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Journal of Virology, July 2009, p. 7004-7014, Vol. 83, No. 14
0022-538X/09/$08.00+0     doi:10.1128/JVI.00377-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Lan Zhang,1,{dagger},{ddagger} Melina A. Agosto,1,2,{dagger},§ Tijana Ivanovic,2,3 David S. King,4 Max L. Nibert,2 and Stephen C. Harrison1,3,5*

Laboratory of Molecular Medicine,1 Howard Hughes Medical Institute, Children's Hospital,5 Departments of Microbiology and Molecular Genetics,2 Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115,3 Department of Molecular and Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, California 947204

Received 19 February 2009/ Accepted 30 April 2009

The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the µ1 protein, each complexed with three copies of the protector protein {sigma}3. Conformational changes in µ1 following the proteolytic removal of {sigma}3 lead to release of the myristoylated N-terminal cleavage fragment µ1N and ultimately to membrane penetration. The µ1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant µ1 trimers and synthetic µ1N peptides with both RBCs and liposomes. The µ1 trimer mediates hemolysis and liposome disruption under conditions that promote the µ1 conformational change, and mutations that inhibit µ1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free µ1 trimer. Autolytic cleavage to form µ1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that µ1N cleavage is not required when steric barriers are removed. Synthetic myristoylated µ1N 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 β-strand secondary structure, suggest a model in which µ1N β-hairpins assemble in the membrane to form a β-barrel pore.

* Corresponding author. Mailing address: Laboratory of Molecular Medicine, Children's Hospital, 320 Longwood Ave., Boston, MA 02115. Phone: (617) 432-5607. Fax: (617) 432-5600. E-mail: harrison{at}crystal.harvard.edu

{triangledown} Published ahead of print on 13 May 2009.

{dagger} These authors contributed equally.

{ddagger} Present address: Vaccine Basic Research, Merck & Co., Inc., West Point, PA 19486.

§ Present address: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX 77030.

Journal of Virology, July 2009, p. 7004-7014, Vol. 83, No. 14
0022-538X/09/$08.00+0     doi:10.1128/JVI.00377-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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