# Deciphering Letermovir's Mode of Action and Resistance Mutation Effects

C. Gourin, T. Flores, C. Lefèvre, S. Alain, G. Ligat, S. Hantz

► To cite this version:

C. Gourin, T. Flores, C. Lefèvre, S. Alain, G. Ligat, et al. Deciphering Letermovir's Mode of Action and Resistance Mutation Effects. Antiviral Research, 2025, 244, pp.106289. 10.1016/j.antiviral.2025.106289. hal-05307832

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# Deciphering letermovir's mode of action and resistance mutation effects

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C. Gourin<sup>a, ID</sup>, T. Flores<sup>b</sup>, C. Lefèvre<sup>a, ID</sup>, S. Alain<sup>a,c ID</sup>, G. Ligat<sup>b,1,* ID</sup>, S. Hantz<sup>a,c,1,** ID</sup>

## ARTICLE INFO

Keywords:

Terminase complex

Letermovir

Resistance

NanoBit PPI

Cytomegalovirus

## ABSTRACT

Human cytomegalovirus is an opportunistic pathogen responsible for severe infections in immunocompromised patients, the leading cause of congenital infections worldwide, and potentially implicated in carcinogenesis. The HCMV terminase complex (pUL56-pUL89-pUL51) has emerged as a key target for antiviral drug development. Letermovir, an antiviral agent targeting this complex, inhibits viral DNA packaging, but resistance-associated mutations have already been identified within subunits. Moreover, the precise mechanism of action of letermovir remains incompletely understood.

We investigated interactions among terminase subunits in presence or absence of letermovir. Wild-type and mutant forms of these proteins (including resistance mutations V236M, L241P, L257I, C325Y, R369M in pUL56 and A95V in pUL51) were cloned into NanoBiT® PPI and pCI-neo vectors. Letermovir was added after transfection in HEK293T cells, and protein-protein interactions were assessed.

Our results show letermovir does not disrupt interactions between wild-type terminase subunits. Resistance-associated mutations modulate the strength of these interactions, with certain mutations (such as pUL56 V236M and L257I) significantly enhancing or reducing binding. Notably, double mutants exhibited synergistic effects. Structural analysis using the AlphaFold3 platform revealed differences between the mutation site of pUL56 and its HSV-1 counterpart pUL28. A hypothetical 3D analysis based on the cryo-EM structure of the HSV-1 terminase complex showed that resistance mutations were oriented outside the complex.

These findings suggest letermovir does not act by directly inhibiting interactions among HCMV terminase subunits. Analysis of resistance-associated mutations provides insight into the molecular basis of HCMV resistance to letermovir and may inform development of novel antiviral strategies targeting the terminase complex.

## 1. Introduction

Human cytomegalovirus (HCMV) is an opportunistic pathogen with a significant risk of severe infection in immunocompromised individuals, including AIDS patients and transplant recipients. It is the leading cause of congenital infections worldwide and increasing evidences also implicate HCMV in the pathogenesis of glioblastoma and acute lymphoblastic leukemia (Gourin et al., 2024; Karagas et al., 2025). Currently, available antiviral treatments, such as ganciclovir and its

prodrugs, target the viral polymerase pUL54. However, these treatments are often associated with adverse effects, including neutropenia and nephrotoxicity, and the emergence of mutations has led to cross-resistance among polymerase inhibitors (Santos Bravo et al., 2021). To address these limitations, alternative antiviral strategies have been developed, including letermovir and maribavir. Letermovir targets the viral terminase complex (pUL56, pUL51 and pUL89), a component unique to the virus with no cellular counterpart in mammalian cells, making it an attractive therapeutic target (Ligat et al., 2020, p. 201,

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

<footnote><sup>b</sup> Univ Toulouse, INSERM, CNRS, Infinity - Toulouse Institute for Infections and Inflammatory Diseases, Toulouse, Hôpital Purpan-BP 3028, 1 place du Dr Baylac, 31024, Toulouse, France</footnote>

