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New discovery on the mode of action of letermovir and the resistance mechanism of human cytomegalovirus
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International audience. Background Human cytomegalovirus (HCMV) is an opportunistic pathogen responsible for potentially fatal infections in immunocompromised patients. It is also the main cause of congenital infections worldwide. The HCMV terminase complex (pUL56-pUL89-pUL51) has been described as a target of choice for the development of antiviral strategies (Ligat et al.,2017). Letermovir, an antiviral targeting the terminase complex, approved in 2017 (Marty et al., 2017) has been shown to block viral DNA encapsidation (Goldner et al.2011). Nevertheless, resistance mutations have already been found in the terminase subunits (Chou and Kleiboeker, 2022; Muller et al.,2022). However, the mode of action of letermovir has not yet been fully elucidated. Methods To better understand the mode of action of letermovir, we used NanoBiT PPI system (Promega) to study the interaction between pUL56 and pUL89 in the presence or absence of letermovir. Wild type pUL56 and pUL89 were cloned into vectors containing the long or small subunit of luciferase respectively. Mutated pUL56 clones harbouring resistance mutations (V236M, L241P, C325Y and R369M) were also built. Both plasmids were then co-transfected into HEK293T cells. Letermovir was added during transfection. After 72h of production, the luciferase substrate was added and luminescence emission was measured. Results Our results showed that a concentration of 50nM of letermovir during transfection was not sufficient to inhibit the interaction between wild type pUL56 and pUL89. In addition, some preliminary results suggest that the high-level letermovir resistance mutation pUL56 C325Y allows the two proteins to continue interacting. Additional data will be added, about the other resistance mutations at different letermovir concentrations. Conclusions Our initial results are encouraging in terms of resolving the mode of action of letermovir. Indeed, this antiviral seems to not act by direct inhibition of the interaction between the HCMV terminase subunits pUL56 and pUL89. In addition, the study of resistance mutations will provide a better understanding of the resistance mechanism of HCMV and perhaps enable clinicians to adapt the treatment more effectively. Moreover, these experiments could help to the development of new antiviral strategies targeting the terminase complex.
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