Mammalian reovirus, a nonfusogenic nonenveloped virus, forms size-selective pores in a model membrane Melina A. Agosto†‡, Tijana Ivanovic†§, and Max L. Nibert†‡§¶ †Department of Microbiology and Molecular Genetics, ‡Biological and Biomedical Sciences Training Program, and §Training Program in Virology, Harvard Medical School, Boston, MA 02115 Edited by John J. Mekalanos, Harvard Medical School, Boston, MA, and approved September 11, 2006 (received for review July 11, 2006) During cell entry, reovirus particles with a diameter of 70–80 nm metastable and can be promoted to undergo conformational must penetrate the cellular membrane to access the cytoplasm. The rearrangements leading to the ISVP* particle (19). mechanism of penetration, without benefit of membrane fusion, is ISVP3ISVP* conversion is correlated with membrane- not well characterized for any such nonenveloped animal virus. perforation activities both in vivo and in vitro (19, 20). Other Lysis of RBCs is an in vitro assay for the membrane perforation hallmarks of this conversion include ␮1 rearrangement to a activity of reovirus; however, the mechanism of lysis has been protease-sensitive conformer and elution of the adhesion protein unknown. In this report, osmotic-protection experiments using ␴1 (18, 19, 21, 22). In addition, a myristoylated N-terminal PEGs of different sizes revealed that reovirus-induced lysis of RBCs fragment of ␮1, ␮1N, is generated by autocleavage and released occurs osmotically, after formation of small size-selective lesions or from particles (23–26). Several lines of evidence suggest that ␮1 ‘‘pores.’’ Consistent results were obtained by monitoring leakage directly engages in membrane perforation (12, 19, 20, 24, 25, of fluorophore-tagged dextrans from the interior of resealed RBC 27–31), but little is yet known about this mechanism. ghosts. Gradient fractionations showed that whole virus particles, The membrane-perforation activity of ISVP*s can be assayed as well as the myristoylated fragment ␮1N that is released from in vitro by lysis of RBCs (12, 19, 32, 33). In this study we particles, are recruited to RBC membranes in association with pore demonstrated that hemolysis occurs osmotically, after formation formation. We propose that formation of small pores is a discrete, of small size-selective lesions, or ‘‘pores,’’ in the RBC membrane. intermediate step in the reovirus membrane-penetration pathway, Both ISVP* particles and the myristoylated fragment ␮1N were which may be shared by other nonenveloped animal viruses. recruited to the membranes, implicating one or both of them in pore formation. We propose that the capability of reovirus to cell entry ͉ hemolysis ͉ membrane penetration ͉ reoviridae form small pores in target membranes represents a discrete, intermediate step in the membrane-penetration pathway during rossing the cellular membrane is a key step in the infectious cell entry. Clife cycle of all viruses. Although the understanding of enveloped-virus entry via membrane fusion has seen many Results advances (1–3), the entry mechanisms of nonenveloped animal Reovirus-Induced Hemolysis Is Inhibited by Osmotic Protection. PEGs viruses remain elusive. Mammalian orthoreovirus (reovirus) is a inhibit hemolysis but not ␮1 conformational change. We previously large nonenveloped virus with a 10-segmented dsRNA genome. thought that reovirus-mediated hemolysis (12, 19, 32, 33) re- During entry, it must penetrate the cellular membrane to deliver flects gross disruption of the RBC membrane. An alternative icosahedral particles of 70–80 nm in diameter to the cytoplasm, hypothesis is that viral components form pores, leading to an where particle-encased polymerases synthesize the viral mRNAs influx of water down the osmotic gradient and consequent cell (4–6). The manner in which this large ‘‘payload’’ (7) is delivered swelling and lysis. To test this hypothesis, hemolysis reactions across the membrane, without benefit of membrane fusion, were performed in the presence of PEGs (Fig. 1), which serve remains largely unknown. as osmotic protectants and have been used in numerous systems Enveloped viruses mediate membrane fusion by virally en- to demonstrate pore formation in RBC membranes (34–36). coded fusion proteins, which undergo conformational rearrange- Purified virions were first primed for hemolysis (19, 20) by ments that are often promoted by low pH or receptor binding protease digestion to yield ISVPs (18), then mixed with bovine and lead to exposure of membrane-interacting sequences. After RBCs and hemolysis buffer, with or without 30 mM PEG. insertion into the target membrane, further rearrangements Reactions were incubated at 37°C to promote ISVP3ISVP* bring the lipid bilayers into proximity, favoring fusion. The conversion and the associated ␮1 rearrangement and hemolysis paradigm of promoted conformational rearrangements is re- (12, 19). After chilling on ice and centrifugation to remove flected in