Immune cytokines tumor necrosis factor alpha and type I interferons provide front-line defense against viral infection and are regulated in part by ubiquitin (Ub) and Ub-like molecules. Ubiquitin and Ub-like molecule ISG15 share a conserved C-terminal motif where a terminal glycine residue becomes attached to cellular target proteins. Nairoviruses and arteriviruses contain an ovarian tumor domain-containing protease (OTU protease) that was found to corrupt pathways by removing Ub or ISG15 from target proteins. This broad substrate specificity is unlike mammalian deubiquitinating enzymes, which cannot recognize both substrates. To understand how viral OTU domain-containing proteases remove Ub and ISG15, the crystal structure of the Crimean-Congo Heamorhaggic Fever nairovirus (CCHFV) was determined with Ub to 2.5 Å resolution. A computational model was built of the CCHFV Otu protease bound to ISG15 as well. The CCHFV Otu protease has several structural differences from known OTU proteases, manifesting in its broad substrate recognition capability.

The great variation displayed by nucleic acid molecules in human cells, and the continuous discovery of their impact on life, consequently require continuous refinements of molecular analysis techniques. Padlock probes and rolling circle amplification offer single nucleotide discrimination in situ, a high signal-to-noise ratio and localized detection within cells and tissues. In this thesis, in situ detection of nucleic acids with padlock probes and rolling circle amplification was applied for detection of DNA in the single cell gel electrophoresis assay to detect nuclear and mitochondrial DNA. This assay is used to measure DNA damage and repair.  The behaviour of mitochondrial DNA in the single cell gel electrophoresis assay has earlier been controversial, but it was shown herein that mitochondrial DNA diffuses away early in the assay. In contrast, Alu repeats remain associated with the nuclear matrix throughout the procedure. A new twelve gel approach was also developed with increased throughput of the single cell gel electrophoresis assay. DNA repair of three genes OGG1, XPD and HPRT and of Alu repeats after H2O2 induced damage was further monitored. All three genes and Alu repeats were repaired faster than total DNA. Finally, padlock probes and rolling circle amplification were applied for detection of the single stranded RNA virus Crimean Congo hemorrhagic fever virus. The virus was detected by first reverse transcribing RNA into cDNA.. The virus RNA together with its complementary RNA and the nucleocapsid protein were detected in cultured cells. The work presented here enables studies of gene specific damage and repair as well as viral infections in situ. Detection by ligation offers high specificity and makes it possible to discriminate even between closely related molecules. Therefore, these techniques will be useful for a wide range of applications within research and diagnostics.

Le virus de la fièvre hémorragique de Crimée-Congo (VFHCC) est un Nairovirus appartenant à la famille des Bunyaviridae, responsable d’une maladie hémorragique sévère chez l’Homme, associée à des symptômes non spécifiques et à une forte mortalité. La transmission se fait par morsure de tique ou par contact direct avec des fluides corporels contaminés. N’ayant ni vaccin ni traitement spécifique, un apport de connaissances sur les interactions cellulaires VFHCC-hôte ainsi que sur les mécanismes développés en réponse à l’infection est nécessaire.Nous avons tout d’abord étudié le potentiel antiviral de molécules sur la réplication du VFHCC. La chloroquine et la chlorpromazine ont été identifiées et inhibent efficacement la réplication virale avec une protection induite chez la souris contre l’infection, en particulier en combinaison avec la ribavirine.De nombreux virus sont connus pour être ciblés par, ou pour détourner la voie de l’autophagie. Nous avons regardé si l’infection par le VFHCC était associée à une modulation de l’autophagie et si la réplication virale était impactée par l’activité autophagique. L’étude de cellules hépatocytaires et épithéliales a montré une mobilisation massive du LC3, principal marqueur des vésicules autophagiques, par le VHFCC. Celle-ci reflète une induction du flux autophagique d’un nouveau type, n’impliquant pas les voies classiques de recrutement du LC3. La réplication virale n’est pas directement modulée par cette autophagie atypique mais des effets indirects sont à étudier. La plupart de ces observations ont été montrées pour le Nairovirus Dugbe avec cependant une cinétique différente.Le dernier axe étudié porte sur l’analyse de l’impact des IFITMs, facteurs de restriction virale connu pour interférer avec les processus de fusion membranaire, sur la réplication du virus Dugbe. L’étude a révélé une inhibition de la réplication virale par certains IFITMs.Des études supplémentaires portant sur l’interaction virus-cellule hôte et les mécanismes moléculaires associés sont nécessaires pour mieux comprendre la physiopathologie induite par le VFHCC et mettre au point de nouvelles stratégies thérapeutiques. / Crimean-Congo hemorrhagic fever virus (CCHFV) belongs to Nairovirus genus and to Bunyaviridae family. It is responsible for a severe hemorrhagic disease in humans, associated with non-specific symptoms and high lethality. Transmission is made by tick’s bite or by direct contact with contaminated body fluids. Since no vaccines or treatments are available, there is a need to accumulate knowledge on all aspects of CCHFV-host cell interaction as well as on response mechanisms that are taking place during infection.We first investigated pharmacological ways to interfere with CCHFV replication. Chloroquine and chlorpromazine (known modulators of some viral infections) were efficiently inhibiting viral replication and induce a protection in mice against CCHFV infection, particularly in the presence of ribavirin. Since several viruses are targeted by, or take advantage of, the autophagy response of infected cells, we explored whether CCHFV infection was associated with modulation of autophagy and whether virus replication was impacted by the autophagic activity of infected cells. By using hepatocytes and epithelial cells, we found that CCHFV induced a massive mobilization of the major marker of autophagic vesicles LC3. This mobilization reflected an induced autophagy flux and was of a novel type since known pathways of LC3 recruitment were not involved. The replication of CCHFV was indeed not directly modulated by this atypical form of autophagy but indirect effects remain to be studied. Most of these observations were found to be valid for the related, Dugbe virus (DUGV) with however, a distinct kinetic.Finally, we analyzed whether DUGV was sensitive to the IFITMs, restriction factors that can interfere with membrane fusion processes. Studies revealed that DUGV replication could be inhibited by some IFITMs. Additional studies on virus host-cell interactions and their associated molecular mechanisms should help to better understand the physiopathology induced by CCHFV and to devise therapeutic strategies.