Putative Autocleavage of Reovirus μ1 Protein in Concert with Outer-capsid Disassembly and Activation for Membrane Permeabilization

Nibert, Max L.; Odegard, Amy L.; Agosto, Melina A.; Chandran, Kartik; Schiff, Leslie A.

doi:10.1016/j.jmb.2004.10.026

Cited by 85 publications

(118 citation statements)

References 51 publications

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“…Myristoylated fragment 1N is also recruited to RG membranes. During ISVP3ISVP* conversion, 1 undergoes an autocatalytic cleavage, generating a 4-kDa N-terminally myristoylated peptide, 1N, that is released from particles (23,24). This cleavage appears to be required for hemolysis activity as well as for cell entry during infection (24).…”

Section: Isvp* Particles and Released Component 1n Are Recruited To Rbcmentioning

confidence: 99%

“…Other hallmarks of this conversion include 1 rearrangement to a protease-sensitive conformer and elution of the adhesion protein 1 (18,19,21,22). In addition, a myristoylated N-terminal fragment of 1, 1N, is generated by autocleavage and released from particles (23)(24)(25)(26). Several lines of evidence suggest that 1 directly engages in membrane perforation (12,19,20,24,25,(27)(28)(29)(30)(31), but little is yet known about this mechanism.…”

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confidence: 99%

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Mammalian reovirus, a nonfusogenic nonenveloped virus, forms size-selective pores in a model membrane

Agosto

Ivanovic

Nibert

2006

Proc. Natl. Acad. Sci. U.S.A.

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109

173

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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

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Section: Isvp* Particles and Released Component 1n Are Recruited To Rbcmentioning

confidence: 99%

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confidence: 99%

Mammalian reovirus, a nonfusogenic nonenveloped virus, forms size-selective pores in a model membrane

Agosto

Ivanovic

Nibert

2006

Proc. Natl. Acad. Sci. U.S.A.

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109

173

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“…Membrane penetration does not appear to require the production of , as cleavageresistant variants of 1 are still infectious (9,12). An autolytic N-terminal cleavage in 1 also occurs at some point in the virion3ISVP* transition, creating fragment 1N (residues 2 to 42 plus the N-terminal myristoyl moiety); its complement is known as 1C, and the large central fragment between the autolytic cleavage and the cleavage is known as ␦ (40,41). The cleaved 1N peptide is released from the particle during the ISVP3ISVP* transition (3,29,42).…”

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confidence: 99%

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

Zhang

Agosto

Ivanovic

et al. 2009

J Virol

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The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the 1 protein, each complexed with three copies of the protector protein 3. Conformational changes in 1 following the proteolytic removal of 3 lead to release of the myristoylated N-terminal cleavage fragment 1N and ultimately to membrane penetration. The 1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant 1 trimers and synthetic 1N peptides with both RBCs and liposomes. The 1 trimer mediates hemolysis and liposome disruption under conditions that promote the 1 conformational change, and mutations that inhibit 1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free 1 trimer. Autolytic cleavage to form 1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that 1N cleavage is not required when steric barriers are removed. Synthetic myristoylated 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 ␤-strand secondary structure, suggest a model in which 1N ␤-hairpins assemble in the membrane to form a ␤-barrel pore.

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“…Following formation of ISVPs, 1 is shed and the 1 cleavage fragments undergo conformational rearrangement, yielding the ISVP* (15,16). ISVP*s penetrate endosomes to deliver transcriptionally active viral cores into the cytoplasm (40,42). Although the precise mechanism of membrane penetration is not well understood, ISVP*s are thought to breach the endosomal membrane by a process that requires 1-mediated formation of small pores (1).…”

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confidence: 99%

NPXY Motifs in the β1 Integrin Cytoplasmic Tail Are Required for Functional Reovirus Entry

Maginnis

Mainou

Derdowski

et al. 2008

J Virol

112

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Reovirus cell entry is mediated by attachment to cell surface carbohydrate and junctional adhesion molecule A (JAM-A) and internalization by ␤1 integrin. The ␤1 integrin cytoplasmic tail contains two NPXY motifs, which function in recruitment of adaptor proteins and clathrin for endocytosis and serve as sorting signals for internalized cargo. As reovirus infection requires disassembly in the endocytic compartment, we investigated the role of the ␤1 integrin NPXY motifs in reovirus internalization. In comparison to wild-type cells (␤1؉/؉ cells), reovirus infectivity was significantly reduced in cells expressing mutant ␤1 integrin in which the NPXY motifs were altered to NPXF (␤1؉/؉Y783F/Y795F cells). However, reovirus displayed equivalent binding and internalization levels following adsorption to ␤1؉/؉ cells and ␤1؉/؉Y783F/Y795F cells, suggesting that the NPXY motifs are essential for transport of reovirus within the endocytic pathway. Reovirus entry into ␤1؉/؉ cells was blocked by chlorpromazine, an inhibitor of clathrin-mediated endocytosis, while entry into ␤1؉/؉ Y783F/Y795F cells was unaffected. Furthermore, virus was distributed to morphologically distinct endocytic organelles in ␤1؉/؉ and ␤1؉/؉Y783F/Y795F cells, providing further evidence that the ␤1 integrin NPXY motifs mediate sorting of reovirus in the endocytic pathway. Thus, NPXY motifs in the ␤1 integrin cytoplasmic tail are required for functional reovirus entry, which indicates a key role for these sequences in endocytosis of a pathogenic virus.

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Putative Autocleavage of Reovirus μ1 Protein in Concert with Outer-capsid Disassembly and Activation for Membrane Permeabilization

Cited by 85 publications

References 51 publications

Mammalian reovirus, a nonfusogenic nonenveloped virus, forms size-selective pores in a model membrane

Mammalian reovirus, a nonfusogenic nonenveloped virus, forms size-selective pores in a model membrane

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

NPXY Motifs in the β1 Integrin Cytoplasmic Tail Are Required for Functional Reovirus Entry

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