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

<footnote>* Corresponding author.</footnote>

<footnote>** Corresponding author. CHU Limoges, Laboratoire de Bactériologie-Virologie-Hygiène, Centre National de Référence pour les Herpèsvirus, 2 avenue Martin Luther King, 87042, Limoges, France.</footnote>

<footnote>E-mail addresses: claire.gourin@unilim.fr (C. Gourin), thelma-nicole.flores-aguilar@inserm.fr (T. Flores), camille.lefevre@etu.unilim.fr (C. Lefèvre), sophie.alain@unilim.fr (S. Alain), gaetan.ligat@inserm.fr (G. Ligat), sebastien.hantz@unilim.fr (S. Hantz).</footnote>

<footnote n="1">¹ co-senior author.</footnote>
2018; Lischka et al., 2010). It was approved for prophylaxis of he-
matopoietic stem cell recipients (Marty et al., 2017). Maribavir inhibits
the viral kinase pUL97 and was approved for refractory-resistant in-
fections (Avery et al., 2021). As with polymerase inhibitors, resistance
mutations have already been identified for both letermovir and mar-
ibavir (Chou, 2015, 2017; Chou et al., 2004, 2007). In the case of
letermovir, most resistance mutations are located in the pUL56 subunit,
although mutations have also been detected in pUL51 and pUL89 (Chou,
2015, p. 56; Muller et al., 2022). These mutations confer varying level of
resistance ranging from 1.8 to 4 fold higher than WT strain for the
mutation V236M to more than 3000 fold for the mutation of the amino
acid C325F (Chou, 2015; Chou et al., 2018). Moreover, this residue was
described as involved in the formation of a binding pocket of letermovir
in pUL56 (Kmetsch et al., 2025). To a lesser extent, some mutations as
pUL56 L257I, R369M, V236M or L241P were found to confer a resis-
tance between 10 and 250 fold higher (Chou, 2015). Moreover, the
addition of mutations in pUL51 or pUL89 was shown to have a syner-
gistic effect and increase resistance levels of pUL56 mutations (Muller
et al., 2022, p. 51).

Regarding the mechanism of action of letermovir, it has been re-
ported to prevent DNA cleavage into concatemeric genomic units
(Goldner et al., 2011). However, its mode of action, particularly its
interaction with the terminase complex is not fully elucidated at present.
In this study, we hypothesized that letermovir might disrupt the inter-
action between terminase subunits. Therefore, the NanoBit® PPI tech-
nology (Promega, USA) was used to assess interactions among pUL56,
pUL51 and pUL89, both in the presence and absence of letermovir. We
also examined how various resistance mutations could affect these in-
teractions. Our findings indicate that letermovir does not interfere with
terminase subunit interactions, although resistance mutations can
modulate the strength of these interactions.

**2. Materials and methods**

**2.1. Cells, plasmids, bacterial strains and HCMV strains**

HEK293-T cells were cultivated in Dulbecco's modified eagle me-
dium (DMEM) with L-glutamine supplemented with 10 % fetal bovine
serum (FBS), 1 % penicillin and 1 % gentamicin. They were incubated at
37 °C in a 5 % atmosphere. *E.coli* MG1656 and *E. coli* Stellar were used
for production of plasmid constructs and were cultivated in Lysogeny
Broth medium supplemented with 25 µg/µL of ampicillin. AD169 and
HCMV-BAC mutants previously built in our laboratory were used to
amplify genes of interest with primers available in Table S1. NanoBit®
PPI vectors and pCI-neo vector (Promega, USA) were used to clone the
terminase genes.

**2.2. Antiviral compound**

Letermovir (PREVYMIS, AIC246, MedChemExpress, #HY-15233) was reconstituted with DMSO at a concentration of 10 mM then diluted in culture medium to reach a final concentration of 50 nM. It was added immediately after the transfection of HEK293-T cells and the selective pressure was kept during all the experiment until the luminescence measurement. DMSO was always used at a concentration of less than 0.1 %.

