Expression of the nucleoprotein and phosphoprotein genes of pneumonia virus of mice and specific interactions of the gene products

Doctoral thesis English OPEN
Barr, John Nicholas
  • Subject: QH301 | QH426
    mesheuropmc: parasitic diseases

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