*2.2. Peptides*

The inhibitory peptide used in this study comprises amino acids 36 to 69 of the CPXV012 protein (QEGISRFKICPYHWYKQHMSLLFRRYYHKLDSII). The control peptide is derived from the pseudorabies virus TAP inhibitor UL49.5 and comprises amino acid residues 28 to 59 of this protein (sequence: STEGPLPLLREESRINFWNAAUAARGVPVDQP). U in amino acid sequence refers to alpha-amino-n-butyric acid, see below.

Synthetic peptides were prepared by normal Fmoc chemistry using preloaded Tentagel resins, using PyBop/N-methylmorpholine for in situ activation and 20% piperidine in N-methylpyrrolidinone for Fmoc removal [25]. Couplings were performed for 75 min. After final Fmoc removal, peptides were cleaved with trifluoroacetic acid/H2O 19:1 *v*/*v* containing additional scavengers when C or W residues were present in the peptide sequence. Peptides were isolated by ether/pentane precipitation. Peptides were checked for purity using reversed-phase HPLC–mass spectrometry and for integrity using MALDI-TOF mass spectrometry, showing the calculated molecular masses. All peptides were synthesized with an N-terminal biotin moiety and a C-terminal amide. Cysteines were replaced by the isosteric alpha-amino-n-butyric acid. Lyophilized peptides were dissolved in DMSO. Peptide concentrations were confirmed by the NanoDrop spectrophotometer (Thermo Scientific, Amsterdam, The Netherlands) using the absorbance of Trp at 280 nm. Peptides were aliquoted in Eppendorf LoBind microcentrifuge tubes (Sigma-Aldrich, Zwijndrecht, The Netherlands) and kept at −20 ◦C for short-term storage.

### *2.3. Cell Viability Assays*

Cells were incubated with CPXV012 peptide, control peptide, or DMSO at the indicated concentrations for at 37 ◦C for 20 to 24 h. Thereafter, cell viability was measured using different assays. DAPI staining was performed to discriminate between live and dead cells using flow cytometry. Cell Titer-Blue Cell Viability Assay (Promega, Leiden, The Netherlands) was performed according to the manufacturer's instructions. Neutral Red uptake (Applichem, Darmstadt, Germany) was performed as described previously [26]. WST-1 salt conversion assay (Roche, Woerden, The Netherlands) was performed according to the manufacturer's instructions at 7 or 20 h post exposure to the peptides. No difference was observed between 7 and 20 h post exposure.

### *2.4. Virus Inhibition Assays*

### 2.4.1. MVA, VACV, and Cowpox

MJS, BHK21, HeLa, and HEK-293T cells (1 × 105) were seeded in 24-well plates in a total volume of 400 μL and incubated overnight at 37 ◦C. Cells were infected with MVA-eGFP, VACV-eGFP, or CPXV-RFP/eGFP at 37 ◦C for 18 to 20 h in medium containing 0.1% DMSO and the indicated concentrations of either CPXV012 peptide or control peptide UL49.5 or 0.1% DMSO only as vehicle control. Flow cytometric analysis was performed to quantify the percentage of infected cells as indicated by eGFP or RFP expression.

Where mentioned, MVA-eGFP was pretreated with 50, 100, or 150 μg/mL CPXV012 peptide, control peptide, or DMSO vehicle at 37 ◦C for 1 h. Thereafter, the virus-CPXV012 peptide mixture was diluted 1:10 by volume and used to infect MJS cells (corresponding to an MOI of 10) resulting in a final concentration of 5, 10, or 15 μg/mL peptide in the culture medium. As a control, MJS cells were incubated with the same concentrations of CPXV012 peptide (5, 10, and 15 μg/mL) during infection with MVA-eGFP that was not pretreated with peptides. After 20 h, the cytometric analysis was performed to quantify the percentage of infected cells indicated by eGFP-expression.

For qPCR analysis (Figure 1B), MJS cells were infected with MVA-eGFP (MOI 10) in the presence of 100 μg/mL of either CPXV012 peptide or control peptide in medium with 0.1% DMSO or in 0.1% DMSO only as vehicle control at 37 ◦C for 20 h. RNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). A total of 3 μg of RNA was digested with 10 U of DNase I (Roche, Woerden, The Netherlands) and cDNA synthesis was performed by using 200 U Superscript III RNase H reverse transcriptase (Invitrogen, Amsterdam, The Netherlands), 7.5 pmol oligo(dT)12–18 (Invitrogen), 20 U of RNasin (Promega), and 10 mM each deoxynucleoside triphosphate (Qiagen). For semiquantitative analysis of viral mRNAs, cDNA was used as the template for a PCR with the indicated primers: B8R (fwd ATCCGCATTTCCAAAGAATG, rev ACATGTCACCGCGTTTGTAA), H3L (fwd GTCTTGAAGGCAATGCATGA, rev TCCCGATGATAGACCTCCAG) and G8R (fwd ATC GAT AAA CTG CGC CAA AT), and rev CTC CGC GGT AGA ACA CTG AT). Quantitative RT-PCR analysis was performed by using the LightCycler DNA Master SYBR green I kit (Roche) and LightCycler 1.5 (Roche). Gene expression was determined using the 2−ΔΔCT method. The results are quantified relative to the housekeeping gene GAPDH [27,28].
