Études structurales de nouvelles cibles thérapeutiques du CMVH révélées par les dérivés benzimidazolés ribonucléosides.

The Human Cytomegalovirus is a virus affecting all populations regardless of their socio-professional environment. Harmless in healthy subjects, the damage caused by the virus in the host proved disastrous when it becomes immunocompromised. Thus, people with HIV, people who have undergone organ transplants or infants are particularly susceptible to HCMV. The first inhibitors formulated for the viral polymerase have been ineffective due to the observation of multiple resistance mutations. A new class of molecule, benzimidazoles ribonucleoside derivatives, for stages of maturation and encapsidation, has emerged for the terminase complex proteins, pUL89 and pUL56, and the phosphotransferase pUL97 and its accessory protein, pUL27. Through the use of nuclear magnetic resonance, UV / Visible spectroscopy and circular dichroism, and homology modelling, we collected structural information on target of derivatives ribobenzimidazoles. We have proposed hypotheses about the mode of action of these molecules. Moreover, the discovery of a domain capable of interacting with the viral double-stranded DNA of HCMV has been described.Le Cytomégalovirus Humain est un virus touchant toutes les populations quelque soit leur milieu socioprofessionnel. Inoffensif chez des sujets sains, les dégâts causés par le virus chez l’hôte se révèlent désastreux dès que celui-ci devient immunodéprimé. Ainsi, les porteurs de VIH, les personnes ayant subi une greffe d’organe ou les nourrissons sont particulièrement exposés à CMVH. Les premiers inhibiteurs formulés visant la polymérase virale se sont avérés inefficaces suite à l’observation de multiples mutations de résistance. Une nouvelle classe de molécule, les dérivés benzimidazolés ribonucléosides, visant les étapes de maturation et d’encapsidation, a donc émergé visant les protéines du complexe terminase, pUL89 et pUL56, et la phosphotransférase pUL97 ainsi que sa protéine accessoire, pUL27.Grâce à l’utilisation de la résonance magnétique nucléaire, des spectroscopies UV/Visible et de dichroïsme circulaire, et de la modélisation par homologie, nous avons collecté des informations structurales relatives aux cibles des dérivés ribobenzimidazolés. Nous avons pu proposer des hypothèses sur le mode d’action de ces molécules. Par ailleurs, la découverte d’un domaine capable d’interagir avec l’ADN viral double brin de CMVH a été décrite

Etudes structurales de nouvelles cibles thérapeutiques du CMVH révélées par les dérivés benzimidazolés ribonucléosides

Le Cytomégalovirus Humain est un virus touchant toutes les populations quelque soit leur milieu socioprofessionnel. Inoffensif chez des sujets sains, les dégâts causés par le virus chez l hôte se révèlent désastreux dès que celui-ci devient immunodéprimé. Ainsi, les porteurs de VIH, les personnes ayant subi une greffe d organe ou les nourrissons sont particulièrement exposés à CMVH. Les premiers inhibiteurs formulés visant la polymérase virale se sont avérés inefficaces suite à l observation de multiples mutations de résistance. Une nouvelle classe de molécule, les dérivés benzimidazolés ribonucléosides, visant les étapes de maturation et d encapsidation, a donc émergé visant les protéines du complexe terminase, pUL89 et pUL56, et la phosphotransférase pUL97 ainsi que sa protéine accessoire, pUL27. Grâce à l utilisation de la résonance magnétique nucléaire, des spectroscopies UV/Visible et de dichroïsme circulaire, et de la modélisation par homologie, nous avons collecté des informations structurales relatives aux cibles des dérivés ribobenzimidazolés. Nous avons pu proposer des hypothèses sur le mode d action de ces molécules. Par ailleurs, la découverte d un domaine capable d interagir avec l ADN viral double brin de CMVH a été décrite.The Human Cytomegalovirus is a virus affecting all populations regardless of their socio-professional environment. Harmless in healthy subjects, the damage caused by the virus in the host proved disastrous when it becomes immunocompromised. Thus, people with HIV, people who have undergone organ transplants or infants are particularly susceptible to HCMV. The first inhibitors formulated for the viral polymerase have been ineffective due to the observation of multiple resistance mutations. A new class of molecule, benzimidazoles ribonucleoside derivatives, for stages of maturation and encapsidation, has emerged for the terminase complex proteins, pUL89 and pUL56, and the phosphotransferase pUL97 and its accessory protein,pUL27. Through the use of nuclear magnetic resonance, UV / Visible spectroscopy and circular dichroism, and homology modelling, we collected structural information on target of derivatives ribobenzimidazoles. We have proposed hypotheses about the mode of action of these molecules. Moreover, the discovery of a domain capable of interacting with the viral double-stranded DNA of HCMV has been described.PARIS-BIUP (751062107) / SudocSudocFranceF

