Reovirus Virion-Like Particles Obtained by Recoating Infectious Subvirion Particles with Baculovirus-Expressed ς3 Protein: an Approach for Analyzing ς3 Functions during Virus Entry

ABSTRACT Structure-function studies with mammalian reoviruses have been limited by the lack of a reverse-genetic system for engineering mutations into the viral genome. To circumvent this limitation in a partial way for the major outer-capsid protein ς3, we obtained in vitro assembly of large numbers of virion-like particles by binding baculovirus-expressed ς3 protein to infectious subvirion particles (ISVPs) that lack ς3. A level of ς3 binding approaching 100% of that in native virions was routinely achieved. The ς3 coat in these recoated ISVPs (rcISVPs) appeared very similar to that in virions by electron microscopy and three-dimensional image reconstruction. rcISVPs retained full infectivity in murine L cells, allowing their use to study ς3 functions in virus entry. Upon infection, rcISVPs behaved identically to virions in showing an extended lag phase prior to exponential growth and in being inhibited from entering cells by either the weak base NH4Cl or the cysteine proteinase inhibitor E-64. rcISVPs also mimicked virions in being incapable of in vitro activation to mediate lysis of erythrocytes and transcription of the viral mRNAs. Last, rcISVPs behaved like virions in showing minor loss of infectivity at 52°C. Since rcISVPs contain virion-like levels of ς3 but contain outer-capsid protein μ1/μ1C mostly cleaved at the δ-φ junction as in ISVPs, the fact that rcISVPs behaved like virions (and not ISVPs) in all of the assays that we performed suggests that ς3, and not the δ-φ cleavage of μ1/μ1C, determines the observed differences in behavior between virions and ISVPs. To demonstrate the applicability of rcISVPs for genetic studies of protein functions in reovirus entry (an approach that we call recoating genetics), we used chimeric ς3 proteins to localize the primary determinants of a strain-dependent difference in ς3 cleavage rate to a carboxy-terminal region of the ISVP-bound protein.

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Putative Autocleavage of Outer Capsid Protein μ1, Allowing Release of Myristoylated Peptide μ1N during Particle Uncoating, Is Critical for Cell Entry by Reovirus

Journal of Virology ◽

10.1128/jvi.78.16.8732-8745.2004 ◽

2004 ◽

Vol 78 (16) ◽

pp. 8732-8745 ◽

Cited By ~ 96

Author(s):

Amy L. Odegard ◽

Kartik Chandran ◽

Xing Zhang ◽

John S. L. Parker ◽

Timothy S. Baker ◽

...

Keyword(s):

Capsid Protein ◽

Structural Changes ◽

General Mechanism ◽

Membrane Penetration ◽

Outer Capsid Protein ◽

Core Proteins ◽

Peptide Release ◽

Outer Capsid

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.

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Structural Protein VP2 of African Horse Sickness Virus Is Not Essential for Virus Replication In Vitro

Journal of Virology ◽

10.1128/jvi.01328-16 ◽

2016 ◽

Vol 91 (4) ◽

Cited By ~ 5

Author(s):

René G. P. van Gennip ◽

Sandra G. P. van de Water ◽

Christiaan A. Potgieter ◽

Piet A. van Rijn

Keyword(s):

