# First evidence of efficacy of peptides targeting the pUL56-pUL89 interaction domain of the human cytomegalovirus terminase complex

Sarah Mafi, Jean-Luc Poyet, Sophie Alain, Gaëtan Ligat, Sébastien Hantz

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Sarah Mafi, Jean-Luc Poyet, Sophie Alain, Gaëtan Ligat, Sébastien Hantz. First evidence of efficacy of peptides targeting the pUL56-pUL89 interaction domain of the human cytomegalovirus terminase complex. Antiviral Research, 2025, 242, pp.106259. 10.1016/j.antiviral.2025.106259. hal-05217501

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# First evidence of efficacy of peptides targeting the pUL56-pUL89 interaction domain of the human cytomegalovirus terminase complex

Sarah Mafi a,b,*, Jean-Luc Poyet c, D, Sophie Alain a,b, Gaëtan Ligat d, Sébastien Hantz a,b, D

## ARTICLE INFO

Keywords:

Cytomegalovirus

Peptides

Terminase complex

pUL56

pUL89

Antivirals

## ABSTRACT

Human cytomegalovirus (HCMV) is a major cause of morbidity in immunocompromised patients and the leading viral cause of congenital infection. The toxicity and emergence of resistance associated with current antivirals underscore the need for alternative therapeutic strategies. The viral terminase complex (pUL56-pUL89-pUL51), essential for genome cleavage and packaging and without homologs in mammalian cells, represents a promising antiviral target. This study evaluated the antiviral potential of peptides targeting the pUL56-pUL89 interaction domain. Peptides derived from the minimal interaction domain between pUL56 and pUL89 (WF10: 671WMVVKYMGF680) or an extended sequence (PD17: 668PSEWMVVKYMGFNFSD684) were synthesized to interfere with this interaction. To optimize intracellular delivery, peptides were conjugated to a cell-penetrating peptide (CPP) derived from either the HIV-1 transactivator of transcription (TAT) or the Antennapedia homeodomain of *Drosophila melanogaster* (penetratin; PT). Peptide candidates- PT-WF10, TAT-WF10, and PT-PD17 were evaluated in cellular models for cytotoxicity, hemolysis, antiviral activity, and intracellular distribution. TAT-WF10 and PT-PD17 significantly reduced the cytopathic foci in HCMV-infected cells, with IC50 values of 58 µM and 39 µM, respectively. PT-WF10 lacked antiviral activity, induced significant cytotoxicity and hemolysis, and was mainly localized in the cytoplasm, with only minimal nuclear signal. TAT-WF10 showed cytoplasmic and nuclear distribution, no hemolysis, but induced long-term cytotoxicity from 40 µM. PT-PD17 exhibited cytoplasmic and nuclear distribution, with no significant cytotoxicity or hemolysis up to 80 µM. This study provides the first proof of concept that a peptide targeting the pUL56-pUL89 interaction domain can inhibit HCMV replication. PT-PD17 demonstrated antiviral activity, intracellular distribution, and a favorable safety profile.

## 1. Introduction

Human cytomegalovirus (HCMV) is a widespread herpesvirus that can cause severe complications in immunocompromised patients, including solid organ or hematopoietic stem cell transplant recipients and individuals with AIDS (Fishman, 2007; Goodrum et al., 2012; Griffiths and Reeves, 2021; Ljungman et al., 2010). Moreover, HCMV infection affects 1 % of newborns worldwide, with sequelae occurring mostly following infections during the first trimester of pregnancy and leading to severe neurodevelopmental disabilities (Rawlinson et al., 2017). Increasing evidence also implicates HCMV in the pathogenesis of

glioblastoma (El Baba et al., 2023; Guyon et al., 2024). Current antiviral strategies primarily interfere with the activity of the viral DNA polymerase pUL54 (Britt and Prichard, 2018). However, these treatments are associated with significant hematotoxicity and nephrotoxicity, limiting their long-term use and underlining the need for alternative therapeutic strategies (Gourin et al., 2023). Among the recently developed drugs, letermovir (a terminase complex inhibitor) (Goldner et al., 2011; Marty et al., 2017) and maribavir (an inhibitor of the viral pUL97 kinase) (Avery et al., 2022; Biron, 2006) represent promising alternatives for the prophylaxis of HCMV infection and for the treatment of refractory or resistant infections, respectively. Nevertheless, as with polymerase

<footnote>ª Université de Limoges, INSERM, RESINFIT, U1092, 2 rue du Dr Marcland, 87025, Limoges, France</footnote>

