Following the molecular cloning of the PVM genome, the opportunity to the individual genes and proteins of PVM has arisen. This study investigated nucleocapsid (N) gene and the phosphoprotein (P) gene of PVM and attempted to characterise the polypeptide products expressed from the N and P genes both in vitro in PVM-infected cells. The ability of the PVM N and P proteins to interact with other was also investigated. The nucleotide sequence of the PVM P gene was determined to be 903 nucleotides in length and shown to comprise a long open reading frame capable of encoding the 295 amino acid long P protein and also a smaller second ORF with the potential to express a polypeptide 137 amino acids in length. The PVM P protein shows overall amino acid homology of 35.3%, 35.6% and 28.3% to the P proteins of pneumovirus members HRSV, BRSV and TRTV respectively. The PVM P gene contrasts with the P genes of other pneumovirus genus members which do not possess extensive alternative ORFs. Both the N and P genes of PVM were shown to be capable of directing the synthesis of more than one polypeptide product both in vitro and in PVM-infected BSC1 cells. mRNA transcribed from the PVM P gene long ORF directed the in vitro expression of the 39 kDa P protein and four additional polypeptides. By constructing transcription plasmids that contained 5' terminally truncated P gene cDNA insets, these polypeptides were determined to be expressed by translational initiation on internal P gene initiation codons. Western blot analysis determined that in addition to PVM P protein, two of these in vitro expressed P protein species, with molecular weights of 26 kDa and 23 kDa, were expressed in PVM-infected BSC 1 cells and this observation was supported by the results of anti-P protein monoclonal antibody epitope mapping studies. The ability of the PVM P gene to direct the expression of P protein related polypeptides from internal initiation codons is a feature not yet described for any other pneumovirus member. By immunising rats with a synthetic peptide, antiserum specific for the second polypeptide product (P2) was generated. Western blot analysis using this anti-P2 antiserum identified a species thought to represent P2 in PVM-infected BSC 1 cell material. The ability of the PVM P gene to express a polypeptide from an alternative is a feature common to the P genes of most other morbilliviruses and paramyxoviruses. mRNA transcribed from PVM N gene cDNA was able to direct the in vitro translation of the 43 kDa N protein and also a highly abundant polypeptide with a molecular weight of 24 kDa which was shown to be expressed by way of internal initiation on the fifth N gene AUG codon of the N gene sequence. The 24 kDa N protein related polypeptide was expressed in E. coli, purified, and used to immunise a rabbit for the production of anti-24 kDa polypeptide antiserum. Western blot analysis using this antiserum with PVM-infected BSC1 cells detected the 43 kDa N protein, a highly abundant 30 kDa N protein related species, but not the 24 kDa polypeptide. precise identity of the 30 kDa polypeptide was not determined. Possible mechanisms which could account for the expression of the protein products of the N P genes are discussed. By using a protein blotting technique the interaction that occurs between the N P proteins of PVM was investigated. The P protein binding affinities of in vitro expressed truncated N proteins suggested that many regions of the N protein are co- operatively involved in the binding process, although some regions contributed more than others. The N protein of Sendai virus is believed to bind to the Sendai virus P protein in a similar way. It was also determined that both the amino and the carboxyl- terminal regions of the PVM P protein were found to be essential for binding to N protein. This contrasts with the situation determined for Sendai virus in which 344 P protein amino-terminal amino acids were found to be dispensable for binding N protein.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:241310 |
| Date | January 1993 |
| Creators | Barr, John Nicholas |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/55456/ |
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