Utilising transmission electron microscopy to better understand viral replication cycles: the characterisation of cytoplasmic replication in Tula virus, rotavirus and herpesvirus

Viruses are intracellular parasites that can subvert host cellular systems to enable the production of progeny viruses, which are then able to infect more cells. Transmission electron microscopy is one the main ways in which viral replication within cells can be directly visualised in a way that reveals ultrastructural details. Throughout this project I have utilised transmission electron microscopy to elucidate aspects of viral replication cycles within the cytoplasm of infected cells. In chapter three I investigated the effects of Tula virus infection on cellular architecture in collaboration with Katherine Davies. Tula virus belongs to the hantavirus genus along with several other hantaviruses which are responsible for zoonotic illness in humans. From our investigation it was found that Tula virus forms large filamentous structures within the cytoplasm of infected cells that increase in size as infection progresses. These filamentous structures have been observed in New World hantavirus infected cells before, but this is the first time that the filaments have been identified in an Old World hantavirus. Furthermore, it was found that Tula virus infection results in the enlargement of the cell endoplasmic reticulum, potentially through inducing endoplasmic reticulum stress. In chapter four I explored the role of liquid-liquid phase separation in rotavirus viroplasm formation. Rotaviruses are the most common cause of diarrhoeal disease in children, and they form viral factories within infected cells known as viroplasms. I found that when rotavirus infected cells were treated with an inhibitor of liquid-liquid phase separation, viroplasms reduced in size. This indicated that liquid-liquid phase separation is an important aspect of rotavirus viroplasm development. Finally, in chapter five I examined the role of the host guanine nucleotide exchange factor DOCK5 on herpesvirus cellular egress. Herpesviruses are known for causing life-long infections in humans in over half the global population. I found that the gammaherpesvirus Kaposi’s sarcoma associated herpesvirus causes endoplasmic reticulum remodelling in infected cells, potentially in a manner similar to herpes simplex virus 1. I also found that evidence that DOCK5 is located within the nucleus of fibroblast and lymphocytes, despite the literature describing it as a cytoplasmic protein. The experiments above demonstrate how transmission electron microscopy can be utilised to better understand viral replication cycles.