Caliciviruses are single-stranded positive-sense RNA viruses. The viral genome is polyadenylated and 7-8 kb in length. Non-structural polypeptide coding sequences are located in the 5' region of the genome whereas structural polypeptide coding sequences are located at the 3' end. In addition the replication cycle involves the synthesis of at least one 3' co-terminal subgenomic RNA. The most important feature of calicivirus mRNA is the lack of a "cap" structure at the 5' end. The viral mRNA bears a 10-15 IcDa VPg protein linked to both genomic and sub-genomic RNAs. The lack of a 'cap' structure suggests that calicivirus mRNA is translated by a cap-independent mechanism. The aim of this project was to investigate the mechanism of translation initiation on feline calicivirus (FCV) mRNA. This was achieved by examining (i) the proposed role for the 15 IcDa VPg protein, as a 'cap analogue', and (ii) the interactions of cellular proteins to the 5' and 3' ends of the FCV genome. Firstly, expressed FCV VPg was purified and used to raise antisera in rabbits. The antisera were subsequently used to analyse proteins from FCV-infected CRFK cells. Secondly, pull-down and ELISA-based binding assays suggested interaction of recombinant FCV VPg with the canonical initiation factor eIF4E. Thirdly, studies of the interactions between cellular proteins and the 5' and 3' terminal ends of the FCV genome by UV cross-linking and oligo(dT) RNA-protein binding assays were conducted. Results suggested interaction of the 5' end of the FCV genome with proteins eIF4A, polypyrimidine tract-binding protein (PTB) and La, and eIF4A with the 3' end of the FCV genome. Based on the results of this work, this thesis proposes a model of the interactions between the FCV genome, VPg and cellular proteins in translation initiation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:274337 |
| Date | January 2003 |
| Creators | Gioldasi, Ioanna |
| Publisher | University of Surrey |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://epubs.surrey.ac.uk/844326/ |
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