Highlighting of a LAGLIDADG and a Zing Finger Motifs Located in the pUL56 Sequence Crucial for HCMV Replication

The human cytomegalovirus (HCMV) terminase complex is part of DNA-packaging machinery that delivers a unit-length genome into a procapsid. Sequence comparison of herpesvirus homologs allowed us to identify a potential LATLNDIERFL and zinc finger pattern in N-terminal part of pUL56. Recombinant viruses were generated with specific serine or alanine substitutions in these putative patterns. We identified a LATLNDIERFL pattern characteristic of LAGLIDADG homing endonucleases and a metal-binding pattern involving the cysteine and histidine residues C191-X2-C194-X22-C217-X-H219 (CCCH) close to the region conferring letermovir resistance. These patterns are crucial for viral replication, suggesting that they are essential for pUL56 structure and function. Thus, these patterns represent potential targets for the development of new antivirals such as small molecules or peptides and may allow to better understand the letermovir mechanism of action

Identification of a short sequence in the HCMV terminase pUL56 essential for interaction with pUL89 subunit

International audienceThe human cytomegalovirus (HCMV) terminase complex consists of several components acting together to cleave viral DNA into unit length genomes and translocate them into capsids, a critical process in the production of infectious virions subsequent to DNA replication. Previous studies suggest that the carboxyl-terminal portion of the pUL56 subunit interacts with the pUL89 subunit. However, the specific interacting residues of pUL56 remain unknown. We identified a conserved sequence in the C-terminal moiety of pUL56 (671 WMVVKYMGFF 680). Overrepresentation of conserved aromatic amino acids through 20 herpesviruses homologues of pUL56 suggests an involvement of this short peptide into the interaction between the larger pUL56 terminase subunit and the smaller pUL89 subunit. Use of Alpha technology highlighted an interaction between pUL56 and pUL89 driven through the peptide 671 WMVVKYMGFF 680. A deletion of these residues blocks viral replication. We hypothesize that it is the consequence of the disruption of the pUL56-pUL89 interaction. These results show that this motif is essential for HCMV replication and could be a target for development of new small antiviral drugs or peptidomimetics. Human cytomegalovirus (HCMV), a beta herpesvirus, can cause serious diseases in immunocompromised patients. Current antiviral inhibitors (ganciclovir, cidofovir and foscarnet) all target the viral DNA polymerase. They have adverse effects and prolonged treatment can select for drug resistance mutations either in the viral pol-ymerase pUL54, the kinase pUL97 or both of them 1, 2. Thus, we need new drugs targeting others stages of repli-cation. The terminase complex is highly specific for HCMV, has no counterpart in the human organism, and thus represents a target of choice for new antivirals development. This has been confirmed by the recent development of letermovir in the transplant setting 3, 4. DNA packaging process requires several proteins such as pUL56 and pUL89, the large and small termi-nase subunits, respectively. Recently, four additional proteins were shown to be also implicated in this process, namely, pUL51, pUL52, pUL77, pUL93 5-10. This process is driven by specific interactions of protein-DNA and protein-protein to cleave and package unit length genomic DNA into an empty capsid. Evidence suggests that the large subunit pUL56 has a crucial role in DNA cleavage/packaging, containing many of the functional sites required for this process like interaction with the portal protein pUL104, endonu-clease activity, and more interestingly an ATP-binding site (amino acids 709 to 723) 11. Although the association between pUL56 and pUL89 has already been reported, the residues of pUL56 involved in the terminase complex integrity are still unknown 12, 13. Nevertheless, co-immunoprecipitation experiments showing an interaction between the C-terminal half of pUL56 (pUL56-Cter) and pUL89 were confirmed by other results 12, 13. Because knowledge of terminase functional and interaction domains is important both for the development of drugs targeting the DNA packaging stage and for the improvement of existing ones such as letermovir, the aim of the current study is to identify a minimum peptide of pUL56 with a putative key role in its interaction with pUL89. Sequence alignments encouraged us to focus on the putative involvement of one part of the pUL5

