadly-acting antiviral agen<sup>t</sup> that probably interacts with the anionic phospholipid PS. The antiviral potency of the CPXV012 peptide can be considered modest, ye<sup>t</sup> we believe that this peptide may be a candidate for biochemical improvement. The interaction between the CPXV012 peptide and PS is likely mediated by opposing electrostatic forces determined by cationic residues within the peptide and the anionic head groups of PS. In addition, hydrophobic and aromatic amino acid residues (which make up more than 30% of CPXV012 peptide) may facilitate penetration of the peptide into the lipid phase of the membrane. As shown for other peptides, such membrane interactions may promote the formation of an alpha-helical secondary structure of otherwise unstructured peptides [33]. Due to the distribution of charged and hydrophobic residues over the length of the CPXV012 peptide, the alpha-helix can be expected to have somewhat of an amphipathic character. This may allow it to bind with an orientation that is roughly parallel to the membrane surface [34] or to assemble into oligomeric transmembrane pores [35]. These biophysical properties of CPXV012 peptide are shared with many antimicrobial host defense peptides [36], that form a crucial innate immune barrier by targeting the negatively charged surfaces of bacteria and certain fungi. Like CPXV012 peptide, these small peptides (up to 50 residues) have a net positive charge and high a ffinity for anionic phospholipids. The structural rearrangements occurring upon membrane binding are crucial for the antimicrobial activity and can trigger pore formation, membrane depolarization, and disruption of the bacterial membrane [37,38].

Although less studied, antimicrobial peptides may also a ffect virus infection by di fferent mechanisms. These mechanisms include interference with viral entry (binding to receptors, fusion), inhibition of viral replication, reduction/suppression of viral gene expression, immunomodulation, viral aggregation, and disruption of the integrity of the viral membrane (reviewed in [6,7]). While a list of peptides is reported to disrupt viral envelopes, their mode of action has not been formally tested. Still, a number of them were shown to directly a ffect viral membranes, although the specific interacting partners remain elusive. Examples include the human cathelicidin LL-37 and its murine equivalent mCRAMP, which disrupt the membranes of RSV, influenza A virus and VACV, and can prevent virus infection in vivo [39–41]. Although their e ffect on the infectivity of other viruses is not known, the structural similarities between these cationic peptides and CPXV012 peptide may sugges<sup>t</sup> that these and other antimicrobial peptides possess a broader antiviral activity. Electron microscopy has also provided indications of envelope disruption with the cationic peptides Tachyplesin (VSV) [7], Temporin B (HSV-1) [42], and more recently Urumin (H1-bearing influenza A viruses) [43].

Membrane leakage induced by CPXV012 peptide was preceded by a lag phase, as was also observed for human IAPP. For human IAPP, this lag phase originates from the formation of transient oligomeric intermediates prior to the assembly of membrane-disruptive aggregates [32]. Likewise, disruption of membranes by CPXV012 peptides may be linked to the formation of (transient) oligomeric structures. The shorter lag time in the presence of PS is possibly due to more e fficient binding of the peptide to the membrane: upon favorable electrostatic interactions, CPXV012 peptides achieve a higher local concentration and therefore the assembly process is supported. The lag phase of several hours appears to be inconsistent with the almost immediate e ffect observed in virus infection assays. There can be several reasons for this apparent discrepancy. First, the local peptide concentration at the membrane surface in the virus assays is likely to be much higher than in the leakage experiments, facilitating the oligomerization process. Second, antiviral activity may not require the formation of pores that are large enough to allow passage of calcein. Instead, binding to the viral membrane and/or insertion of hydrophobic amino acid side chains between the lipid acyl chains by itself already may be su fficient for an antiviral e ffect. Third, other factors may be involved in the virus infection assays, such as interactions with viral (glycol)proteins in addition to PS, that help accelerate peptide oligomerization on the viral surface.

Our data indicate that the CPXV012 peptide neutralizes the infectivity of viruses from diverse families. In vitro assays indicate that the CPXV012 peptide directly interacts with membranes. The disruptive behavior of the CPXV012 peptide on artificial membrane vesicles suggests that the CPXV012 peptide may similarly affect viral membranes. As viral particles do not have a membrane repair mechanism, this damaging effect may particularly destabilize and disrupt viral particles. Extensive membrane damage may also hamper virus fusion to the host cell membrane by impacting the fluidity and curvatures of lipid membranes. Lipids including PS play an essential role in the induction of these membrane curvatures and subsequent membrane fusion [44,45]. Even in the absence of membrane damage, the interaction between CPXV012 peptide and PS in the viral envelope may interfere with virus infectivity by sterically blocking the interaction between viral PS and host cell PS receptors. A similar inhibitory mechanism was observed using the PS-specific antibody Bavituximab [3,46], or the PS-binding protein Annexin V [47,48]. CPXV012 peptide may also indirectly affect virus infection by binding to PS on the host cell membrane. Specific microdomains of the host cell plasma membrane are enriched for PS, including lipid rafts [49]. These sites are used by certain viruses as an entry point [50]. Although PS typically would reside in the inner leaflet of the plasma membrane, there are indications that some viruses induce redistribution of PS to the outer membrane to promote/facilitate entry [50]. In addition, some viruses use lipid rafts as budding sites when exiting the host cell [51].

The effect of CPXV012 peptide on the viruses used in this study and potential inhibitory mechanism(s) are discussed in more detail.
