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Following Cell-fate in E. coli After Infection by Phage Lambda
Published on: October 14, 2011
Characterization of a dual-function domain that mediates membrane insertion and excision of Ff filamentous
Nicholas J Bennett1, Dragana Gagic, Andrew J Sutherland-Smith
1Institute of Molecular BioSciences, Massey University, Palmerston North 4442, New Zealand.
Abstract:
The filamentous phage Ff (f1, fd, or M13) of Escherichia coli is assembled at the cell membranes by a process that is morphologically similar to that of pilus assembly. The release of the filament virion is mediated by excision from the membrane; conversely, entry into a host cell is mediated by insertion of the virion coat proteins into the membrane. The N-terminal domains of the minor virion protein pIII have the sole role of binding to host receptors during infection. In contrast, the C domain of pIII is required for two opposite functions: insertion of the virion into the membrane during infection and excision at the termination step of assembly/secretion. We identified a 28-residue-long segment in the pIII C domain, which is required for phage entry but dispensable for release from the membrane at the end of assembly. This segment, which we named the infection-competence segment (ICS), works only in cis with the N-terminal receptor-binding domains and does not require the equivalent ICS sequences in other subunits within the virion cap. The ICS contains a predicted amphipathic α-helix and is rich in small amino acids, Gly, Ala, and Ser, which are arranged as a [small]XXX[small]XX[small]XXX[small]XXX[small] motif. Scanning Ala/Gly mutagenesis of ICS showed that small residues are compatible with infection. Overall, organization of the C domain is reminiscent of α-helical pore-forming toxins' membrane insertion domains. The unique ability of pIII to mediate both membrane insertion and excision allowed us to compare these two fundamental membrane transactions and to show that receptor-triggered insertion is a more complex process than excision from membranes.
Insights
A specific segment of filamentous phage protein pIII, the infection-competence segment (ICS), is crucial for viral entry into host cells but not for phage release. This finding clarifies complex membrane interactions during viral infection and assembly.
Area of Science:
- Molecular Biology
- Virology
- Membrane Biology
Background:
- Filamentous phages (Ff group, including M13) assemble at Escherichia coli cell membranes, mirroring pilus assembly.
- Phage release involves membrane excision, while entry requires coat protein insertion into the host membrane.
- The minor phage protein pIII mediates host receptor binding (N-terminal) and membrane insertion/excision (C-terminal).
Purpose of the Study:
- To identify specific domains within phage protein pIII responsible for distinct membrane interaction functions.
- To elucidate the molecular mechanisms underlying viral entry and assembly/secretion processes.
- To compare the complexity of membrane insertion versus excision in viral lifecycle.
Main Methods:
- Identification and characterization of a 28-residue segment within the pIII C domain, termed the infection-competence segment (ICS).
- Analysis of ICS function in cis with N-terminal domains.
- Site-directed mutagenesis (Ala/Gly scanning) of the ICS to assess the role of small amino acids and predicted α-helical structure.
Main Results:
- A 28-residue infection-competence segment (ICS) in pIII's C domain is essential for phage entry but not for membrane release during assembly.
- The ICS functions in cis with N-terminal receptor-binding domains and does not require homologous sequences in other pIII subunits.
- ICS contains a predicted amphipathic α-helix rich in Gly, Ala, and Ser, with small residues being compatible with infection.
Conclusions:
- The pIII C domain possesses distinct functional regions for membrane insertion and excision.
- The ICS is a novel element critical for receptor-triggered viral membrane insertion, highlighting its role in phage entry.
- Receptor-triggered membrane insertion is a more complex process than membrane excision during phage assembly.
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