1.A.60 The Mammalian Reovirus Pre-forming Peptide, Mu-1 (Mu-1) Family
During cell entry, reovirus particles with a diameter of 70-80 nm must penetrate the cellular membrane to access the cytoplasm. The mechanism of penetration, without benefit of membrane fusion, is poorly characterized for nonenveloped animal viruses. Lysis of RBCs is an in vitro assay for the membrane perforation activity of reovirus. Reovirus-induced lysis of RBCs occurs osmotically, after formation of small size-selective lesions or 'pores.' Consistent results were obtained by monitoring leakage of fluorophore-tagged dextrans from the interior of resealed RBC ghosts.
The mechanism of penetration involves pore formation via protein A1 following proteolysis (Agosto et al., 2006). Osmotic-protection experiments using polyethylene glycols and dextrans of different sizes revealed that reovirus-induced lysis of RBCs occurs after formation of small size-selective pores. Whole virus particles, as well as the myristoylated fragment, µ1N, that is released from particles, are recruited to RBC membrane-forming pores. Formation of small pores by µ1N thus occurs in discrete steps as part of the reovirus membrane-penetration pathway (Agosto et al., 2006).
While the parent protein, µ1, is 708aas long with a hydrophobic N-terminus, the 41aa (5000 Da) pore-forming N-terminally myristoylated peptide is derived from the N-terminus of µ1. The pore formed by µ1N is ~4-6nm in diameter and allows passage of the double stranded RNA genome of reovirus (present in ~20 short pieces) into the cell cytoplasm. µ1 is a wound trimer with ~200 copies/virion. µ1N and myristoylation are required for pore formation and membrane penetration. The pore forms first, and viral particle association follows. Thus, the oligomeric µ1N may be the docking site for the viral particle (M. Nibert, personal communication).
Noneveloped animal viruses must disrupt or perforate a cell membrane during entry. Work with reoviruses have shown formation of size-slective pores in RBC membranes in concert with structural changes in capsid protein mu1. Ivanovic et al. (2008) demonstrated that mu1 fragments released from reovirus particles are sufficient for pore formation. Both myristoylated N-terminal fragment mu1N and C-terminal fragment φ are released from particles. Both also associate with RBC membranes and contribute to pore formation in the absence of particles, but mu1N has the primary and sufficient role. Particles with a mutant form of mu1, unable to release mu1N or form pores, lack the ability to associate with membranes. They are, however, recruited by pores preformed with peptides released from wild-type particles or with synthetic mu1N. These facts suggest that docking to membrane pores by virus particles may be a next step in membrane penetration after pore formation by released peptides.
The transport reaction catalyzed by Mu-1 is:
ions and small molecules (out) ions and small molecules (in).
Core protein Mu-1 (42aas; 1TMS) (Agosto et al., 2006). The reovirus myristoylated µ1N pore forming peptide derived from the N-terminus of the µ1 viral capsid protein (708aas). Permeability order: Cs+ > Rb+ > K+ > Na+ > Li+ (crystal structures are available for chains A-U).
Mu-1 of mammalian reovirus (P12397)