Heath, Colin Malcolm; (2002) The role of proteolytic processing of the 220kDa polyprotein of African swine fever virus and aggresomes during virion assembly. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

African swine fever virus (ASFV) is a large double stranded DNA virus causing a lethal haemorrhagic disease in domestic pigs. The 170kbp genome encodes 150 open reading frames and virions contain more than 50 different proteins. ASFV uses a 220kDa polyprotein (pp220) to produce major structural proteins p150, p37, p34 and p14. This thesis investigates the subcellular distribution and regulation of pp220 processing. Antibodies specific for p150 and p34 were generated and characterised. Western blot analysis showed that approximately 35% of the intracellular pool of pp220 associated with membranes, the remainder was cytosolic. The membrane bound pool was processed via intermediates to p34 and p150. p34 and p150, but not the polyprotein, or the processing intermediates, were enveloped and released in virions. The cytosolic pool was processed incorrectly and failed to produce structural proteins. The correct processing of pp220 occurs at 4 Gly-Gly-X motifs, however the polyprotein contains 19 Gly-Gly-X motifs. It was concluded that membrane association is required for correct processing. In the absence of membrane association the polyprotein maybe cleaved at all 19 Gly-Gly-X motifs. Aggresomes are a major site of proteolysis in cells and their role in ASFV assembly was investigated. Aggresomes are pericentriolar structures enriched for chaperones, proteasomes and ubiquitin. They are enclosed in a vimentin cage surrounded by mitochondria. ASFV assembly occurs in perinuclear structures called viral factories and the possibility that these derive from aggresomes was investigated. Immunofluorescence experiments showed viral factories close to the centriole and surrounded by vimentin cages and mitochondria. Furthermore, inhibitors of aggresome proteolysis and the over expression of misfolded proteins in the cytosol blocked viral replication. It is possible that ASFV uses aspects of the aggresomes to concentrate viral proteins during assembly.

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