the mechanisms of nonenveloped viruses as well. For unlysed RBCs, hemoglobin release into the supernatants was example, the poliovirus capsid is promoted by receptor binding measured by absorbance at 405 nm (12, 33). PEG with a mean to undergo conformational rearrangements exposing the N molecular weight of 10,000 (10K-PEG) was found to inhibit terminus of capsid protein VP1 and the myristoylated autocleav- hemolysis completely (Fig. 1A), even though ␮1 rearrangement age peptide VP4; these newly exposed sequences are then thought to form a transmembrane pore, through which the genomic RNA may be extruded into the cytoplasm (8). Similarly, Author contributions: M.A.A., T.I., and M.L.N. designed research; M.A.A. and T.I. performed adenovirus capsids can be promoted by low pH to undergo research; M.A.A., T.I., and M.L.N. analyzed data; and M.A.A., T.I., and M.L.N. wrote the rearrangements that expose the membranolytic protein VI, paper. which is thought to mediate endosome rupture (9). The authors declare no conflict of interest. During reovirus infection, the outermost capsid protein ␴3is This article is a PNAS direct submission. digested by intestinal or endosomal proteases (10–15), leaving Abbreviations: ISVP, infectious subvirion particle; RG, resealed RBC ghost; CF, carboxyfluo- capsid protein ␮1 exposed on the particle surface (16). The rescein; VB, virion buffer; CHT, chymotrypsin. resulting infectious subvirion particles (ISVPs) can also be ¶To whom correspondence should be addressed. E-mail: [email protected]. generated in vitro by protease digestion (17, 18). ISVPs are © 2006 by The National Academy of Sciences of the USA 16496–16501 ͉ PNAS ͉ October 31, 2006 ͉ vol. 103 ͉ no. 44 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0605835103 Downloaded by guest on September 24, 2021 500 A C A CF 3K +I, 37°C 400 +I, 0°C 100 1st sup 3rd sup 300 ° 80 -I, 37 C 75 200 -I, 0°C 60 # of RGs 50 100 NO PEG 40 0 25 PEG 10000 1 3 1 3 20 10 10 10 10 0 % Hemolysis 500 % Hemolysis 0 10K 40K 70K -25 ISVP: +++ + ++ 400 010203040 37°C: +++ + + + Time (min) PEG: none 8000 10000 300 200 # of RGs B Time (min) D 100 2.5 5 10 20 30 40 0 λ's 100 101 103 101 103 101 103 µ1 75 fluoresc. (au) fluoresc. (au) fluoresc. (au) σ1 50 100 u 25 B NO PEG 0 % Hemolysis 75 λ's -25 µ1 no 46810 σ1 PEG PEG avg. MW 50 (x1000) % Leakage u 25 PEG 10000 0 Fig. 1. Inhibition of hemolysis by osmotic protection. (A) Hemolysis time CF 3K 10K 40K 70K courses were performed with or without 30 mM 10K-PEG. (B) Aliquots of supernatants from each time point in A were tested for protease-sensitivity of Fig. 2. Leakage of fluorescent dextrans from RGs. (A) Hemolysis-like reac- ␮1. Arrowhead, trypsin-stable ␮1 fragment. (C) Hemolysis reactions contain- tions with RGs containing CF, or fluorescein-labeled dextran of the indicated ing no PEG, 8K-PEG, or 10K-PEG were washed and resuspended in buffer with size, were analyzed by flow cytometry. (B) Percentage of leakage was as Ϯ no PEG. (D) Hemolysis reactions were performed with PEGs of a range of sizes. described in Materials and Methods. Means SD of three experiments are Means Ϯ SD of three experiments are shown. shown. as assayed by trypsin sensitivity (12, 19) was not affected (Fig. Reovirus Induces Size-Selective Leakage of Dextrans from Resealed 1B). Essentially identical results were obtained with 8K-PEG Ghosts. To assay for pores by a complementary approach, (data not shown). These results indicate that 8K- and 10K-PEGs leakage of fluorescently labeled dextrans from resealed RBC do not interfere with ISVP3ISVP* conversion but inhibit ghosts (RGs) was examined by flow cytometry (Figs. 2 and 3). hemolysis at a later step. Because RGs do not contain enough solutes to create an osmotic Washing out PEG restores hemolysis. When hemolysis reactions gradient, and therefore are not expected to lyse osmotically, we MICROBIOLOGY performed in the presence of 8K- or 10K-PEG were washed and hypothesized that virus-induced pores would allow small dex- resuspended in cold hemolysis buffer without PEG, lysis oc- trans to leak out while large ones would remain trapped inside. curred without further incubation (Fig. 1C). The background Accordingly, RGs were loaded with carboxyfluorescein (CF) or levels of lysis in control reactions incubated on ice, or without fluorescein-conjugated dextran with mean molecular weights of virus particles, reflected nonspecific damage by PEG to the RBC 3,000, 10,000, 40,000, and 70,000. membranes; however, nearly complete hemolysis was restored Reovirus induces size-selective leakage of dextrans from singly labeled only in reactions containing virus particles that had been incu- RGs. RGs were mixed with ISVPs in hemolysis-like conditions bated in conditions that promote ISVP3ISVP* conversion. and incubated at 37°C (Fig. 2A). Control reactions were included These findings suggest that ISVP3ISVP* conversion and mem- to assess the levels of initial loading and nonspecific leakage.