<footnote>ᵇ CHU Limoges, Laboratoire de Bactériologie-Virologie-Hygienne, Centre National de Référence pour les Herpèsvirus, 2 avenue Martin Luther King, 87042, Limoges, France</footnote>

<footnote>ᶜ INSERM UMR1342, Université Paris Cité, Institut de Recherche Saint-Louis, Hôpital Saint-Louis, 1 avenue Claude Vellefaux, 75010, Paris, France</footnote>

<footnote>ᵈ Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université de Toulouse, INSERM, CNRS, UT, Hôpital Purpan-BP 3028, 1 place du Dr Baylac, 31024, Toulouse, France</footnote>

<footnote>* Corresponding author. Université de Limoges, INSERM, RESINFIT, U1092, 2 rue du Dr Marcland, 87025, Limoges, France.</footnote>

<footnote>E-mail addresses: sarah.mafi@unilim.fr (S. Mafi), jean-luc.poyet@inserm.fr (J.-L. Poyet), sophie.alain@unilim.fr (S. Alain), gaetan.ligat@inserm.fr (G. Ligat), sebastien.hantz@unilim.fr (S. Hantz).</footnote>
inhibitors, the emergence of resistance mutations may compromise their long-term effectiveness (Chou, 2015; Chou et al., 2025; Gentry et al., 2019; Hantz et al., 2010; Kmetsch et al., 2025).

The terminase complex (pUL56-pUL89-pUL51), highly specific to HCMV, is an attractive therapeutic target owing to its crucial role in viral genome cleavage and packaging, and the absence of cellular homologs in mammalian cells (Gentry et al., 2019; Ligat et al., 2018a). Among the antiviral strategies under investigation, targeting viral protein-protein interactions (PPIs) has emerged as a promising approach against HCMV. Disruption of the interaction between the catalytic subunit pUL54 and the accessory protein pUL44 of the viral DNA polymerase, as well as inhibition of pUL44 dimerization, has been shown to reduce viral replication (Ghassabian et al., 2021; Loregian and Coen, 2006). These studies support the rationale of designing molecules that target critical interactions within viral replication machinery.

Our previous research, together with other studies, has focused on understanding the structure-function relationships within the terminase complex to identify key domains that could serve as therapeutic targets (Ligat et al., 2017, 2019; Muller et al., 2023; Scholz et al., 2003; Theiß et al., 2019). In particular, we have identified a short sequence, 671WMVVKYMGFF680 (named WF10), located in the C-terminal region of pUL56 and predicted to form an alpha-helix, which is essential for the pUL56-pUL89 interaction (Ligat et al., 2017). This discovery represents a promising target for peptide-based antiviral strategies. Therefore, peptides derived from the WF10 interaction domain were designed to interfere with this interaction. Peptides offer several advantages over small molecules (Habault et al., 2022; Habault and Poyet, 2019). Due to their physicochemical properties, they can be designed to mimic or disrupt essential viral PPIs, thereby inhibiting viral replication (Monteiro et al., 2021). However, efficient intracellular delivery remains a challenge. To address this, we optimized peptide delivery using cell-penetrating peptides (CPP). TAT, derived from the HIV-1 transactivator of transcription, and penetratin, derived from the Antenapedia homeodomain of *Drosophila melanogaster*, are widely used CPP (Habault et al., 2020; Madani et al., 2011). These short cationic sequences interact with cell membranes via electrostatic interactions, facilitating cellular uptake, improving bioavailability, and reducing toxicity.

This study aims to provide a proof of concept for the antiviral potential of these WF10 interaction domain-derived peptides by evaluating their antiviral efficacy, cytotoxicity, and intracellular uptake.

## 2. Materials and methods

### 2.1. Cells and virus

Human embryonic lung fibroblasts (MRC-5 cells) were obtained from bioMérieux (Lyon, France) and maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, and 1% gentamicin. Cells were incubated at 37 °C in 5% CO₂.

The recombinant strain AD169-GFP of HCMV, which expresses green fluorescent protein (GFP), was described by Borst and Messerle in 2000 (Borst and Messerle, 2000) and used to infect MRC-5 cells.

<table><caption>**Table 1**

Peptide names and sequences.</caption><thead><tr><td>Name</td><td>Amino acid sequence</td></tr></thead><tbody><tr><td>PT-WF10</td><td>AC-RQIKIWQNRRMKWKKWMVVKYMGFF-NH<sub>2</sub></td></tr><tr><td>TAT-WF10</td><td>AC-GRKKRRQRRRPPQWMVVKYMGFF-NH<sub>2</sub></td></tr><tr><td>PT-PD17</td><td>AC-RQIKIWQNRRMKWKK<font color="red">PSEWMVVKYMGFFNFSD</font>-NH<sub>2</sub></td></tr></tbody></table>

PT: penetratin; TAT: human immunodeficiency virus (HIV) transactivator of transcription.

