Étude des mécanismes biochimiques et moléculaires de la résistance du cytomégalovirus humain et du virus herpès simplex 1 au foscarnet
|Abstract:||The structure of the human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) DNA polymerase (pol), belonging to the Herpesviridae family, is associated to a right hand with palm, thumb and fingers domains. The viral DNA pol adopts different conformations (open and closed) that implies a move of the fingers domain to facilitate the interaction between the nucleotide and the elongating DNA. It has been shown that the antiviral foscarnet (FOS) which targets the HCMV and HSV-1 DNA pol binds to the enzyme in its closed conformation and mutations confering resistance to this antiviral and localised in the fingers domain would favor a more open conformation of the enzyme for which FOS has a lower affinity. The aim of this thesis was to analyse whether this hypothesis could be extended to mutations localised in the NH2-terminal and the palm domains which interact with the fingers domain during the conformational changes of the enzyme during the polymerization process. Our hypothesis is that mutations localized in the helix K (NH2-terminal domain) and region II (palm domain) that participate in the conformational changes of the enzyme could favor a more open conformation of the viral DNA pol, and thus, decrease the susceptibility of viruses to FOS. We selected theoretical substitutions using a strategy based on amino acid sequences alignement of the DNA pol of HCMV and HSV-1 (susceptible to FOS) with those of RB69 and T4 bacteriophages (naturally resistants to this antiviral). We tried to generate recombinant HCMV and HSV-1 containing the different theoretical substitutions that we selected. However, the introduction of some theoretical substitutions [Q578P, R581T, L587F (helix K), P712Y, F718L (region II) for HCMV and Q617P, R620T, L626F (helix K), F718L (region II) for HSV-1] was so detrimental for the DNA pol that recombinant viruses were not able to grow in cell culture. Among the substitutions selected in the helix K, the substitution I619K confers resistance of HSV-1 to FOS. In the helix K, we also characterized the theoretical Q579I substitution that confers hypersusceptibility of HCMV to FOS. We compared this substitution with the K805Q substitution located in the helix P (fingers domain), already known to induce hypersusceptibility of HCMV to FOS. In region II, substitutions V715S and A719T confer resistance of both viruses to FOS whereas the Q697P substitution was associated with resistance of HCMV to FOS but not for HSV-1. The susceptibility profiles of recombinant viruses to FOS were confirmed by enzymatic assays that allowed us to determine the inhibition of the recombinant mutated DNA pol activity by this antiviral compound. We observed a decrease of the replicative capacities of recombinant HCMV and HSV-1 harboring these mutations compared to their wild-type counterparts. Tri-dimensional modeling was also performed to better understand the impact of these substitutions on the DNA pol of HCMV and HSV-1. On the one hand, substitutions confering resistance to FOS were associated to a destabilization of the closed conformation of the DNA pol and would favor a more open conformation for which the antiviral has a lower affinity. On the other hand, substitutions associated to a hypersusceptibility profile would favor a more closed conformation of the DNA pol for which FOS has a higher affinity. The characterization of the theoretical substitution V715S of HCMV and HSV-1 (FOSR/GCVR and FOSR/ACVR, respectively) was compared to the substitutions V715G of HSV-1 (FOSR/ACVR), V715M of HCMV and HSV-1 (FOSR/GCVS and FOSS/ACVR, respectively), already described in the literature and that were, thus, associated with different antiviral susceptibility phenotypes compared to those of V715S. Briefly, we showed that the introduction of these different substitutions could induce varying changes of the hydrophobic environment of the valine at position 715 influencing the antiviral susceptibility profile. Altogether, these results support our hypothesis that substitutions in helix K and region II could influence the susceptibility of HCMV and HSV-1 to FOS by modifiying the protein structure and impacting the correct conformational changes of the enzyme.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||13 September 2021|
|Collection:||Thèses et mémoires|
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