**2.3. NanoBit PPI cloning and assays**

UL56, UL89 and UL51 and mutant genes were amplified with primers
in Table S1, pUL56 R369M and L241P mutations were reproduced by
primers in Table S2 with an assembling PCR. The first amplification was
as follow: first denaturation at 98 °C for 5 min followed by 35 cycles of
30s denaturation at 98 °C, 30s hybridization at 58 °C and 10s elongation
with a final elongation at 72 °C for 5 min. The second PCR for assem-
bling fragments or only amplifying was as described: first denaturation

at 98 °C for 5 min followed by 35 cycles of 30s denaturation at 98 °C, 45s
hybridization at 58 °C and 20s elongation with a final elongation at
72 °C for 5 min. Digestion of inserts and vectors were done using EcoRI/
NheI enzymes. Ligation was conducted at 16 °C overnight using T4 ligase
(New England Biolabs, France). Constructs were transformed in E. coli
strains by thermal shock at 42 °C. Colonies were screened using primers
C-Sm F/R in Table S1 and sequenced using primers in Table S3. Plasmids
were purified using Xtra Midi kit (Macherey Nagel™, Germany). Then,
2.5 µg of each construct was transfected into HEK293-T cells at 125
000c/mL in a 6-well plate using Fugene 4K reagent (Promega, USA).
After 72 h of production, cells were trypsinized and transferred into a
96-well plate at 500 000c/mL in triplicates. NanoGlo® substrate was
added and luminescence emission was recorded using FluoStar® Omega
reader plate (BMG LabTech, Germany).

**2.4. Structure prediction and interaction modelling**

AI-based 3D model of terminase complex was obtained with the AlphaFold3 platform. Model was obtained with constraints based on already known data concerning HSV-1 terminase complex (PDB IDs: 6M5R and 6M5S) (Yang et al., 2020) such as presence of Mg²⁺ and Zn²⁺ ions and double stranded DNA helices. Five models were obtained with pLDDt scores ranging around 76 for each. The best model with higher ipTM (interface predicted template modeling) and pTM (predicted template modeling) scores was chosen for the study. Structures were analyzed and visualized thanks to Visual Molecular Dynamics 1.9.4α software (VMD). For structure alignments, PyMOL software was used. The unresolved structures of the pUL56 and pUL51 models are not shown (residues 425 to 485 and 778 to 850 for pUL56 and 1 to 80 for pUL51).

**2.5. Statistical analysis**

To ensure reproducibility of experiments, all assays were done in triplicate at different times. Obtained results were submitted in GraphPad PRISM 8.4.3 software. A t-test and a two-way Anova mixed-model test were used to analyse differences between values and significance was expressed as described: (* *p* < 0.05, ** *p* < 0.01, *** *p* = 0.001, **** *p* < 0.0001).

**3. Results**

**3.1. The interaction between terminase subunits remains unaffected by letermovir treatment**

To perform the NanoBit® PPI experiments, we tested the following
interaction pairs: C-Lg-pUL56/C-Sm-6his-pUL89, C-Lg-pUL56/C-Sm-
6his-pUL89/pCI-neo-pUL51, C-Lg-pUL56/N-Sm-6his-pUL51, C-Lg-
pUL56/N-Sm-6his-pUL51/pCI-neo-pUL89, C-Lg-6his-UL51/N-Sm-6his-
UL89 and C-Lg-6his-UL51/N-Sm-6his-UL89/pCI-neo-pUL56. Lumines-
cence was measured for each pair to evaluate the level of interaction.
The pair C-Lg-pUL56/C-Sm-6his-pUL89/pCI-neo-pUL51 showed signif-
icantly higher luminescence emission compared to C-Lg-pUL56/C-Sm-
6his-pUL89 (*p* < 0.0001) (Fig. 1B), suggesting an enhancing effect of
pUL51. Similarly, the interaction pair C-Lg-pUL56/N-Sm-6his-pUL51
displayed significantly greater interaction in the absence of pCI-neo-
pUL89 (*p* = 0.004) (Fig. 1C). For all other pairs, there was no signifi-
cant difference (Fig. 1).