### 2.2. Peptides

Peptides were synthesized by Proteogenix (Strasbourg, France) and guaranteed to be more than 95 % pure by HPLC and mass spectrometry analysis (Table 1). Rhodamine B (RhB)-labeled peptides were used for confocal microscopy to analyze cellular internalization. Stock solutions of peptides were prepared at 1 mM in water.

Peptides were designated according to the cell-penetrating peptide (CPP) used (PT or TAT), followed by two letters representing the first and last amino acids of the sequence of interest, and the number of amino acids in this sequence. For PT-PD17, the sequence 668PSEWMVVKYMGFFNFSD684 corresponds to an extended version of the minimal interaction domain of pUL56 with pUL89 (671WMVVKYMGFF680), with the addition of amino acids (in red: UL56-668 to 670 and UL56-681 to 684) to reduce the peptide hydrophobicity.

### 2.3. Cytotoxicity assays

The cytotoxicity of each peptide was assessed using the CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA) following the manufacturer's instructions. MRC-5 cells were seeded into a sterile 96-well plate (250 µL per well, $1 \times 10^5$ cells/mL) in DMEM, supplemented with 10% fetal bovine serum, 1% penicillin, 1% gentamicin and incubated at 37 °C in 5% CO₂ for 24-48 h. MRC-5 cells were then treated with peptide solutions (1.25-80 µM, 100 µL per well) and incubated at 37 °C in 5% CO₂ for 3 h (short-term exposure) or 5 days (long-term exposure). Untreated cells (cultured in medium only) served as the negative control, while 0.9% Triton X-100 was used as the positive control for 100% cytotoxicity by lysing cells for 50 min at 37 °C.

After incubation, plates were centrifuged at 250×g for 4 min, and 50 µL of supernatant from each well was transferred to a 96-well plate. Fifty microliters of reconstituted substrate mix were added to each well, and the plate was incubated for 30 min at room temperature (protected from light). The reaction was stopped by adding 50 µL of stop solution, and absorbance was measured at 490 nm using a Multiskan™ Go microplate reader (Thermo Scientific, Waltham, MA, USA). All values were normalized to Triton X-100-induced lysis. Each cytotoxicity assay was performed in triplicate and independently repeated three times.

### 2.4. Hemolysis assays

Hemolysis assays were performed to evaluate potential membrane-damaging effects of the peptides on human erythrocytes. Fresh human blood was collected in heparinized tubes and immediately centrifuged at 500×g for 10 min at 4 °C. The plasma fraction was carefully aspirated, and the remaining pellet was resuspended in 1X Dulbecco's phosphate-buffered saline (1X PBS, pH = 7.4). The suspension was gently mixed by inversion and centrifuged again at 500×g for 10 min at 4 °C. This washing step was repeated twice, removing the supernatant after each centrifugation. Following the final wash, the pellet was resuspended in 1X PBS to prepare a 4% red blood cell (RBC) suspension. One hundred twenty-five microliters of the 4% RBC suspension were added to a sterile 96-well plate, followed by 125 µL of peptide solution (final concentration ranging from 1.25 to 80 µM). The plate was then incubated for 1 h at 37 °C in 5% CO₂. 1X PBS served as the negative control (0%
hemolysis), while 1 % Triton X-100 was used as the positive control for
100 % hemolysis. The plate was subsequently centrifuged at 4000 rpm
for 5 min, and the absorbance of the supernatants was measured at 540
nm using a Multiskan™ Go microplate reader (Thermo Scientific, Wal-
tham, MA, USA). All values were normalized to Triton X-100-induced
hemolysis. Each hemolysis assay was performed in triplicate and inde-
pendently repeated three times.

2.5. *Antiviral assays*

The antiviral activity of peptides was evaluated using a plaque
reduction assay in MRC-5 cells infected with HCMV strain AD169-GFP.
Monolayer cultures of MRC-5 cells were seeded into a 48-well plate
(500 µL per well, 1 × 10⁵ cells/mL) in DMEM supplemented with 10 %
fetal bovine serum, 1 % penicillin, and 1 % gentamicin, then incubated
at 37 °C in 5 % CO₂ until confluence. Infection was then initiated by
adding 2.5 × 10² AD169-GFP-infected MRC-5 cells per well. Virus
adsorption was enhanced by centrifugation at 2876×g for 45 min at
37 °C. This process was calibrated in our laboratory for performing
antiviral assays to obtain IC₅₀ in the range of those described in litera-
ture for each antiviral. After removing the medium, infected cells were
incubated with 500 µL of peptide solution at final concentrations
ranging from 1.25 to 40 µM. If antiviral activity was observed at 40 µM,
the concentration range was extended to 80 µM. Foci of infected cells
were visualized using an Eclipse Ti2 inverted fluorescence microscope
(Nikon, Japan), and the number of GFP-positive foci was quantified.
Infected cells without peptide treatment served as the control condition.
Results were expressed as a percentage relative to this control (set at
100 %), reflecting the level of residual infection.

