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.