A dimeric holin/antiholin complex controls lysis by phage T4.

Area of Science:

• Bacteriophage biology
• Molecular mechanisms of viral lysis
• Protein-protein interactions in viral replication

Background:

• Lytic phages control host cell lysis via holin-mediated endolysin release.
• Phage T4 antiholin RI inhibits holin T, preventing premature lysis and endolysin release.
• Antiholin RI mediates lysis inhibition (LIN) during phage superinfection, increasing survival in low phage concentrations.

Purpose of the Study:

• To investigate the functional relevance of the T4 holin T and antiholin RI complex in lysis inhibition (LIN).
• To determine the structural basis and mechanism of LIN mediated by the T/RI interaction.

Main Methods:

• Reconstitution of LIN in a phage-free system using purified RI, T, and endolysin.
• Targeted mutagenesis of RI and T proteins.
• Functional analyses of lysis inhibition and protein interactions.
• AlphaFold2 prediction of protein complex structure.

Main Results:

• Inactivation of the RI signal peptide cleavage site did not abolish LIN, indicating membrane-bound RI function.
• Only one of the two T/RI interfaces in the tetramer crystal structure is physiologically relevant.
• Mutations at the relevant interaction site prevented lysis, suggesting inhibition of holin oligomerization.
• LIN is mediated by a dimeric T/RI complex, which forms readily when both proteins are membrane-anchored.

Conclusions:

• Lysis inhibition (LIN) in phage T4 is mediated by a dimeric T/RI complex.
• The RI interaction likely inhibits lysis by blocking essential holin oligomerization for pore formation.
• The functional T/RI complex is dimeric and membrane-anchored, distinct from the previously observed tetrameric crystal structure.