Capsid Protein ◽

Virus Replication ◽

Hemorrhagic Disease ◽

African Horse Sickness ◽

Structural Protein ◽

Outer Capsid Protein ◽

African Horse Sickness Virus ◽

Content Type ◽

Outer Capsid

ABSTRACT The Reoviridae family consists of nonenveloped multilayered viruses with a double-stranded RNA genome consisting of 9 to 12 genome segments. The Orbivirus genus of the Reoviridae family contains African horse sickness virus (AHSV), bluetongue virus, and epizootic hemorrhagic disease virus, which cause notifiable diseases and are spread by biting Culicoides species. Here, we used reverse genetics for AHSV to study the role of outer capsid protein VP2, encoded by genome segment 2 (Seg-2). Expansion of a previously found deletion in Seg-2 indicates that structural protein VP2 of AHSV is not essential for virus replication in vitro. In addition, in-frame replacement of RNA sequences in Seg-2 by that of green fluorescence protein (GFP) resulted in AHSV expressing GFP, which further confirmed that VP2 is not essential for virus replication. In contrast to virus replication without VP2 expression in mammalian cells, virus replication in insect cells was strongly reduced, and virus release from insect cells was completely abolished. Further, the other outer capsid protein, VP5, was not copurified with virions for virus mutants without VP2 expression. AHSV without VP5 expression, however, could not be recovered, indicating that outer capsid protein VP5 is essential for virus replication in vitro. Our results demonstrate for the first time that a structural viral protein is not essential for orbivirus replication in vitro, which opens new possibilities for research on other members of the Reoviridae family. IMPORTANCE Members of the Reoviridae family cause major health problems worldwide, ranging from lethal diarrhea caused by rotavirus in humans to economic losses in livestock production caused by different orbiviruses. The Orbivirus genus contains many virus species, of which bluetongue virus, epizootic hemorrhagic disease virus, and African horse sickness virus (AHSV) cause notifiable diseases according to the World Organization of Animal Health. Recently, it has been shown that nonstructural proteins NS3/NS3a and NS4 are not essential for virus replication in vitro, whereas it is generally assumed that structural proteins VP1 to -7 of these nonenveloped, architecturally complex virus particles are essential. Here we demonstrate for the first time that structural protein VP2 of AHSV is not essential for virus replication in vitro. Our findings are very important for virologists working in the field of nonenveloped viruses, in particular reoviruses.

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Thermolabilizing Pseudoreversions in Reovirus Outer-Capsid Protein μ1 Rescue the Entry Defect Conferred by a Thermostabilizing Mutation

Journal of Virology ◽

10.1128/jvi.02720-06 ◽

2007 ◽

Vol 81 (14) ◽

pp. 7400-7409 ◽

Cited By ~ 14

Author(s):

Melina A. Agosto ◽

Jason K. Middleton ◽

Elaine C. Freimont ◽

John Yin ◽

Max L. Nibert

Keyword(s):

Capsid Protein ◽

General Mechanism ◽

Compensatory Mutation ◽

Outer Capsid Protein ◽

Viral Protein Synthesis ◽

Animal Virus ◽

Isolation And Characterization ◽

Outer Capsid

ABSTRACT Heat-resistant mutants selected from infectious subvirion particles of mammalian reoviruses have determinative mutations in the major outer-capsid protein μ1. Here we report the isolation and characterization of intragenic pseudoreversions of one such thermostabilizing mutation. From a plaque that had survived heat selection, a number of viruses with one shared mutation but different second-site mutations were isolated. The effect of the shared mutation alone or in combination with second-site mutations was examined using recoating genetics. The shared mutation, D371A, was found to confer (i) substantial thermostability, (ii) an infectivity defect that followed attachment but preceded viral protein synthesis, and (iii) resistance to μ1 rearrangement in vitro, with an associated failure to lyse red blood cells. Three different second-site mutations were individually tested in combination with D371A and found to wholly or partially revert these phenotypes. Furthermore, when tested alone in recoated particles, each of these three second-site mutations conferred demonstrable thermolability. This and other evidence suggest that pseudoreversion of μ1-based thermostabilization can occur by a general mechanism of μ1-based thermolabilization, not requiring a specific compensatory mutation. The thermostabilizing mutation D371A as well as 9 of the 10 identified second-site mutations are located near contact regions between μ1 trimers in the reovirus outer capsid. The availability of both thermostabilizing and thermolabilizing mutations in μ1 should aid in defining the conformational rearrangements and mechanisms involved in membrane penetration during cell entry by this structurally complex nonenveloped animal virus.

