The classical description of the innate antiviral response involves the production of type I interferon (IFN) and the subsequent expression of hundreds of interferon stimulated genes (ISGs), which cooperatively repress viral replication and spread. More recently, an IFN-independent antiviral response has also been described, in which the entry of an enveloped virus induces a subset of ISGs without requiring the production of IFN, although the details of this response remain unclear. In this work, multiple approaches were used to further characterize antiviral signaling pathways. Initially, the potential involvement in the IFN-independent response of the small GTPase Rac1, which has been implicated in both viral entry and antiviral signaling, was investigated. Here, Rac1 was shown to have a possible function in the negative regulation of ISG expression, although technical complications prevented definitive conclusions. As an alternative strategy to identify novel aspects of antiviral signaling, the mechanism of action of ICP0, a herpes simplex virus (HSV) protein involved in innate immune evasion, was investigated. Although ICP0 is generally thought to perform its actions in the nucleus, by tagging proteins for proteasome-mediated degradation via the E3 ubiquitin ligase activity of its RING finger domain, here it was shown that not only does cytoplasmic ICP0 have a RING-dependent but proteasome-independent ability to block antiviral signaling, but also that ICP0 has a previously unknown RING-independent function in the promotion of viral replication in the cytoplasm. To further investigate the cytoplasmic activities of ICP0, proteins interacting with ICP0 in the cytoplasm were identified using quantitative mass spectrometry. This revealed several intriguing binding partners for ICP0, including WDR11, a poorly-characterized cellular protein which was shown to undergo a dramatic relocation during HSV infection, although it was not required for viral replication in cultured cells. Therefore, this study has uncovered several new and unexpected insights into ICP0 behavior. / Thesis / Doctor of Philosophy (PhD)

Human norovirus (HuNoV) is the leading cause of gastroenteritis worldwide. Despite the significant disease and economic burden, currently there are no licensed vaccines or antivirals. The understanding of norovirus biology has been hampered by the inability to cultivate HuNoV in cell culture. To establish a tissue culture system, infectious HuNoVs were purified from clinical stool samples. HuNoV replication was tested in different cell types. The B-cell and intestinal organoids culture systems were validated. In addition, using organoids culture a DNA-based reverse genetic system was shown to recover infectious HuNoV. Due to the challenges associated with cultivating HuNoV, murine norovirus (MNV) was used as a surrogate system to understand the role of eIF4E phosphorylation in norovirus pathogenesis, and VP1-RdRp interaction in regulating viral genome replication. MNV infection results in the phosphorylation of the translation initiation factor eIF4E, re-programming host-cell translation during infection. Inhibiting eIF4E phosphorylation reduces MNV replication in cell culture suggesting a role in viral replication. A mouse model with eIF4E S209A, a phosphor-ablative mutation, was established to understand the role of eIF4E phosphorylation in MNV pathogenesis. In vitro and in vivo characterisations demonstrated that eIF4E phosphorylation may have multiple roles in norovirus-host interactions, but overall has little impact on MNV pathogenesis. The shell domain (SD) of norovirus major capsid protein VP1 interacts with viral RNA-dependent RNA polymerase (RdRp) in a genogroup-specific manner to enhance de novo initiation of RdRp, and to promote negative-strand RNA synthesis. To understand how VP1 regulates norovirus genome replication, chimeric MNVs with genogroup-specific residues mutagenised were characterised in vitro and in vivo. A single amino acid mutation was shown to destabilise viral capsid. SDs with reduced VP1-RdRp interaction showed less capacity to stimulate RdRp, resulting in delayed virus replication. In vivo, the replication of an MNV-3 with homologous mutations was abolished, highlighting the crucial role of this interaction.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 205-212.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 17, 2009). Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed June 23, 2008). Includes bibliographical references.