Nonenveloped animal viruses must disrupt or perforate a cell membrane during entry. Recent work with reovirus has shown formation of size-selective pores in RBC membranes in concert with structural changes in capsid protein l1. Here, we demonstrate that l1 fragments released from reovirus particles are sufficient for pore formation. Both myristoylated N-terminal fragment l1N and C-terminal fragment / are released from particles. Both also associate with RBC membranes and contribute to pore formation in the absence of particles, but l1N has the primary and sufficient role. Particles with a mutant form of l1, unable to release l1N 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 l1N. The results provide evidence that docking to membrane pores by virus particles may be a next step in membrane penetration after pore formation by released peptides.

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 not well characterized for any such nonenveloped animal virus. Lysis of RBCs is an in vitro assay for the membrane perforation activity of reovirus; however, the mechanism of lysis has been unknown. In this report, osmotic-protection experiments using PEGs of different sizes revealed that 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. Gradient fractionations showed that whole virus particles, as well as the myristoylated fragment 1N that is released from particles, are recruited to RBC membranes in association with pore formation. We propose that formation of small pores is a discrete, intermediate step in the reovirus membrane-penetration pathway, which may be shared by other nonenveloped animal viruses.cell entry ͉ hemolysis ͉ membrane penetration ͉ reoviridae

Rhodopsin recycling via the retromer, rather than degradation through lysosomes, can alleviate light-induced photoreceptor degeneration in Drosophila.

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