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The VP7 Outer Capsid Protein of Rotavirus Induces Polyclonal B-Cell Activation

Journal of Virology ◽

10.1128/jvi.78.13.6974-6981.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 6974-6981 ◽

Cited By ~ 36

Author(s):

Sarah E. Blutt ◽

Sue E. Crawford ◽

Kelly L. Warfield ◽

Dorothy E. Lewis ◽

Mary K. Estes ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Capsid Protein ◽

Cell Activation ◽

B Cell Activation ◽

Outer Capsid Protein ◽

Rotavirus Infection ◽

Virus Like Particles ◽

Outer Capsid

ABSTRACT The early response to a homologous rotavirus infection in mice includes a T-cell-independent increase in the number of activated B lymphocytes in the Peyer's patches. The mechanism of this activation has not been previously determined. Since rotavirus has a repetitively arranged triple-layered capsid and repetitively arranged antigens can induce activation of B cells, one or more of the capsid proteins could be responsible for the initial activation of B cells during infection. To address this question, we assessed the ability of rotavirus and virus-like particles to induce B-cell activation in vivo and in vitro. Using infectious rotavirus, inactivated rotavirus, noninfectious but replication-competent virus, and virus-like particles, we determined that neither infectivity nor RNA was necessary for B-cell activation but the presence of the rotavirus outer capsid protein, VP7, was sufficient for murine B-cell activation. Preincubation of the virus with neutralizing VP7 antibodies inhibited B-cell activation. Polymyxin B treatment and boiling of the virus preparation were performed, which ruled out possible lipopolysaccharide contamination as the source of activation and confirmed that the structural conformation of VP7 is important for B-cell activation. These findings indicate that the structure and conformation of the outer capsid protein, VP7, initiate intestinal B-cell activation during rotavirus infection.

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Development and efficacy of a grass carp reovirus (GCRV) outer capsid protein VP7 subunit vaccine

JOURNAL OF HUNAN AGRICULTURAL UNIVERSITY ◽

10.3724/sp.j.1238.2011.00659 ◽

2012 ◽

Vol 37 (6) ◽

pp. 659-664 ◽

Cited By ~ 1

Author(s):

Shi-ying XU ◽

Jing-hui LI ◽

Yong ZOU ◽

Lin LIU ◽

Cheng-liang GONG ◽

...

Keyword(s):

Capsid Protein ◽

Grass Carp ◽

Subunit Vaccine ◽

Outer Capsid Protein ◽

Grass Carp Reovirus ◽

Outer Capsid

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Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

Journal of Virology ◽

10.1128/jvi.00847-11 ◽

2011 ◽

Vol 85 (16) ◽

pp. 8141-8148 ◽

Cited By ~ 39

Author(s):

A. Fokine ◽

M. Z. Islam ◽

Z. Zhang ◽

V. D. Bowman ◽

V. B. Rao ◽

...

Keyword(s):

Capsid Protein ◽

Outer Capsid Protein ◽

Outer Capsid

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Cryo-EM structure of the bacteriophage T4 isometric head at 3.3-Å resolution and its relevance to the assembly of icosahedral viruses

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1708483114 ◽

2017 ◽

Vol 114 (39) ◽

pp. E8184-E8193 ◽

Cited By ~ 34

Author(s):

Zhenguo Chen ◽

Lei Sun ◽

Zhihong Zhang ◽

Andrei Fokine ◽

Victor Padilla-Sanchez ◽

...