The 50 % inhibitory concentration (IC50), defined as the concentra-
tion required to reduce the number of cytopathic foci by 50 % compared
to untreated infected cells, was determined. Antiviral efficacy of pep-
tides was compared to ganciclovir (GCV). Each antiviral assay was
performed in triplicate and independently repeated at least three times.

2.6. *Analysis of intracellular distribution of labeled peptides*

MRC-5 cells were seeded on glass coverslips in a 24-well plate (1 × 10⁴ cells/mL) in DMEM supplemented with 10 % fetal bovine serum, 1 % penicillin, 1 % gentamicin, and incubated at 37 °C in 5 % CO₂. After reaching 10–20 % confluency, the medium was removed, and cells were incubated with RhB-labeled peptides (40 µM) for 1 h at 37 °C in 5 % CO₂. Following incubation, cells were washed twice with 1X PBS, fixed with 3 % paraformaldehyde in 1X PBS for 15 min at room temperature (protected from light), and washed again twice with 1X PBS. Nuclei were stained with 1 µg/mL DAPI in 1X PBS for 5 min at room temperature (protected from light), followed by a final wash with 1X PBS. Coverslips were mounted on glass slides using Mowiol® mounting medium. Images were acquired using an LSM 880 inverted confocal microscope (Zeiss, Germany).

2.7. Statistical analysis

Statistical analyses were performed with Prism 9 software (Graph-
Pad Software, USA) and MedCalc statistical software (version 22.026,
MedCalc Software Ltd., Ostend, Belgium). Data are presented as the
mean ± standard deviation from experiments performed in triplicate
and independently repeated at least three times. Statistical comparisons
were performed using the Student's t-test. For cytotoxicity and hemo-
lysis, peptide-treated groups were compared to untreated cells. For
antiviral activity, infected cells treated with peptides were compared to
infected cells treated with ganciclovir (GCV). Statistical differences were
considered significant at *p* < 0.05. The intracellular distribution of the
peptides was assessed by confocal microscopy. ImageJ2 software
(version 2.9.0/1.53t, NIH, USA) was used to delineate nuclear and
cytoplasmic regions and to measure the fluorescence signal of the

peptides labeled with RhB in each compartment.

3. Results

3.1. Cytotoxicity assays

3.1.1. Short-term cytotoxicity assays

No significant short-term cytotoxicity was observed for TAT-WF10 and PT-PD17 in MRC-5 cells (3 h of incubation) between 1.25 and 80 µM (Fig. 1A, Table S1A). For PT-WF10, short-term cytotoxicity was 20.85 % ± 7.22 at 80 µM, approaching but not reaching the significance threshold (p = 0.0531), representing a 2 to 3-fold increase compared to untreated cells.

3.1.2. Long-term cytotoxicity assays

For PT-WF10, significant long-term cytotoxicity was observed in MRC-5 cells (5 days of incubation), starting at 2.5 µM (20.67 % ± 2.06) and reaching 45.83 % ± 13.42 at 80 µM, representing a 2 to 3-fold increase compared to untreated cells (Fig. 1B–Table S1B). For TAT-WF10, significant long-term cytotoxicity was detected at 40 µM (28.18 % ± 6.15), showing a 1.5- to 2-fold increase compared to untreated cells. At 80 µM, cytotoxicity reached 49.40 % ± 6.40, corresponding to a 3-fold increase relative to untreated cells. Interestingly, for PT-PD17, no significant long-term cytotoxicity was observed between 1.25 and 80 µM.

3.2. Hemolysis assays

We next evaluated the hemolytic effects of the constructed peptides
in human erythrocytes. PT-WF10 exhibited significant hemolytic activ-
ity starting at 10 µM (7.04 % ± 0.39), with increasing hemolysis
measured at 20 µM (8.38 % ± 1.89), 40 µM (13.33 % ± 5.26), and 80 µM
(16.95 % ± 5.33) (Fig. 2, Table S2). For TAT-WF10 and PT-PD17, no
significant hemolytic activity was observed, with mean hemolysis values
remaining below 6 % across all tested concentrations. At 80 µM, TAT-
WF10 induced a slight increase in hemolysis (4.37 % ± 0.44),
approaching but not reaching the significance threshold (p = 0.0505).

