An examination of NP-P and NP-V interactions within the simian virus 5 (SV5) replication complex
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The aim of this study was to examine the mechanisms of transcription and replication of the paramyxovirus, simian virus type 5 (SV5). This was initially attempted using reverse genetics techniques and subsequently examining specific viral protein: protein interactions within the replication complex. A cDNA clone encoding a synthetic negative-sense RNA genome analogue was constructed. Reverse genetics techniques were used to attempt to characterise conditions which supported the transcription and replication of this genome analogue, with or without the use of wild-type helper virus but were unsuccessful. During the course of these studies, a number of mammalian cell lines inducibly expressing SV5 proteins were isolated. These cell lines were subsequently used to examine viral protein: protein interactions within the replication complex. When expressed alone, both P and V proteins exhibited diffuse cytoplasmic fluorescence and V was also found in the nucleus. However, when NP was expressed alone, it was seen as punctate and granular cytoplasmic fluorescence. The distribution patterns of the proteins changed when expressed in combination. Large cytoplasmic aggregates similar to those at late times in an SV5 infection were seen in cells which co-expressed NP and P. When NP was co-expressed with V, however, NP was partially redistributed to give diffuse cytoplasmic and nuclear fluorescence. This showed that both P and V proteins could interact with NP and suggested that V may play a role in keeping NP soluble prior to an ordered encapsidation process. Extracts from these cell lines were then used in a novel protein: protein capture assay and demonstrated that NP could interact with both P and V proteins. NP expressed by the cell line was shown to contained both soluble and polymeric forms of NP. P was shown to bind both forms of NP, while V could only bind soluble NP. Since P and V proteins are amino co-terminal, the site of interaction between P and polymeric NP was predicted to be in the P unique C-terminus. This was strengthened when a P-specific C- terminal mAb was found to block the binding of P with polymeric NP. Deletion mutant analysis in the C-terminus of the P protein showed that the mAb binding site was at the extreme C-terminus of the protein suggesting this is the point of interaction between P and polymeric NP. Possible roles for these protein: protein interactions and implications for the paramyxovirus replication complex are discussed.
Thesis, PhD Doctor of Philosophy
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