Examining orthohantavirus host cell interactions

Orthohantaviruses are negative sense single stranded RNA viruses that are capable of causing severe respiratory and haemorrhagic disease in humans. Currently, many aspects of the orthohantavirus lifecycle are poorly understood predominantly due to difficulties in detecting and quantifying orthohantavirus components. Here, I developed new and efficient techniques to quantify orthohantavirus proteins, RNA and infectious viral particles. These techniques were used to provide a detailed understanding of the orthohantavirus lifecycle kinetics. Additionally, I examined the interactions of the orthohantavirus nucleocapsid protein (NP) with the host cell. The NP has functions in viral transcription, translation and immune evasion as well as encapsidation of viral RNAs. As orthohantaviruses have been found to form persistent infections in vitro the molecular mechanisms behind this were of particular interest I examined the interaction of the Tula orthohantavirus (TULV) NP with host-cell components using co-localisation assays at early, peak and persistent infections. Furthermore, to explore the sites of viral replication within the host cell, RNA labelling techniques were utilised in combination with immunofluorescent analysis. RNA fluorescent in situ hybridisation probes designed against the sense and anti-sense S segment RNA were used to assess the localisation of viral RNA in vitro in conjunction with TULV NP and previously identified interacting host cell proteins. Here, I identified distinct localisation of TULV NP in Vero E6 cells at 36 hpi, 7 dpi and 30 dpi notably the formation of large filamentous structures in the perinuclear region of infected cells in addition to establishing multinucleate cells during persistent infection. These filamentous structures appear to be maintained through the vimentin intermediate network. Additional TULV NP was found to show high levels of co-localisation with the Golgi and the stress granule marker, TIA-1. These markers also showed high-levels of colocalisation with sense and anti-sense TULV S segment RNA which may indicate these sites to be areas of viral replication and accumulation.