Rinderpest virus (RPV) is a morbillivirus which causes a highly contagious disease affecting members of the order Artiodactyla. The viral L protein is the catalytic subunit of the RNA-dependent RNA polymerase, but requires the P protein for activity. In previous studies it was found that, in addition to a direct L-P interaction, both the C and V non-structural proteins bind to L. The L proteins of morbilliviruses consist of three long highly conserved domains separated by short unconserved sequences. The interaction of P, C and V with these three domains was studied. Using co-immunoprecipitation, it was shown that P interacts with the first domain, whilst C and V were each shown to interact with the central domain. Further mutational analysis using the yeast two-hybrid system (Y2HS), showed that the P binding site lies in the amino-proximal domain of L, between amino acids 1 and 233, which fits with the co-immunoprecipitation data. However, the Y2HS suggested that the binding site for C and V includes a region between amino acids 1 and 363 of L, i.e. within the first domain. These data indicate (i) that the P binding site is distinct from that ofC and V, and (ii) that the C and V binding site(s) may be complex. To search for host cell proteins with which L interacts, a library screen was performed using the Y2HS and a porcine macrophage cDNA library. Three host cell proteins were recovered from the library screen as putative L interactors. The interaction with one of these, striatin, was confirmed by co-immunoprecipitation, and co-localisation of the two proteins was observed by confocal microscopy. The L sequence with which striatin interacts was investigated. Like the C and V proteins, striatin was shown to interact with the second conserved domain of L by co-immunoprecipitation and Y2HS data indicated that a possible second binding site for striatin includes a region of L sequence between amino acids 1 and 363.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:403098 |
| Date | January 2003 |
| Creators | Sleeman, Katrina |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/79583/ |
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