Keyword(s):

Capsid Protein ◽

Major Capsid Protein ◽

Bacteriophage T4 ◽

Hexagonal Lattice ◽

Scaffolding Proteins ◽

Outer Capsid Protein ◽

Head Assembly ◽

Icosahedral Viruses ◽

End Caps ◽

Outer Capsid

The 3.3-Å cryo-EM structure of the 860-Å-diameter isometric mutant bacteriophage T4 capsid has been determined. WT T4 has a prolate capsid characterized by triangulation numbers (T numbers) Tend= 13 for end caps and Tmid= 20 for midsection. A mutation in the major capsid protein, gp23, produced T=13 icosahedral capsids. The capsid is stabilized by 660 copies of the outer capsid protein, Soc, which clamp adjacent gp23 hexamers. The occupancies of Soc molecules are proportional to the size of the angle between the planes of adjacent hexameric capsomers. The angle between adjacent hexameric capsomers is greatest around the fivefold vertices, where there is the largest deviation from a planar hexagonal array. Thus, the Soc molecules reinforce the structure where there is the greatest strain in the gp23 hexagonal lattice. Mutations that change the angles between adjacent capsomers affect the positions of the pentameric vertices, resulting in different triangulation numbers in bacteriophage T4. The analysis of the T4 mutant head assembly gives guidance to how other icosahedral viruses reproducibly assemble into capsids with a predetermined T number, although the influence of scaffolding proteins is also important.

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Infection immunity of piglets to either VP3 or VP7 outer capsid protein confers resistance to challenge with a virulent rotavirus bearing the corresponding antigen.

Journal of Virology ◽

10.1128/jvi.62.3.744-748.1988 ◽

1988 ◽

Vol 62 (3) ◽

pp. 744-748 ◽

Cited By ~ 73

Author(s):

Y Hoshino ◽

L J Saif ◽

M M Sereno ◽

R M Chanock ◽

A Z Kapikian

Keyword(s):

Capsid Protein ◽

Outer Capsid Protein ◽

Outer Capsid

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Cell Entry-Independent Role for the Reovirus μ1 Protein in Regulating Necroptosis and the Accumulation of Viral Gene Products

Journal of Virology ◽

10.1128/jvi.00199-19 ◽

2019 ◽

Vol 93 (11) ◽

Cited By ~ 5

Author(s):

Katherine E. Roebke ◽

Pranav Danthi

Keyword(s):

Cell Death ◽

Capsid Protein ◽

Apoptotic Cell ◽

Guanidine Hydrochloride ◽

Viral Gene ◽

Gene Products ◽

Minus Strand ◽

Outer Capsid Protein ◽

Infected Cells ◽

Outer Capsid

ABSTRACTThe reovirus outer capsid protein μ1 regulates cell death in infected cells. To distinguish between the roles of incoming, capsid-associated, and newly synthesized μ1, we used small interfering RNA (siRNA)-mediated knockdown. Loss of newly synthesized μ1 protein does not affect apoptotic cell death in HeLa cells but enhances necroptosis in L929 cells. Knockdown of μ1 also affects aspects of viral replication. We found that, while μ1 knockdown results in diminished release of infectious viral progeny from infected cells, viral minus-strand RNA, plus-strand RNA, and proteins that are not targeted by the μ1 siRNA accumulate to a greater extent than in control siRNA-treated cells. Furthermore, we observed a decrease in sensitivity of these viral products to inhibition by guanidine hydrochloride (GuHCl) (which targets minus-strand synthesis to produce double-stranded RNA) when μ1 is knocked down. Following μ1 knockdown, cell death is also less sensitive to treatment with GuHCl. Our studies suggest that the absence of μ1 allows enhanced transcriptional activity of newly synthesized cores and the consequent accumulation of viral gene products. We speculate that enhanced accumulation and detection of these gene products due to μ1 knockdown potentiates receptor-interacting protein 3 (RIP3)-dependent cell death.IMPORTANCEWe used mammalian reovirus as a model to study how virus infections result in cell death. Here, we sought to determine how viral factors regulate cell death. Our work highlights a previously unknown role for the reovirus outer capsid protein μ1 in limiting the induction of a necrotic form of cell death called necroptosis. Induction of cell death by necroptosis requires the detection of viral gene products late in infection; μ1 limits cell death by this mechanism because it prevents excessive accumulation of viral gene products that trigger cell death.

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