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Sequence of the phosphoprotein gene of pneumonia virus of mice: expression of multiple proteins from two overlapping
J Barr1, P Chambers, P Harriott
1Department of Biological Sciences, University of Warwick, Coventry, England.
Abstract:
The gene encoding the phosphoprotein of the pneumovirus pneumonia virus of mice (PVM) has been cloned and sequenced. The gene is 903 nucleotides in length and contains a long open reading frame (ORF) capable of encoding a polypeptide of 295 amino acid residues. A smaller, second, overlapping ORF encoding a polypeptide 137 amino acids in length was also present. The large ORF directed the synthesis of a 39-kDa polypeptide and four additional polypeptides with masses of 37 kDa, 26 kDa, 23 kDa, and 16 kDa in vitro. The smaller polypeptides were generated by internal initiation on in-frame AUG initiation codons to generate carboxy co-terminal products. Western immunoblot analysis indicated that at least two of these proteins and several other related polypeptides are present in infected cells, and the possible origins of these are discussed. Western blot analysis using antiserum raised against a synthetic peptide and specific for the predicted second ORF product identified a polypeptide of 23 kDa in PVM-infected cells. The pattern of PVM P gene expression is unlike that of the closely related respiratory syncytial virus and is reminiscent of that of paramyxoviruses such as Sendai virus. This is the first example of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.
Insights
Researchers cloned and sequenced the pneumonia virus of mice (PVM) phosphoprotein gene. This gene produces multiple proteins from a single mRNA, a unique finding for pneumoviruses.
Area of Science:
- Virology
- Molecular Biology
- Genetics
Background:
- Pneumonia virus of mice (PVM) is a significant respiratory pathogen.
- Understanding PVM gene expression is crucial for developing antiviral strategies.
Purpose of the Study:
- To clone and sequence the PVM phosphoprotein gene.
- To investigate the polypeptide products encoded by the PVM phosphoprotein gene.
- To compare PVM gene expression with related viruses.
Main Methods:
- Gene cloning and sequencing of the PVM phosphoprotein gene.
- In vitro synthesis of polypeptides using the cloned gene.
- Western immunoblot analysis of infected cells to detect viral proteins.
Main Results:
- The PVM phosphoprotein gene is 903 nucleotides long with two open reading frames (ORFs).
- The large ORF encodes a 295-amino acid polypeptide, and a smaller, overlapping ORF encodes a 137-amino acid polypeptide.
- In vitro studies produced multiple polypeptides, with evidence of internal initiation generating carboxy-terminal products.
- Western blot confirmed the presence of several PVM phosphoprotein products in infected cells, including a 23 kDa protein from the second ORF.
- PVM gene expression differs from respiratory syncytial virus but resembles paramyxoviruses.
Conclusions:
- This study presents the first evidence of a pneumovirus encoding multiple polypeptide products from a single mRNA in vivo.
- The complex expression pattern of the PVM phosphoprotein gene has implications for viral replication and pathogenesis.
- The findings provide a basis for further research into PVM molecular biology and host interactions.