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Ribosomal frameshifting during translation of measles virus P protein mRNA is capable of directing synthesis of a
1Department of Microbiology and Immunology, McGill University, Montreal, Quebec, Canada.
Abstract:
Members of the Paramyxoviridae family utilize a variety of different strategies to increase coding capacity within their P cistrons. Translation initiation at alternative 5'-proximal AUG codons is used by measles virus (MV) to express the virus-specific P and C proteins from overlapping reading frames on their mRNAs. Additional species of mRNAs are transcribed from the MV P cistron by the insertion of extra nontemplated G residues at a specific site within the P transcript. Addition of only a single nontemplated G residue results in the expression of the V protein, which contains a unique carboxyl terminus. We have used an Escherichia coli system to express MV P cistron-related mRNAs and proteins. We have found that ribosomal frameshifting on the MV P protein mRNA is capable of generating a previously unrecognized P cistron-encoded protein that we have designated R. Some ribosomes which have initiated translation of the P protein mRNA use the sequence TCC CCG AG (24 nucleotides upstream of the V protein stop codon) to slip into the -1 reading frame, thus translating the sequence as TC CCC GAG. The resulting R protein terminates five codons downstream of the frameshift site at the V protein stop codon. We have gone on to use a chloramphenicol acetyltransferase reporter system to demonstrate that this MV-specific sequence is capable of directing frameshifting during in vivo translation in eukaryotic cells. Analysis of immunoprecipitated proteins from MV-infected cells by two-dimensional gel electrophoresis allowed detection of a protein species consistent with R protein in MV-infected cells. Quantitation of this protein species allowed a rough estimation of frameshift frequency of approximately 1.8%. Significant stimulation of ribosomal frameshift frequency at this locus of the MV P mRNA was mediated by a downstream stimulator element which, although not yet fully defined, appeared to be neither a conventional stem-loop nor an RNA pseudoknot structure.
Insights
Measles virus (MV) uses ribosomal frameshifting to create a new protein, R, from its P cistron mRNA. This discovery reveals an additional mechanism for MV coding capacity and protein diversity.
Area of Science:
- Molecular Biology
- Virology
- Genetics
Background:
- Paramyxoviridae family members employ diverse strategies to expand coding capacity within their P cistrons.
- Measles virus (MV) expresses P and C proteins from overlapping reading frames via alternative translation initiation.
- MV P cistron transcription yields additional mRNA species through nontemplated G residue insertions, including V protein expression.
Purpose of the Study:
- To investigate novel protein expression mechanisms within the MV P cistron.
- To identify and characterize previously unrecognized proteins encoded by the MV P cistron.
- To elucidate the role of ribosomal frameshifting in MV protein synthesis.
Main Methods:
- Expression of MV P cistron-related mRNAs and proteins in an Escherichia coli system.
- Utilizing a chloramphenicol acetyltransferase reporter system to assess frameshifting in eukaryotic cells.
- Analyzing immunoprecipitated proteins from MV-infected cells using two-dimensional gel electrophoresis.
Main Results:
- Discovery of a novel P cistron-encoded protein, designated R, generated by ribosomal frameshifting on MV P protein mRNA.
- Identification of a specific mRNA sequence (TCC CCG AG) mediating -1 ribosomal frameshifting.
- Demonstration of MV-specific sequence-directed frameshifting in vivo and detection of R protein in infected cells.
- Estimation of frameshift frequency at approximately 1.8%, stimulated by an undefined downstream element.
Conclusions:
- Ribosomal frameshifting is a significant mechanism for generating protein diversity from the MV P cistron.
- The newly identified R protein contributes to the coding capacity of MV.
- The frameshift stimulator element warrants further investigation for its structural and functional properties.