Putative autocleavage of outer capsid protein μ1, allowing release of myristoylated peptide μ1N during particle uncoating, is critical for cell entry by reovirus

Amy L. Odegard, Kartik Chandran, Xing Zhang, John S L Parker, Timothy S. Baker, Max L. Nibert

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

Original languageEnglish (US)
Pages (from-to)8732-8745
Number of pages14
JournalJournal of Virology
Volume78
Issue number16
DOIs
StatePublished - Aug 2004
Externally publishedYes

Fingerprint

Reoviridae
Capsid Proteins
coat proteins
Mammalian Orthoreovirus
peptides
Peptides
Membranes
Capsid
Virion
cells
Viral Core Proteins
Viruses
Picornaviridae
capsid
Asparagine
Amino Acid Substitution
DNA-Directed RNA Polymerases
Hemolysis
virion
Alanine

ASJC Scopus subject areas

  • Immunology

Cite this

Putative autocleavage of outer capsid protein μ1, allowing release of myristoylated peptide μ1N during particle uncoating, is critical for cell entry by reovirus. / Odegard, Amy L.; Chandran, Kartik; Zhang, Xing; Parker, John S L; Baker, Timothy S.; Nibert, Max L.

In: Journal of Virology, Vol. 78, No. 16, 08.2004, p. 8732-8745.

Research output: Contribution to journalArticle

Odegard, Amy L. ; Chandran, Kartik ; Zhang, Xing ; Parker, John S L ; Baker, Timothy S. ; Nibert, Max L. / Putative autocleavage of outer capsid protein μ1, allowing release of myristoylated peptide μ1N during particle uncoating, is critical for cell entry by reovirus. In: Journal of Virology. 2004 ; Vol. 78, No. 16. pp. 8732-8745.
@article{99ee8d403723432ca755652a9106949b,
title = "Putative autocleavage of outer capsid protein μ1, allowing release of myristoylated peptide μ1N during particle uncoating, is critical for cell entry by reovirus",
abstract = "Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.",
author = "Odegard, {Amy L.} and Kartik Chandran and Xing Zhang and Parker, {John S L} and Baker, {Timothy S.} and Nibert, {Max L.}",
year = "2004",
month = "8",
doi = "10.1128/JVI.78.16.8732-8745.2004",
language = "English (US)",
volume = "78",
pages = "8732--8745",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "16",

}

TY - JOUR

T1 - Putative autocleavage of outer capsid protein μ1, allowing release of myristoylated peptide μ1N during particle uncoating, is critical for cell entry by reovirus

AU - Odegard, Amy L.

AU - Chandran, Kartik

AU - Zhang, Xing

AU - Parker, John S L

AU - Baker, Timothy S.

AU - Nibert, Max L.

PY - 2004/8

Y1 - 2004/8

N2 - Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

AB - Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein μ1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments μ1N (4 kDa; myristoylated) and μ1C (72 kDa). In this study, we found that μ1 cleavage to yield μ1N and μ1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective μ1 (asparagine → alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including μ1 conformational change and σ1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin α-sarcin). In addition, after these particles were allowed to enter cells, the δ region of μ1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective μ1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of μ1N/μ1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal μ1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

UR - http://www.scopus.com/inward/record.url?scp=3543115472&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=3543115472&partnerID=8YFLogxK

U2 - 10.1128/JVI.78.16.8732-8745.2004

DO - 10.1128/JVI.78.16.8732-8745.2004

M3 - Article

VL - 78

SP - 8732

EP - 8745

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 16

ER -