NanoBit® PPI experiments were conducted using letermovir at a
concentration ten times higher than the IC50 determined for the AD169
strain. The following interaction pairs were studied: C-Lg-pUL56/C-Sm-
6his-pUL89, C-Lg-pUL56/C-Sm-6his-pUL89/pCI-neo-pUL51, C-Lg-
pUL56/N-Sm-6his-pUL51, C-Lg-6his-UL51/N-Sm-6his-UL89 and C-Lg-
6his-UL51/N-Sm-6his-UL89/pCI-neo-pUL56. Luminescence measure-
ment indicated that 50 nM of letermovir did not significantly reduce the
interaction levels of any of the tested pairs compared to their respective
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** NanoBiT® protein-protein interaction system and analysis of HCMV terminases with and without letermovir treatment. **A.** Principle of NanoBiT® PPI technology applied to terminase subunits. UL56 and UL89 are cloned into the C-LgBiT and C-SmBiT vectors, respectively. pUL89 has a 6his tag in its N-terminal region. The UL51 gene has been cloned into the pCI-neo vector to complete the terminase complex. When pUL56 and pUL89 come into contact, this brings together the two luciferase subunits and forms a functional luciferase capable of consuming the NanoGlo Live Cell substrate. Its consumption produces a luminescent emission that can be recorded using a plate reader. **B.** Luminescence measurement to evaluate pUL56 and pUL89 interaction in presence or in absence of letermovir. **C.** Interaction between pUL56 and pUL51 with and without letermovir. **D.** pUL51 and pUL89 interaction with and without letermovir. Interactions were recorded after 72h in HEK293T cells. 50 nM of letermovir were added at transfection and during measurement of luminescence. Luminescence was measured as a percentage of the 100 % control, i.e. the whole complex without letermovir for each condition tested. Three independent replicates were done for each experiment and a two-way Anova mixed model was used for statistical analysis: \* p < 0.05, \*\* p < 0.01, \*\*\* p = 0.001, \*\*\*\* p < 0.0001, ns: non-significant.</figcaption></figure>

controls without the letermovir (Fig. 1).

## 3.2. Resistance mutations to letermovir modulate the interaction levels between terminase subunits

Letermovir resistance mutations in pUL56 and pUL51 were introduced into our experimental model and protein-protein interactions were assessed using the same pairs described above. For pUL56, we analyzed V236M, L241P, L257I, R369M and C325Y, as they are known to confer varying degrees of resistance to letermovir. Additionally, we investigated the pUL56 double mutant L257I-R369M to evaluate potential additive or synergistic effects. The A95V mutation in pUL51 was also generated and tested both individually and in combination with the pUL56 L257I mutation, as this combination decreased letermovir susceptibility in previous reports (Muller et al., 2022).

Interaction measurements revealed a significant decrease in the interaction between pUL56 and pUL89 when mutations L257I-R369M and C325Y were present (p = 0.03 and p = 0.03 respectively). In contrary, the mutation V236M significantly increased the interaction between the two subunits (p = 0.01). None of the other mutations tested had a significant impact on the pUL56-pUL89 interaction in our model (Fig. 2A).

Regarding the interaction between pUL56 and pUL51, the mutations

pUL56 V236M, L257I and the combination of pUL56 L257I with pUL51 A95V, led to a significant increase in interaction levels (p < 0.001; p = 0.001; p = 0.003 respectively). The remaining mutations did not significantly affect the interaction level of the studied pair. Notably, association of mutations L257I and A95V showed a synergistic effect (Fig. 2B).

