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Title: Localisation of human respiratory syncytial virus proteins during infection
Author: Droniou, Magali Eliane
ISNI:       0000 0004 2667 9875
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2007
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Human respiratory syncytial vilUs (hRSV) is an important pathogen causing respiratory disease, affecting predominantly the infant and elderly populations. No effective vaccine or anti-viral treatment is yet available against this virus. hRSV is a negative-sense, nonsegmented RNA virus, which encodes II proteins. The N, P, Land M2-1 proteins constitute the viral polymerase and the M2-2 protein acts as a regulatory element in the balance between transcription and replication. The G protein allows attachment of the virus to cellular glycosaminoglycans receptors, and the F protein mediates entry of the virus into the cell. The M protein drives virus assembly and budding of progeny virions, and the non-structural proteins NSI and NS2 act as antagonists of the interferon system. The SH protein has been shown to possess anti-apoptotic properties. Some of hRSV proteins have been shown to play additional roles to the ones described. Since protein function and intracellular localisation are linked, analysis of viral protein movement within infected cells may lead to information regarding the functions of the proteins studied. hRSV protein localisation was elucidated by immunostaining of viral proteins for which antibodies were available. This confirmed the hRSV N, P and M2-1 proteins distribution to cytoplasmic inclusion bodies and uncovered the additional diffuse presence of these viral proteins in the cytoplasm and into small cytoplasmic inclusions with distinct morphological characteristics. The M2-1 protein was also faintly detected in the nucleus of infected cells. To acquire an insight into the dynamics of viral proteins during infection in live cells, the hRSV proteins were tagged with either the green fluorescent protein or its spectral variants, for introduction into virus by reverse genetics. Initially, a codon-optimized synthetic G gene was generated and introduced in the pre-existing G-deleted hRSV strain RS-S2 antigenomic. eDNA clone. Infectious recombinant rRS-S2syG virus, expressing the synthetic G gene, was successfully recovered and exhibited altered growth kinetics compared to both wild-type virus hRSV strain RS-S2 and recombinant rRS-S2 vilUs bearing a deletion of the G gene. Since the G protein was correctly expressed and localised, this suggested that the sequence or structure of the authentic genomic RNA is important for virus replication. The hRSV N, P, L, M2-1, NSI and NS2 proteins were tagged at both amino- and carboxy-termini with a fluorescent marker. The N, P and L fusion proteins retained some binding activities, as characterised by their correct localisation to inclusion bodies in hRSV superinfected cells. The localisation observed within the inclusion bodies was shown to follow specific patterns which varied within the same cell population, indicative of a dynamic rearrangement of protein distribution within the inclusion bodies. However, the loss of activity in minigenome transcription experiments observed for all tagged N, P and L proteins prevented their introduction into recombinant virus. In contrast, the carboxytagged M2-1 protein retained the majority of the wild-type transcriptional enhancement properties. The NSI protein, when expressed alone, was found to follow two distinct localisation patterns within the cell population, being principally observed as present in the nucleus and cytoplasm or in a lesser proportion of cells, exclusively in the cytoplasm. The NS2 protein, when expressed alone, showed a perinuclear accumulation of granular appearance. However, the aberrant intracellular localisation of fluorescentlytagged NSI and NS2 proteins, compared to wild-type NS1/2 prevented the introduction into recombinant viruses. Investigating the role of the ubiquitin-proteasome pathway in hRSV infection, it was found that the previously described rapid turnover of the NS2 protein was due its degradation by the proteasome complex. The NS1 protein was also found to be targeted to the proteasome, while the rate of ubiquitination was fomid to be increased during hRSV infection.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available