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Tracking Adenovirus DNA in Entry and Replication Provides Insight into Virion Uncoating and Assembly


Gomez Gonzalez, Alfonso. Tracking Adenovirus DNA in Entry and Replication Provides Insight into Virion Uncoating and Assembly. 2024, University of Zurich, Faculty of Science.

Abstract

Adenoviruses (AdV) are double-stranded DNA (dsDNA) viruses with an icosahedral proteinaceous capsid lacking a lipid envelope. AdV initiate infection by traversing from the cell surface to the nucleus and release their genome into the nucleus at the nuclear pore complex (NPC). The proteins and RNA encoded in the viral DNA (vDNA) counteract the host defense machinery and activate viral gene expression, genome replication and progeny formation. During the infection cycle, the vDNA undergoes significant rearrangements. Although some of these changes have been described in the literature, several central questions remain unanswered due to methodological limitations, in particular vDNA uncoating from the incoming virion, and vDNA packaging into progeny particles. The first part of this thesis characterizes the role of protein V during virus entry and its influence on the intracellular delivery of the vDNA, while the second part focuses on the characterization of the overall spatiotemporal dynamics of the AdV DNA throughout infection. To track the vDNA we use low invasive bioorthogonal chemistry. Results show that incoming protein V is ubiquitinated by the cellular RING E3-ubiquitin-ligase Mib1 during virus entry. Viruses lacking protein V, or carrying a non-ubiquitinable protein V, where all lysine residues were changed to arginine, uncoated in the absence of Mib1, but had a higher chance to misdeliver the vDNA to the cytoplasm. Cytoplasmic vDNA did not lead to infection, but to the activation of the innate immune response in a cGAS DNA sensor-dependent manner. Later on, by adapting the inverse electron-demand Diels-Alder (IEDDA) reaction for AdV infection, we showed major DNA rearrangements occurring during vDNA replication in a time-resolved manner. While early-replicated vDNA dissociated from the viral replication compartment (VRC) upon time in a replication-dependent manner, the intermediate- and late-replicated vDNA pool accumulated at the VRC and assembled into infectious progeny. Using an extensive antibody panel, we assessed the functional differences in replication, transcription and packaging of the different vDNA pools. Ultimately, we were able to identify viral assembly intermediates bubbling off the VRC in live cells, and giving rise to infectious virus particles. Together, the data in this thesis shed light on multiple steps of the adenovirus infection cycle and offer new tools to study viral processes which have been understudied so far due to technical limitations. The results from this work will impact on viral disease processes and the use of viruses as gene vectors in therapeutic settings.

Abstract

Adenoviruses (AdV) are double-stranded DNA (dsDNA) viruses with an icosahedral proteinaceous capsid lacking a lipid envelope. AdV initiate infection by traversing from the cell surface to the nucleus and release their genome into the nucleus at the nuclear pore complex (NPC). The proteins and RNA encoded in the viral DNA (vDNA) counteract the host defense machinery and activate viral gene expression, genome replication and progeny formation. During the infection cycle, the vDNA undergoes significant rearrangements. Although some of these changes have been described in the literature, several central questions remain unanswered due to methodological limitations, in particular vDNA uncoating from the incoming virion, and vDNA packaging into progeny particles. The first part of this thesis characterizes the role of protein V during virus entry and its influence on the intracellular delivery of the vDNA, while the second part focuses on the characterization of the overall spatiotemporal dynamics of the AdV DNA throughout infection. To track the vDNA we use low invasive bioorthogonal chemistry. Results show that incoming protein V is ubiquitinated by the cellular RING E3-ubiquitin-ligase Mib1 during virus entry. Viruses lacking protein V, or carrying a non-ubiquitinable protein V, where all lysine residues were changed to arginine, uncoated in the absence of Mib1, but had a higher chance to misdeliver the vDNA to the cytoplasm. Cytoplasmic vDNA did not lead to infection, but to the activation of the innate immune response in a cGAS DNA sensor-dependent manner. Later on, by adapting the inverse electron-demand Diels-Alder (IEDDA) reaction for AdV infection, we showed major DNA rearrangements occurring during vDNA replication in a time-resolved manner. While early-replicated vDNA dissociated from the viral replication compartment (VRC) upon time in a replication-dependent manner, the intermediate- and late-replicated vDNA pool accumulated at the VRC and assembled into infectious progeny. Using an extensive antibody panel, we assessed the functional differences in replication, transcription and packaging of the different vDNA pools. Ultimately, we were able to identify viral assembly intermediates bubbling off the VRC in live cells, and giving rise to infectious virus particles. Together, the data in this thesis shed light on multiple steps of the adenovirus infection cycle and offer new tools to study viral processes which have been understudied so far due to technical limitations. The results from this work will impact on viral disease processes and the use of viruses as gene vectors in therapeutic settings.

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Additional indexing

Item Type:Dissertation (cumulative)
Referees:Greber Urs F, Münz Christian, Baubec Tuncay, Suomalainen Maarit
Communities & Collections:07 Faculty of Science > Institute of Molecular Life Sciences
UZH Dissertations
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Place of Publication:Zürich
Date:26 March 2024
Deposited On:27 Mar 2024 11:07
Last Modified:21 May 2024 20:48
Number of Pages:167
OA Status:Green
  • Content: Published Version
  • Language: English