## 3.3. Structural modelling to investigate protein-protein interaction variability

To support our study with structural insights, we generated a 3D interaction model of HCMV terminases using an artificial intelligence (AI)-based approach. Specifically, the FASTA amino acid sequences of the terminase subunits were submitted to the AlphaFold 3 platform (Abramson et al., 2024) which produced five predicted models with the presence of Mg2+ and Zn2+ ions and DNA as for the previously published HSV-1 complex (PDB ID: 6M5R) (Yang et al., 2020). Among the five models calculated during the calculation job, the rank of them obtained ipTM and pTM scores of 0.79 and 0.78, respectively closed to the 0.8 score of good quality models as described in AlphaServer documentation. The top-ranked prediction model was the first one, with a pLDDT score of 76.64, indicating a generally reliable overall structure but with side chains that were not well resolved. In our model we
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Interaction of terminases in presence of resistance mutations in pUL56 or pUL51. **A.** Interaction analysis between pUL56 and pUL89 wild-type (100 % interaction control) or with the following mutations: V236M, L241P, L257I, L257I-R369M, C325Y. **B.** Interaction analysis between pUL56 and pUL51 wild-type (100 % interaction) or with the following mutations: pUL56 V236M, L241P, L257I, L257I-R369M, C325Y and pUL51 A95V. Three independent replicates were done for each experiment and a Student's t-test was used for statistical analysis: \* p < 0.05, \*\* p < 0.01, \*\*\* p = 0.001.</figcaption></figure>

decided to not show pUL56 (425–485 and 778 to 850) and pUL51 (1–80) regions that were non-defined regions with low pLDDT scores.

Using this model, we mapped the studied mutations on both the complex and individual subunit structures to gain insight into the observed interaction variations. Interestingly, all mutations clustered within the same region of the complex and were located close to one another. They were all in alpha-helix in a tunnel-like part of the complex (Fig. 3A and B). Concerning pUL56 mutations, they were all found in a kink formed by alpha-helix (Fig. 3C). We also aligned pUL56 and pUL51 structures with their respective homologs in HSV-1, pUL28 and pUL33, obtained by cryo-EM (PDB ID: 6M5R). This permitted us to visualize structure differences in mutations sites in both proteins to better understand why letermovir is only effective against HCMV. Indeed, the

alignment of pUL56 with its homolog in HSV-1, pUL28, permitted to show some structural differences in the region of resistance mutations. pUL28 alpha-helix were not fully superposed to those of pUL56 (Fig. 3D). Finally, the representation of pUL51 and its superposition with its HSV-1 homolog pUL33 permitted to localize the mutation A95V as in our previous study (Muller et al., 2022). The mutation A95V is located in an alpha-helix of both structures (Fig. 3E and F).

To take our study a step further, we propose here a model with the HSV-1 terminase complex in its hexameric form (PDB ID: 6M5S assembly) (Yang et al., 2020) with one subunit replaced by the HCMV terminase complex (Fig. 4A). All known letermovir resistance mutations were localized in this hypothetical model. As shown in Fig. 4B and D, the pUL56 and pUL51 mutations would be oriented outside the hexameric
<figure><img src="image_7.png" /><figcaption>**Fig. 3.** AI-based modelling of HCMV terminase complex and location of resistance mutations. **A.** Quaternary structure of HCMV terminase complex predicted by AlphaFold3 and represented in NewRibbon with the different sub-units: pUL56 (blue), pUL89 (green) and pUL51 (magenta). Resistance mutations pUL51 A95V and pUL56 V236M, L241P, L257I, C325Y, R369M are represented in Van Der Waals and colored by mass (red). For pUL56, residues 425 to 485 and 778 to 850 and for pUL51, residues 1 to 80 were not shown because their prediction scores (pLDDT) were under 50. To be consistent, same colours are reused in all panels. **B.** Zoom in letermovir mutations site of terminase complex with pUL51 and pUL56 letermovir resistance mutations. **C.** pUL56 tertiary structure with resistance mutations V236M, L241P, L257I, C325Y and R369M. **D.** Structure alignment of pUL56 and its HSV-1 homolog, pUL28 (yellow), with letermovir resistance mutations localization and corresponding zoom. **E.** Tertiary structure of pUL51 protein and visualization of the mutation A95V. **F.** Structural alignments of pUL51 and its HSV-1 homolog, pUL33 (apple green), and mapping of pUL33 A95V.</figcaption></figure>

