Shaw, Andrew Edward; (2009) The role of non-structural proteins in bluetongue virus replication. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bluetongue virus (BTV) is the type species of the genus Orbivirus, family Reoviridae. Bluetongue viruses have genomes composed of ten segments of dsRNA, encoding seven structural and four non-structural (NS) proteins. They cause economically important disease (called bluetongue) in ruminants and are transmitted by Culicoides biting midges. The role of BTV NS proteins during replication in mammalian cells was investigated. Genes encoding NS1, NS2 and NS3/3A were cloned and transiently expressed (from transfected plasmids). Localisation of these proteins in uninfected cells matched their distribution in infected cells. However, BTV infection of mammalian cells restricts translation of 'host-like' polyadenylated mRNAs, and restricted expression of NS-proteins from these plasmids. BTV infection also stimulated translation of 'BTV-like' mRNAs, provided they contain both 3' and 5' untranslated regions (UTRs). Specifically, the 3' terminal conserved-hexanucleotide of BTV mRNAs (3'- CAUUCA....) was essential for translation and must be positioned at the terminus, although the remainder of the 3' UTR was also required. However, unlike rotavirus NSP3, BTV-NS2 did not inhibit host cell translation. BTV infection caused abortive division of mammalian cells, with multiple spindle formation and cell-cycle-arrest in late metaphase. NS2 was seen associated with condensed chromosomes in infected cells displaying aberrant mitosis. NS2 interactions with microtubule-end binding-proteins may prevent association of spindle microtubules with the chromosome kinetochores, blocking mitosis. NS1 was also shown to associate with and disrupt the centrosome. A real-time reverse-transcriptase polymerase chain reaction (rRT-PCR) assay was developed for detection of BTV and is now a primary diagnostic tool for BTV, used by the community reference laboratory (at IAH Pirbright). The assay was adapted to Qiagen rRT-PCR chemistry and is commercially available. Double-stranded RNA synthesised in vitro was digested with dicer, forming siRNAs. RNA silencing of plasmid expressed mRNAs/protein was achieved, but insufficient time remained to complete these studies in infected cells.

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