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To enter and infect cells, viruses must overcome the barrier presented by the cell membrane. Enveloped viruses, which possess their own lipid bilayer, fuse their viral membrane with the cell membrane. Non-enveloped viruses, whose outer surface is composed of proteins, penetrate through the hydrophobic interior of the cell membrane. Viruses accomplish the processes by coupling conformational changes in viral "entry proteins" to membrane disruption. This dissertation investigates the membrane disruption mechanisms of rotavirus, a non-enveloped virus, and vesicular stomatitis virus (VSV), an enveloped virus. Rotavirus uses proteins of its outer capsid to penetrate the membrane and deliver a transcriptionally-active core particle into the cell cytoplasm. \(VP5^*\), an outer capsid protein, undergoes a foldback rearrangement that translocates three clustered hydrophobic loops by \(\sim 180^{\circ}\). This rearrangement resembles the foldback rearrangements of enveloped virus fusion proteins. In the first half of my dissertation, I show that the hydrophobicity of the \(VP5^*\) apex is required for membrane disruption during rotavirus cell entry by mutating hydrophobic residues within the loop to hydrophilic residues. One particular mutation diminishes liposome interaction by the protein, blocks membrane penetration by virus particles in cells, and reduces particle infectivity by 10,000-fold. VSV uses its fusion protein, G, to fuse at low pH. Unlike other viral fusion proteins, pH-induced conformational changes in G are reversible. In the second half of my dissertation, I measure the fusion kinetics of individual VSV particles using a single-particle fusion assay previously developed for influenza virus. I find that hemifusion by VSV consists of at least two steps, an initial step that is pH-dependent and reversible, and a second step that is pH-independent. At low pHs, the second step becomes the sole rate-limiting step. I also show that at pH 6.6, the VSV particle enters a stable intermediate state that binds tightly to membranes but does not precede to fusion. This dissertation uses a variety of experimental approaches to arrive at a more detailed understanding of how viruses use their entry proteins to either penetrate or fuse with the cell membrane.

Les adénovirus sont des virus non enveloppés. Afin de pouvoir se répliquer ils doivent entrer dans leur cellule hôte et être transportés jusqu’au noyau pour pouvoir initier l’expression du génome viral. Pour ce faire le virus utilise les composants de sa capside. Parmi ses composants, la protéine VI, une protéine interne de capside, induit la rupture de l’endosome grâce à son hélice amphipatique en N-terminal de la protéine. Récemment, une autre fonction de cette protéine a été décrite durant l’entrée du virus, impliquant cette fois-ci le motif conservé PPxY de la protéine VI. En effet la mutation de ce motif conservé : mutant M1 (PPxYPGAA), diminue de 20 fois l’infection du virus par rapport au virus sauvage. Cette baisse d’infectiosité est liée à un défaut de transport et d’accumulation du virus au niveau du centre organisateur des microtubules (MTOC). Il se trouve que la mutation du motif PPxY conduit à une perte d’interaction de la protéine VI avec les ubiquitines ligase de la famille Nedd4, mais également à un défaut d’ubiquitylation de la protéine VI. Nous avons ainsi entrepris d’étudier le rôle de cette modification post-traductionnelle lors de l’entrée du virus dans la cellule, mais aussi, de manière plus générale, le rôle de la protéine VI. Ainsi nous avons mis en évidence le rôle de la protéine VI et de son motif PPxY dans l’activation du génome viral. Par ailleurs, nous avons identifié une lysine ubiquitylée de la protéine VI et produit un mutant : mutant M6, pour étudier le rôle de cette ubiquitylation. Nous avons enfin entrepris de caractériser l’entrée du virus en produisant et en utilisant des adénovirus mutants, dont le nouveau mutant M6 / Adenoviruses are non-enveloped viruses. In order to replicate they have to enter their host cell and be transported toward the nucleus to initiate the viral gene expression. This requires the involvement of viral capsids components. Among these components, protein VI, an inner capsid protein, can induce endosomal rupture, thanks to its amphipathic helix located at the N-terminus part of the protein. Recently, the involvement of a conserved PPxY motif in the Protein VI has also been described in viral entry. Indeed, mutation of this motif (PPxY  PGAA) reduced infectivity of the mutant virus (M1 mutant) 20 folds compared to the wild type virus. This reduction of infectivity is related to a defect of transport and accumulation of viruses at the microtubule organizing center (MTOC) during virus entry. The mutation of PPxY motif leads to a loss of interaction between the protein VI and ubiquitin ligases from the Nedd4 family, and to a lack of protein VI ubiquitylation. The aim of this study was therefore to investigate the role of this posttranslational modification during virus entry, but also more generally the role of protein VI. In this work, we highlight the role of protein VI and its PPxY motif in the activation of the viral genome. Moreover, to investigate the ubiquitylation during virus entry we identify a lysine mutant of protein VI that lack ubiquitylation without altering the potential for interaction with ubiquitin ligases: the mutant M6. We then proceed to characterize the entry of the virus by producing and using mutant viruses, including this new mutant.

5-aminolevulinic acid (5-ALA) and derivatives thereof are widely usedprodrugs in treatment of pre-malignant skin diseases of the cancer treatmentmethod photodynamic therapy (PDT). The target molecule in 5-ALAPDTis protoporphyrin IX (PpIX), which is synthesized endogenously from5-ALA via the heme pathway in the cell. This thesis is focused on 5-ALA,which is studied in different perspectives and with a variety of computationalmethods. The structural and energetic properties of 5-ALA, itsmethyl-, ethyl- and hexyl esters, four different 5-ALA enols, and hydrated5-ALA have been investigated using Quantum Mechanical (QM) first principlesdensity functional theory (DFT) calculations. 5-ALA is found to bemore stable than its isomers and the hydrolysations of the esters are morespontaneous for longer 5-ALA ester chains than shorter. The keto-enoltautomerization mechanism of 5-ALA has been studied, and a self-catalysismechanism has been proposed to be the most probable. Molecular Dynamics(MD) simulations of a lipid bilayer have been performed to study themembrane permeability of 5-ALA and its esters. The methyl ester of 5-ALAwas found to have the highest permeability constant (PMe-5-ALA = 52.8 cm/s).The mechanism of the two heme pathway enzymes; Porphobilinogen synthase(PBGS) and Uroporphyrinogen III decarboxylase (UROD), have beenstudied by DFT calculations and QM/MM methodology. The rate-limitingstep is found to have a barrier of 19.4 kcal/mol for PBGS and 13.7kcal/mol for the first decarboxylation step in UROD. Generally, the resultsare in good agreement with experimental results available to date.