3.3. Antiviral assays

The antiviral activity of the peptides was evaluated in MRC-5 cells
infected with the HCMV strain AD169-GFP and compared to GCV. After
5 days of incubation, PT-WF10 did not exhibit antiviral activity within
the tested concentration range (1.25–40 µM), as no reduction in the
number of cytopathic foci was observed (Fig. 3A, Table S3).

In contrast, TAT-WF10 and PT-PD17 induced a reduction in the number of cytopathic foci in a dose-dependent manner (Fig. 3B and C, Table S3), with IC₅₀ values of 58 µM and 39 µM, respectively.

For TAT-WF10, concentrations up to 20 µM led to a limited reduction
in cytopathic foci, with values ranging from 86 % ± 40 % at 1.25 µM to
85 % ± 25 % at 20 µM, expressed relative to untreated infected cells
(Fig. 3B–Table S3). A more substantial reduction was observed at 40 µM
(66 % ± 23 %), reaching 31 % ± 14 % at 80 µM. No significant differ-
ence in cytopathic foci counts was observed between TAT-WF10 at 80
µM and GCV at 5 µM (p = 0.37).

For PT-PD17, cytopathic foci were 89 % ± 20 % of control at 1.25 µM, 86 % ± 17 % at 2.5 µM, and decreased to 80 % ± 22 % at 5 µM (Fig. 3C–Table S3). Values then stabilized around 77 % (± 25 % at 10 µM and ± 26 % at 20 µM) before reaching 50 % ± 19 % at 40 µM (IC₅₀ = 39 µM). At 80 µM, foci further decreased to 11 % ± 12 %. Notably, at 40 µM, PT-PD17 exhibited an antiviral effect comparable to that of GCV at 1.25 µM, with no significant difference at these concentrations (p = 0.82).

3.4. Analysis of intracellular distribution of labeled peptides

We next investigated the intracellular distribution of the peptides.
<figure><img src="image_4.png" /><figcaption>Fig. 1. Evaluation of short-term (A) and long-term (B) cytotoxicity of peptides in MRC-5 cells.</figcaption></figure>

The cytotoxicity of PT-WF10, TAT-WF10, and PT-PD17 was assessed in MRC-5 cells using the CytoTox96® Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). The release of LDH from non-treated (NT) cells or cells exposed to different peptide concentrations was measured after 3 h of incubation in MRC-5 cells (A) and after 5 days of incubation in MRC-5 cells (B). As a positive control, cells were lysed with 0.9 % Triton X-100 for 50 min at 37 °C to induce maximal LDH release. Optical density values at 490 nm were normalized to the signal obtained from cells lysed with 0.9 % Triton X-100 (positive control). Results are expressed as the mean percentage of cytotoxicity ± standard deviation (SD). Each condition was tested in triplicate, and the experiments were independently repeated three times. p-values were calculated using the Student's t-test, comparing each treated condition to its respective NT control. Statistical significance is indicated as follows: \*p < 0.05, \*\*p < 0.01.

<figure><img src="image_5.png" /><figcaption>Fig. 2. Evaluation of hemolytic activity of peptides in human red blood cells.</figcaption></figure>

The hemolytic activity of PT-WF10, TAT-WF10, and PT-PD17 was assessed in human red blood cells (RBC) after 1 h of incubation at 37 °C in 5 % CO₂. Hemoglobin release was measured using spectrophotometry at 540 nm and normalized to the signal obtained from RBC lysed with 1 % Triton X-100 (positive control). Results are expressed as the mean percentage of hemolysis ± standard deviation (SD). Each condition was tested in triplicate, and the experiments were independently repeated three times. Statistical significance is indicated as follows: \*p < 0.05, \*\*p < 0.01, \*\*\*p < 0.001.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

(caption on next page)
<caption>**Fig. 3.** Evaluation of the antiviral activity of peptides in MRC-5 cells infected with the HCMV strain AD169-GFP.</caption>

The antiviral activity of PT-WF10 (A), TAT-WF10 (B), and PT-PD17 (C) was assessed in MRC-5 cells infected with the AD169-GFP reference strain and compared to ganciclovir (GCV). Cells were treated with increasing concentrations of PT-WF10 (A), TAT-WF10 (B), or PT-PD17 (C). After 5 days of incubation, the number of fluorescence cytopathic foci was counted for each peptide concentration and expressed as a percentage relative to non-treated (NT) cells. Curves representing the number of cytopathic foci in the presence of each peptide are shown as black circles, and those in the presence of GCV as grey squares. Data are presented as the mean ± standard deviation from independent experiments performed in triplicate and independently repeated at least three times. Red dashed horizontal lines indicate the 50 % inhibition threshold, and red dashed vertical lines indicate the mean IC₅₀ values for TAT-WF10 (58 µM) and PT-PD17 (39 µM). PT-WF10 did not reach 50 % inhibition within the tested concentration range (up to 40 µM).

