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Peptide display on live MS2 phage: restrictions at the RNA genome level
Dico van Meerten1, Rene C L Olsthoorn1, Jan van Duin1
1Leiden Institute of Chemistry, Department of Biochemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands1.
Abstract:
The potential of the RNA phage MS2 to accommodate extra amino acids in its major coat protein has been examined. Accordingly, a pentapeptide was encoded in the genome as an N-terminal extension. In the MS2 crystal structure, this part of the coat protein forms a loop that extends from the outer surface of the icosahedral virion. At the RNA level, the insert forms a large loop at the top of an existing hairpin. This study shows that it is possible to maintain inserts in the coat protein of live phages. However, not all inserts were genetically stable. Some suffer deletions, while others underwent adaptation by base substitutions. Whether or not an insert is stable appears to be determined by the choice of the nucleic acid sequence used to encode the extra peptide. This effect was not caused by differential translation, because coat-protein synthesis was equal in wild-type and mutants. We conclude that the stability of the insert depends on the structure of the large RNA hairpin loop, as demonstrated by the fact that a single substitution can convert an unstable loop into a stable one.
Insights
Researchers explored inserting extra amino acids into the MS2 phage coat protein. Genetic stability of these inserts in live phages depends on the RNA hairpin loop structure, not translation efficiency.
Area of Science:
- Virology
- Molecular Biology
- Protein Engineering
Background:
- The MS2 phage major coat protein is amenable to modifications.
- Structural analysis reveals N-terminal extensions form surface loops on the virion.
- RNA hairpin loops are key structural elements within the phage genome.
Purpose of the Study:
- To investigate the feasibility of incorporating extra amino acids into the MS2 phage coat protein.
- To determine the genetic stability of peptide inserts within the coat protein of live phages.
- To elucidate the factors influencing the stability of RNA inserts in the MS2 genome.
Main Methods:
- Encoding a pentapeptide as an N-terminal extension in the MS2 phage genome.
- Analyzing the structural impact of inserts on the coat protein and RNA hairpin loops.
- Assessing genetic stability through observation of deletions and base substitutions in live phages.
- Comparing coat protein synthesis levels between wild-type and mutant phages.
Main Results:
- Peptide inserts can be accommodated in the MS2 coat protein of live phages.
- Insert stability is variable, with some phages exhibiting deletions or base substitutions.
- Genetic stability is dictated by the nucleic acid sequence encoding the peptide insert.
- Translation efficiency of coat protein was comparable between wild-type and mutant phages.
Conclusions:
- The successful accommodation of inserts demonstrates the potential for engineering MS2 phage coat proteins.
- Insert stability is primarily determined by the structural integrity of the resulting RNA hairpin loop.
- A single nucleotide substitution can significantly alter the stability of the RNA hairpin loop and the insert.