|Titel:||The late stages of human adenovirus infection - Characterising human adenovirus nuclear reorganisation and egress||Sprache:||Englisch||Autor*in:||Pfitzner, Søren||Erscheinungsdatum:||2021||Tag der mündlichen Prüfung:||2021-06-11||Zusammenfassung:||
Human adenovirus type 5 (HAdV5) infection causes disease of the upper and lower respiratory tract. Most research attention has been focussed on the early steps of the HAdV5 life cycle, including HAdV5 entry up until genome replication in the host cell nucleus. Late events during HAdV5 infection remain poorly characterised. The work of this dissertation aimed at further characterising two viral late events, nuclear reorganisation and nuclear egress. To this end, two viral proteins, pV and pIX, were fluorescently labelled within the HAdV5 genome. The core protein V binds to viral DNA and bridges between viral genome and capsid proteins. The capsid protein IX gives stability to the capsid. During virus progeny production both of these proteins were shown to localise to a membrane-less intranuclear compartment, which was termed ’late virion accumulation compartment’ (LVAC). The LVAC was characterised by multiple light and electron microscopic techniques and showed high impermeability to antibodies. Correlative light and electron microscopy revealed the accumulation of paracrystalline arrays of newly formed capsids, which were tightly packed within a honeycomb-like structure consisting of pV and viral DNA. Live-cell fluorescence microscopy and FRAP measurements showed that the LVAC restricts the movement of viral paracrystalline arrays, an indication that virions are trapped within. The formation of LVACs was shown to be linked to the congregation of viral replication centres.
In the second part of this work, the nuclear egress of progeny virions was analysed with particular focus on the effect of the small adenovirus death protein (ADP). While ADP is generally implicated in the release of virions, its mechanism of action is not known. ADP expression was shown to increase virion levels in the cytoplasm and supernatant but did not have a clear effect on nuclear membrane stability or nuclear membrane permeability. The appearance of the nuclear membrane in infection was studied using fluorescence microscopy and a variety of electron microscopy techniques including classical sectioning, serial block-face scanning electron microscopy and electron cryo-tomography. HAdV5 infection was shown to reduce levels of lamin A at the nuclear envelope while destabilising the nuclear membrane to form large-scale invaginations. Additionally, small nuclear membrane lesions were detected in infection and an overall increase in nuclear membrane permeability was measured.
|Enthalten in den Sammlungen:||Elektronische Dissertationen und Habilitationen|
geprüft am 01.08.2021
geprüft am 01.08.2021