MRC-5 cells treated for 1 h with the RhB-labeled peptides showed red staining in the cytoplasm, with enhanced intensity at the plasma membrane (DAPI and RhB fluorescence channels) (Fig. 4). These results suggest that the peptides rapidly enter the cells (within 1 h), accumulate at the plasma membrane, and subsequently diffuse into the cytoplasm at the tested concentration (40 µM). Of note, RhB-TAT-WF10 and RhB-PT-PD17 were also localized in the nucleus (RhB fluorescence channel), with staining patterns overlapping with that of DAPI. In contrast, PT-WF10 did not exhibit nuclear staining, suggesting that only TAT-WF10 and PT-PD17 efficiently translocate to the nucleus. To support these observations, fluorescence signal areas were analyzed in the nuclear and cytoplasmic compartments. A low nuclear signal was detected for PT-WF10 (6.6 µm²), consistent with its lack of significant nuclear penetration. In contrast, TAT-WF10 and PT-PD17 showed higher nuclear signal areas (61.4 µm² and 42.9 µm², respectively), supporting their penetration into the nuclear compartment.

## 4. Discussion

Despite advances in antiviral therapy, the management of HCMV infection remains challenging, particularly in immunocompromised patients and in the prevention of congenital transmission. Current antivirals are limited by toxicity and the emergence of resistance, emphasizing the need to explore new therapeutic targets. The HCMV terminase complex, essential for viral genome cleavage and packaging, is an attractive target for developing alternative therapeutic strategies. Peptide-based drug development is already used in many areas of medical disorders (Monteiro et al., 2021; Wang et al., 2022). The mode of action for therapeutic peptides is like biologics such as therapeutic antibodies and proteins, with the advantage of having less immunogenicity and production costs (Wang et al., 2022). Peptide therapeutics are also being investigated for their capacity to disturb and disrupt critical tumor anti-apoptosis proteins, as well as their ability to inhibit tumor drug resistance mechanisms through targeting related protein-protein signaling pathways. Additionally, the increased specificity of peptide therapy can also lead to reduced side effects (Warthen and Lueckheide, 2024). In this context, our study focused on the development of peptides that can interfere with the pUL56–pUL89 interaction, which has been previously identified as essential for terminase activity (Thoma et al., 2006). For that purpose, we designed two peptides derived from the minimal interaction domain between pUL56 and pUL89 (671WMVVKYMGFF680), and one from an extended sequence of this region (668PSEWMVVKYMGFFNFSD684) with the addition of amino acids (UL56-668 to 670 and UL56-681 to 684) to reduce hydrophobicity. Each peptide was conjugated to a CPP (penetratin or TAT) in order to address challenges associated with intracellular delivery. This design was based on the hypothesis that peptides mimicking critical binding motifs could act as competitive inhibitors, preventing subunit assembly and thereby disrupting the cleavage-packaging process essential for viral replication. Previous studies have demonstrated that interfering with essential viral PPIs can effectively suppress HCMV replication. In particular, disruption of the interaction between the accessory subunit of the viral DNA polymerase, pUL44, and the C-terminal 22 residues of the catalytic subunit pUL54, or inhibition of pUL44 homodimerization, was sufficient to impair viral replication (Ghassabian et al., 2021; Loregian and Coen, 2006). These studies reinforce the broader rationale for exploring peptide-based approaches to inhibit critical PPIs involved in various

stages of the HCMV life cycle.