complex. As for the pUL89 resistance mutations, they are located inside the complex, close to the DNA. Finally, we mapped all currently described resistance mutations of the three subunits in the complex structure shown in Fig. 4D. All mutations were represented in Van Der Waals and colored in red. All mutations were found in the tunnel-like part of the complex (Fig. 4D).

**4. Discussion and conclusion**

Current antivirals against HCMV primarily target the viral DNA polymerase. However, their clinical use is limited by toxicity and the emergence of resistance mutations, creating an urgent need for novel antiviral agents. In this context, letermovir was developed and approved for the prophylaxis of HSCT recipients (Marty et al., 2017). Letermovir demonstrates strong efficacy against HCMV with high specificity and a favorable safety profile, showing fewer adverse effects compared to traditional antivirals (Chemaly et al., 2014; Lischka et al., 2010, 2016; Stoelben et al., 2014). Notably, letermovir is the first approved antiviral targeting the HCMV terminase complex. Others molecule such as BDCRB, TCRB and tomeglovir were described as targeting the terminase complex but failed to progress in clinical trials (Gourin et al., 2023).

Nevertheless, although letermovir is a promising molecule, its mode of action is currently not fully elucidated. It was shown that letermovir prevents the cleavage of viral DNA concatemers in unit-length genomes and interferes with virion packaging, but the molecular details remain unclear. Resistance mutations were already described in pUL51, pUL56 and pUL89 (Chou, 2017; Chou et al., 2018; Muller et al., 2022), with pUL56 being considered the primary target due to the wide range of resistance levels conferred by its mutations, ranging from 2-fold to over 3000-fold compared to the reference strain. In particular, the mutation of the residue C325 in tyrosine, tryptophane or phenylalanine is responsible for the highest letermovir resistance level currently described. In a recent *in silico* study, this residue was described as forming a binding-pocket for letermovir suggesting that a modification of the molecule could improve efficacy of treatment and bypass this resistance (Kmetsch et al., 2025).

Based on these findings, our study focused on letermovir mode of action and associated resistances. We hypothesized that letermovir inhibits interactions among terminase subunits and that resistance mutations might restore or alter these interactions. Using NanoBiT® PPI technology, we measured interaction between pUL56, pUL51 and pUL89 in absence or in presence of letermovir. Same experiments were
<figure><img src="image_8.png" /><figcaption>**Fig. 4.** Mapping of HCMV terminase complex into HSV-1 hexameric complex (PDB ID: 6M5S) and visualization of resistance mutations. A. HSV-1 terminase complex in its hexameric form (transparent black) with one subunit replaced by HCMV terminase complex. HCMV terminases, pUL56, pUL89 and pUL51, are respectively colored in blue, green and magenta. Double-stranded DNA is multi-colored and is in the middle of the complex. B and C. Zoom of HCMV terminase complex with letermovir resistance mutations (red) in the hexamer. D. HCMV terminase complex monomer and letermovir resistance mutations localization. Resistance mutations are represented in Van Der Waals.</figcaption></figure>

performed with resistance mutations in pUL56 and/or pUL51. Our results demonstrated that a 10-fold higher concentration of letermovir than IC50 of AD169 reference strain was not sufficient to inhibit the interaction between all the subunits of terminase complex. Mechanism of action of letermovir could so lie in the inhibition of the interaction of terminases with viral DNA or with portal protein. Thus, further experiments are necessary to confirm these hypotheses. Furthermore, resistance mutations impact the level of interaction between terminases especially V236M which increased both the level of interaction between UL56 and UL51, but also in the triplex UL56-51-89 studied. Moreover, the mutation L257I increased the interaction of pUL56 with pUL51 but decreased that between pUL56 and pUL89. Interestingly, the high-level resistance mutation C325Y did not have the higher effect on studied interactions as might have been expected. We also demonstrated that coupling mutations could have a synergistic effect on interaction level. To deepen our understanding, we generated an AI-based 3D model of the terminase complex to better understand variations of interaction. This model allowed to show that residues involved in resistance are localized

