*4.5. RVFV*

The inhibition of RVFV infection by CPXV012 peptide suggests that PS is critical for RVFV infection. Although the lipid composition of RVFV is unknown, the viral envelope of the Uukuniemi virus, a related member of the bunyavirus family, is enriched for PS [66]. Like the Uukuniemi virus, RVFV obtains its membrane by budding into the Golgi membrane, suggesting that PS may also be enriched in these virus particles [67,68].

### *4.6. Viruses Not A*ff*ected by CPXV012 Peptide*

No effects of CPXV012 peptide were observed on infection with adenovirus, coxsackie B3, measles virus, and VSV. Although the measles virus and VSV are enveloped viruses, the role of PS during infection is limited for these viruses. For the measles virus, different phospholipids were tested for their ability to reassemble with the viral envelope glycoproteins and to restore hemolytic activity. In contrast to phosphatidylethanolamine and PC, PS completely failed to restore hemagglutination activity [69]. For VSV, early studies considered PS expressed on the host cell important for cell entry [70]. In contrast, a more recent study found no correlation between PS surface levels and VSV binding. Moreover, masking of phosphatidylserine with annexin V during infection did not affect VSV binding to cells, or entry of virus particles pseudotyped with VSV-G [64,71]. Thus, the limited role of PS during entry of VSV and measles virus may explain the lack of inhibition by CPXV012 peptide.

Both adenovirus and Coxsackievirus B3 are non-enveloped viruses and the nucleocapsid does not contain any phospholipids (nor glycoproteins) CPXV012 peptide could bind to. CPXV012 peptide binding to the host cell membrane might not be sufficient for the steric impediment of viral ligand–cellular receptor interaction. Supporting this, the adenoviral elongated fiber protein responsible for binding to the host cell is flexible and large in size (9 to 30 nm), as is the coxsackie and adenovirus receptor (CAR) on the host cell [72].

CAR is also important for the infection of free CVB3 particles [73]. In addition, CVB3 may use a PS-dependent entry strategy for the bulk transmission of virus particles [74]. Infected cells release clusters of virus particles wrapped in PS-enriched membranes. These vesicles enhance subsequent virus entry in a process that highly depends on the presence of PS [74]. As CPXV012 peptide had no effect on CVB3 virus infection, PS may not play a significant role in our study. This discrepancy likely results from our virus production protocol that involves repeated freeze-thawing to liberate virus particles. Although necessary to obtain high virus titers, this method also releases CVB3 from PS-enriched vesicles [74].

In conclusion, the CPXV012 peptide-mediated inhibition of virus infection correlates well with the observed interaction between CPXV012 peptide and PS. The broad inhibitory range may thereby be further extended to other viruses for which PS has a crucial function during the viral life cycle. These viruses include clinically and economically important pathogens such as Ebola virus, Lassa virus, dengue virus, and poliovirus [61].

The differences between viral and host membranes make PS an Achilles' heel that can be targeted by the CPXV012 peptide. It will be worth investigating if other cationic peptides, including certain antimicrobial peptides, can also target PS within the viral envelope, and perhaps fulfill a similar function as broad-range antiviral agents. Analyses of the peptide's biophysical and physicochemical properties can shed some light on how to improve the moderate efficiency of CPXV012 and other cationic peptides. Highly efficient antiviral peptides are interesting candidates for prophylactic treatment and antiviral therapy, as they do not require active replication and target viral components that are less likely to develop drug-resistance [75].

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4409/9/9/1989/s1, Table S1: Inhibitory effect of CPXV012 peptide on enveloped and non-enveloped viruses of different families. Figure S1: CPXV012 peptide does not affect cell viability. Figure S2: CPXV012 peptide (CPX) prevents infection with MVA in different cell lines. Figure S3: CPXV012 peptide does not affect infection with the measles virus.

**Author Contributions:** Conceptualization, R.D.L. and F.T.; methodology, P.P., S.M.S., K.S., C.B., S.M.d.B. and M.H.; validation, S.T., S.M.S., N.C.B. and U.P.; formal analysis, K.S., R.J.L., S.T. and M.H.; investigation, R.D.L., P.P., F.T., S.M.S., K.S., C.B., S.M.d.B., S.T. and M.H.; resources, E.J.H.J.W., J.W.D., J.A.K., R.J.L. and I.D.; data curation, A.I.C., F.T., R.J.L. and N.C.B.; writing—original draft preparation, R.D.L., P.P. and F.T.; writing—review and editing, R.D.L., P.P., I.D., U.P. and A.I.C.; visualization, R.D.L., J.A.K., P.P. and A.I.C.; project administration, E.J.H.J.W. and I.D.; funding acquisition, P.P. and S.M.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** P.P. was supported by the European Commission under the Horizon2020 program H2020 MSCA-ITN GA 675278 EDGE. S.M.S. was supported by the seventh framework program of the European Union (Initial Training Network "ManiFold," Grant 317371), and ID was supported by the DFG funding GRK 1949.

**Conflicts of Interest:** The authors declare no conflict of interest.