Our results showed that TAT-WF10 and PT-PD17 significantly reduced the number of cytopathic foci in plaque reduction assays. In contrast, PT-WF10 - despite sharing the same core sequence as TAT-WF10 - failed to exhibit measurable antiviral activity up to 40 µM, leading us to discontinue further efficacy assessments at higher doses. Furthermore, long-term exposure to PT-WF10 resulted in statistically significant cytotoxicity from 2.5 µM, and short-term exposure at 80 µM approached significance. Hemolysis assays indicated that PT-WF10 induced hemolytic activity from 10 µM. Confocal microscopy revealed cytoplasmic localization of PT-WF10, with enhanced fluorescence at the plasma membrane. Although PT-WF10 shows minimal nuclear signal, this limited localization alone may not fully explain its lack of antiviral activity, especially considering that the terminase complex assembles in the cytoplasm prior to nuclear import (Bogner, 2002). Preliminary aggregation assays further revealed that the peptide sequence 671WMVVKYMGFF680 alone tends to self-aggregate, likely due to its hydrophobicity (unpublished data); such self-aggregation could limit effective cytoplasmic penetration and subsequent nuclear translocation. Although several models have been proposed to explain CPP internalization (Kotadiya et al., 2024), the exact mechanism involved remains poorly understood. Previous studies have demonstrated that penetratin can efficiently translocate across cellular membranes and predominantly accumulate in the nucleus (Derossi et al., 1994). Cellular uptake has been observed between 1 and 100 µM, with no evidence of saturation within this range, supporting a receptor-independent, passive mechanism of internalization. In contrast, our findings indicate that coupling penetratin to the 671WMVVKYMGFF680 sequence appears to limit nuclear access of the conjugated peptide at 40 µM, as only minimal nuclear signal was detected. This suggests that the interaction between the CPP and our target sequence may alter the expected intracellular distribution, underscoring the critical role of both the CPP and the peptide cargo composition in determining subcellular localization and, therefore, antiviral efficacy. Moreover, the observed accumulation of PT-WF10 at the plasma membrane may contribute to its increased cytotoxicity and hemolytic effects. Preferential cytoplasmic and membranous localization likely reflects inherent biochemical differences, such as variations in hydrophobicity and charge, that influence its subcellular partitioning and limit effective engagement with the viral target. Overall, our results indicate that the absence of antiviral activity of PT-WF10 is not solely due to its localization but also arises from its unique physicochemical properties compared with TAT-WF10 and PT-PD17.

TAT-WF10 significantly reduced the number of cytopathic foci in plaque reduction assays, without inducing short-term cytotoxicity or hemolysis. However, long-term cytotoxic effects were observed from 40 µM. Given that the IC₅₀ of TAT WF10 is 58 µM, the observed antiviral effect may, at least partially, result from cytotoxicity rather than specific inhibition of the terminase complex, which is consistent with prior reports of cytotoxicity associated with TAT-conjugated peptides (Jones et al., 2005). On the basis of these results, penetratin was selected as the CPP for subsequent experiments, and the peptide sequence was extended by incorporating amino acids to reduce hydrophobicity, enhance intracellular delivery and reduce potential cytotoxic effects.

PT-PD17 demonstrated consistent antiviral activity in plaque reduction assays, significantly decreasing the number of infectious foci at non-cytotoxic concentrations (IC₅₀ = 39 µM). Notably, no cytotoxicity
<figure><img src="image_9.png" /><figcaption>**Fig. 4.** Cellular localization of RhB-labeled peptides in MRC-5 cells analyzed by confocal microscopy. Cells were incubated with Rhodamine B (RhB)-labeled peptides (40 µM, red) for 1 h at 37 °C in 5 % CO₂, in the presence or absence of peptides (negative control). After fixation with 3 % paraformaldehyde in 1X PBS and nuclear staining with DAPI (1 µg/mL, blue), cells were examined using confocal microscopy with a 63x oil immersion objective (DAPI: λexcitation = 405 nm, λemission = 415–485 nm; RhB: λexcitation = 561 nm, λemission = 605–670 nm). Images were acquired using an LSM 880 inverted confocal microscope (Zeiss, Germany) controlled by Zen Black software and analyzed with ImageJ2 software, displaying individual fluorescence channels (DAPI, blue or RhB, red) and merged images (DAPI + RhB). Fluorescence signal areas were quantified within defined nuclear and cytoplasmic regions. The nuclear signal area was low for PT-WF10 (6.6 µm²), whereas TAT-WF10 and PT-PD17 exhibited higher nuclear signal areas (61.4 µm² and 42.9 µm², respectively), supporting their nuclear penetration.</figcaption></figure>

was observed up to 80 µM under both short- and long-term exposure, and no significant hemolytic activity was detected within the tested range (1.25–80 µM). As the 50 % cytotoxic concentration (CC₅₀) was not reached for any of the peptides within the tested range, selectivity index values could not be determined. However, the absence of significant cytotoxicity up to 80 µM for PT PD17, together with an IC₅₀ of 39 µM, supports a favorable selectivity profile.

Confocal microscopy revealed that PT-PD17 was effectively internalized and distributed within both the cytoplasm and the nucleus. This pattern may result from the addition of proline, serine, glutamic acid, asparagine, and aspartic acid to the sequence of interest to reduce hydrophobicity, likely limiting peptide self-aggregation and facilitating transmembrane transport. These findings support that, beyond the choice of CPP, the sequence composition plays a critical role in
modulating both intracellular distribution and antiviral efficacy (Khayyatnejad Shoushtari et al., 2021). While intracellular distribution was assessed at a single time point (1-h post-incubation), live-cell time-course imaging could provide additional insight into the kinetics of peptide uptake and trafficking.