in the same part of the terminase complex and that they are close to each other. This correlate with our interaction's assays, mutations could refine complex conformation and vary the level of interaction to eject antiviral molecule. These observations are in line with the study of the binding pocket of pUL56 which suggests that the terminase complex may inhibit the action of the letermovir through a mechanism of molecule ejection (Kmetsch et al., 2025). Moreover, structural superimposition of HCMV terminases with their HSV-1 homologs revealed some differences, particularly at mutations sites, suggesting that the physicochemical properties of residues involved in letermovir resistance may be more critical than previously appreciated. This reinforces the need to redesign letermovir derivatives to overcome resistance and potentially expand activity to other herpesviruses. Future studies should also explore how these mutations sites influence terminase interactions with viral DNA.

Our study has limitations, notably the exclusion of other viral proteins as terminase complex partner's pUL52, pUL77, pUL93 and pUL104. While pUL52 was ruled out as a contributor to letermovir
resistance (Muller et al., 2021), it remains important for packaging by linking terminase proteins with viral DNA (Harmening et al., 2025). Moreover, as their respective homologs in HSV-1, pUL77, pUL93 and pUL104 were shown as interacting with the terminase complex or as essential for the packaging step (DeRussy and Tandon, 2015; Dittmer et al., 2005; Köppen-Rung et al., 2016; Scholtes and Baines, 2009; Taus et al., 1998). Thus, the presence of partner proteins could help in maintaining the conformation of the terminase complex or of its interaction with the DNA. Furthermore, HEK293-T cells are not permissive to HCMV infection, even though we have already demonstrated that HCMV immediate early proteins IE1/IE2 can be produced after transfection with wild-type HCMV (Gourin et al., 2025). Nevertheless, this eukaryotic production system is widely known for its high protein yield and high transfection capabilities. Further studies are also needed to definitely confirm our results, such as co-immunoprecipitation assays.

Taken together our results advance the understanding of letermovir mode of action and associated resistance mechanisms in pUL56 and pUL51. Moreover, our study is in line with previous published data. Nevertheless, further studies are needed to pinpoint where exactly letermovir acts during viral genome packaging and whether it forms covalent interactions, which will be crucial to fully decipher resistance pathways and guide next-generation antiviral development.

### CRediT authorship contribution statement

**C. Gourin:** Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **T. Flores:** Formal analysis. **C. Lefèvre:** Investigation. **S. Alain:** Writing – review & editing, Project administration, Funding acquisition, Conceptualization. **G. Ligat:** Writing – review & editing, Supervision, Project administration, Funding acquisition, Conceptualization. **S. Hantz:** Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization.

### Funding

CG has a financial support by the Ministère de l'Enseignement Supérieur et de la Recherche. SA and SH received financial support from INSERM, Santé Publique France (National Reference Center for Herpesviruses) and University of Limoges. GL received financial support from INSERM, CNRS, Toulouse University, Infinity, 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", the Ligue contre le cancer (FoCyG), the foundation "Flavien" and the federation "Grandir sans cancer". The PhD thesis of TF is funded by the Occitanie Region and INSERM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

### Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: SA is an expert for Takeda, MSD, Merck and Biotest with no personal link. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

### Acknowledgments

The authors would like to thank Dr Christophe Sirac (CRIBL laboratory, Limoges) for the loan of the Fluostar Omega reading plate. They would also like to thank Dr Anthony Couvreux for the Python script used for the 3D modelling.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.antiviral.2025.106289.

### Data availability

Data will be made available on request.

### References

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