Requirements for the Formation of Membrane Pores by the Reovirus Myristoylated μ1N Peptide

The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the mu 1 protein, each complexed with three copies of the protector protein sigma 3. Conformational changes in mu 1 following the proteolytic removal of sigma 3 lead to release of the myristoylated N-terminal cleavage fragment mu 1N and ultimately to membrane penetration. The mu 1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant mu 1 trimers and synthetic mu 1N peptides with both RBCs and liposomes. The mu 1 trimer mediates hemolysis and liposome disruption under conditions that promote the mu 1 conformational change, and mutations that inhibit mu 1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free mu 1 trimer. Autolytic cleavage to form mu 1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that mu 1N cleavage is not required when steric barriers are removed. Synthetic myristoylated mu 1N peptide forms size-selective pores in liposomes, as measured by fluorescence dequenching of labeled dextrans of different sizes. Addition of a C-terminal solubility tag to the peptide does not affect activity, but sequence substitution V13N or L36D reduces liposome disruption. These substitutions are in regions of alternating hydrophobic residues. Their locations, the presence of an N-terminal myristoyl group, and the full activity of a C-terminally extended peptide, along with circular dichroism data that indicate prevalence of beta-strand secondary structure, suggest a model in which mu 1N beta-hairpins assemble in the membrane to form a beta-barrel pore.