To investigate the antiviral potential of the designed peptides, we used the recombinant AD169-GFP strain. Although derived from a laboratory-adapted virus with genomic deletions, it remains a widely used and well-established model in experimental virology. This model enabled a robust and reproducible assessment of antiviral activity in a standardized context, appropriate for initial phenotypic evaluation. Nonetheless, to assess the broader relevance of this approach, follow-up studies using clinical isolates and drug-resistant strains will be required. Testing the peptides against ganciclovir- and letermovir-resistant HCMV strains, in particular, would help determine their potential utility in the setting of antiviral resistance.

Taken together, the combination of a significant reduction in cyto-
pathic foci, absence of statistically significant cytotoxicity or hemolytic
effects, and effective intracellular distribution in both the cytoplasm and
nucleus suggests that PT-PD17 may inhibit CMV replication through
direct interference with the terminase complex. Although qPCR could be
used to quantify intracellular viral DNA, inhibition at the genome
cleavage or packaging step may result in the accumulation of non-
packaged or non-functional viral DNA. Consequently, qPCR alone may
not reliably distinguish between productive and abortive replication
events, particularly in the context of terminase inhibition. By contrast,
plaque reduction assays directly quantify infectious virus production
and offer a more relevant functional readout of antiviral efficacy in this
proof-of-concept setting. While our study focused on evaluating the
antiviral efficacy and intracellular distribution of the peptides, future
experiments will aim to determine whether the observed effects result
from a direct inhibition of the pUL56–pUL89 interaction as implied by
the design of our peptides. In this context, complementary approaches
such as co-immunoprecipitation or the NanoBiT® system—which allows
real-time monitoring of PPIs in living cells—could provide valuable
mechanistic insight.

Overall, these results identify PT-PD17 as a promising *in vitro* candidate for the development of innovative therapeutic options targeting the HCMV terminase complex. We hypothesize that the validation of PT-PD17 could provide a basis for novel antiviral strategies against HCMV. Additionally, PT-PD17 may be used in combination with existing therapies- such as DNA polymerase inhibitors (ganciclovir, valganciclovir, cidofovir, foscarnet) or recently developed drugs (maribavir, letermovir)- although the toxicity and pharmacodynamic interactions of such combinations will require careful evaluation (Cai *et al.*, 2014). A current limitation in the rational design of terminase inhibitors is the absence of a crystal structure for pUL56; only HSV-1 homolog pUL28 structure was resolved by cryoEM (Yang *et al.*, 2020). A high-resolution model of this protein would enable detailed visualization of peptide conformation and binding orientation within the targeted interaction domains. Furthermore, the antiviral peptide concept may be extended to other essential viral PPIs, such as those involved in the HCMV helicase-primase complex (Ligat *et al.*, 2018b) or in later stages of capsid assembly (Muller *et al.*, 2021). Finally, further optimization of PT-PD17 is warranted to enhance its cellular uptake, with strategies such as encapsulation in extracellular vesicles, liposomes, or nanoparticles offering potential routes to improve delivery while minimizing toxicity.

CRediT authorship contribution statement

**Sarah Mafi:** Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Jean-Luc Poyet:** Writing – review & editing, Methodology, Formal analysis, Conceptualization. **Sophie Alain:** Writing – review & editing, Resources, Funding acquisition. **Gaëtan Ligat:** Writing – review & editing,

Resources, Methodology, Funding acquisition, Formal analysis, Conceptualization. Sébastien Hantz: Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Funding

SA and SH received financial support from INSERM, Santé Publique France (National Reference Center for Herpesviruses) and Limoges University. GL received financial support from INSERM, CNRS, Toulouse University, the Défi Clé Cell Based Biotherapies Occitanie (INTERCYTE), the association “Des étoiles dans la mer, vaincre le glioblastome”, the foundation “FONROGA”, the association “Eva pour la vie”, the association “Raphaël Coeur Chocolat”, the association “111 Des Arts, Toulouse” and the Ligue contre le cancer (FoCyG).

The funding sources had no role in the study design, data collection, analysis or interpretation, manuscript writing, or the decision to submit the article for publication.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

The authors would like to thank Claire Carrion for her assistance
with confocal microscopy.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.antiviral.2025.106259.

Data availability

The data supporting the findings of this study are available in the supplementary tables submitted with the manuscript.

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