New functional domains of human cytomegalovirus pUL89 predicted by sequence analysis and three-dimensional modelling of the catalytic site DEXDc.

International audienceBenzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89, the large and small subunits of the HCMV terminase respectively, their mechanism of action is not yet fully understood

New functional domains of human cytomegalovirus pUL89 predicted by sequence analysis and three-dimensional modelling of the catalytic site DEXDc.

International audienceBenzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89, the large and small subunits of the HCMV terminase respectively, their mechanism of action is not yet fully understood

Putative functional domains of human cytomegalovirus pUL56 involved in dimerization and benzimidazole D-ribonucleoside activity

International audienceBACKGROUND: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89 (the large and small subunits of the HCMV terminase, respectively) their mechanism of action is not yet fully understood. We aimed here to better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives. METHODS: The HCMV pUL56 protein was studied by sequence analysis of the HCMV UL56 gene and herpesvirus counterparts combined with primary structure analysis of the corresponding amino acid sequences. RESULTS: The UL56 sequence analysis of 45 HCMV strains and counterparts among herpesviruses allowed the identification of 12 conserved regions. Moreover, comparison with the product of gene 49 (gp49) of bacteriophage T4 suggested that the pUL56 zinc finger is localized close to the dimerization site of pUL56, providing a spatial organization of the catalytic site that allows recognition and cleavage of DNA. CONCLUSIONS: This study provides a basis to investigate the mechanism of concatemeric DNA cleavage and a biochemical basis for DNA packaging inhibition by benzimidazole derivatives

New functional domains of human cytomegalovirus pUL89 predicted by sequence analysis and three-dimensional modelling of the catalytic site DEXDc

International audienceINTRODUCTION: Benzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89, the large and small subunits of the HCMV terminase respectively, their mechanism of action is not yet fully understood. METHODS AND RESULTS: To better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives, we studied the HCMV pUL89 protein by a genetic approach combined with primary structure analysis. The pUL89 sequence analysis of 25 HCMV strains and counterparts among herpesviruses allowed identification of 12 conserved regions. We also built a three-dimensional model of the pUL89 ATPasic catalytic site, including ATPase motor motifs 1, II and III, that may facilitate the development of future antiviral drugs active against HCMV. Finally, we identified several putative functional domains in pUL89, such as pUL89 zinc finger (pUL89-ZF), DNA cutting sites and portal binding sites, that are probably involved in CMV DNA cleavage and packaging

Conserved domains and structure prediction of human cytomegalovirus UL27 protein.

International audienceThe human cytomegalovirus (HCMV) nuclear UL27 protein (pUL27) could be involved at the stage of nuclear egress. Maribavir is a new anti-HCMV drug that targets nuclear egress through direct inhibition of the HCMV serine-threonine kinase, UL97 protein (pUL97). Because maribavir-resistance-related mutations are observed in both proteins, pUL27 is thought to interfere with pUL97 activity; however, its mechanism of action remains unclear

Putative functional domains of human cytomegalovirus pUL56 involved in dimerization and benzimidazole D-ribonucleoside activity.

International audienceBenzimidazole D-ribonucleosides inhibit DNA packaging during human cytomegalovirus (HCMV) replication. Although they have been shown to target pUL56 and pUL89 (the large and small subunits of the HCMV terminase, respectively) their mechanism of action is not yet fully understood. We aimed here to better understand HCMV DNA maturation and the mechanism of action of benzimidazole derivatives