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

2016 ◽  
Vol 91 (4) ◽  
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.

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

2004 ◽  
Vol 78 (16) ◽  
pp. 8732-8745 ◽  
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.

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

2007 ◽  
Vol 81 (14) ◽  
pp. 7400-7409 ◽  
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.

MODELACIÓN POR COMPARACIÓN Y ACOPLAMIENTO MULTIMÉRICO DE LA PROTEÍNA EXTERIOR PEQUEÑA DE LA CÁPSIDE DEL BACTERIÓFAGO IME08

2020 ◽  
Vol 5 ◽  
Author(s):  
Maicol Ospina-Bedolla
Keyword(s):  
Capsid Protein ◽  
Protein Docking ◽  
Suitable Model ◽  
Structural Protein ◽  
Ramachandran Plot ◽  
Outer Capsid Protein ◽  
Structure Reliability ◽  
Model Platform ◽  
Outer Capsid

The small outer capsid protein plays a stabilizing role in the viral assembly, adhering to the<br />capsid during the later stages of maturation. This protein acts as glue among adjacent<br />capsomers, protecting the virus against extreme changes. The small outer capsid protein of the<br />bacteriophage IME08 was modelled using structural protein homology. A trimeric protein<br />docking was developed with the best-scored model and important sites of the molecules<br />interfaces were identified. It was used the Swiss Model platform for developing the protein<br />structure. Reliability was assessed by the QMEAN, Verify3D and ERRAT indices. The quality of<br />the whole model was verified by Ramachandran plot and the trimerization model was<br />performed on the platform ClusPro 2.0 Protein-Protein Docking. The structure obtained has a<br />reliability estimator QMEANscore4 of 0.769, rating it as a suitable model. The Z-Score QMEAN<br />value was 0.133, showing that the obtained model is not different from the experimental<br />structures stored in PDB database. The estimators and the Ramachandran plot evaluated<br />positively the model. Finally we identified a loop between two secondary structures as an<br />important site of the interaction of small outer capsid proteins, indicating that from residues 35<br />to 41 are relevant in the trimerization process.

scholarly journals VP2 Exchange and NS3/NS3a Deletion in African Horse Sickness Virus (AHSV) in Development of Disabled Infectious Single Animal Vaccine Candidates for AHSV

2015 ◽  
Vol 89 (17) ◽  
pp. 8764-8772 ◽  
Author(s):  
Sandra G. P. van de Water ◽  
René G. P. van Gennip ◽  
Christiaan A. Potgieter ◽  
Isabel M. Wright ◽  
Piet A. van Rijn
Keyword(s):  
Mammalian Cells ◽  
Reverse Genetics ◽  
African Horse Sickness ◽  
African Horse Sickness Virus ◽  
Vaccine Platform ◽  
Biting Midges ◽  
Content Type ◽  
Vp2 Protein ◽  
Single Animal

ABSTRACTAfrican horse sickness virus (AHSV) is a virus species in the genusOrbivirusof the familyReoviridae. There are nine serotypes of AHSV showing different levels of cross neutralization. AHSV is transmitted by species ofCulicoidesbiting midges and causes African horse sickness (AHS) in equids, with a mortality rate of up to 95% in naive horses. AHS has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates appear to be competent vectors for the related bluetongue virus (BTV). To control AHS, live-attenuated vaccines (LAVs) are used in Africa. We used reverse genetics to generate “synthetic” reassortants of AHSV for all nine serotypes by exchange of genome segment 2 (Seg-2). This segment encodes VP2, which is the serotype-determining protein and the dominant target for neutralizing antibodies. Single Seg-2 AHSV reassortants showed similar cytopathogenic effects in mammalian cells but displayed different growth kinetics. Reverse genetics for AHSV was also used to study Seg-10 expressing NS3/NS3a proteins. We demonstrated that NS3/NS3a proteins are not essential for AHSV replicationin vitro. NS3/NS3a of AHSV is, however, involved in the cytopathogenic effect in mammalian cells and is very important for virus release from cultured insect cells in particular. Similar to the concept of the bluetongue disabled infectious single animal (BT DISA) vaccine platform, an AHS DISA vaccine platform lacking NS3/NS3a expression was developed. Using exchange of genome segment 2 encoding VP2 protein (Seg-2[VP2]), we will be able to develop AHS DISA vaccine candidates for all current AHSV serotypes.IMPORTANCEAfrican horse sickness virus is transmitted by species ofCulicoidesbiting midges and causes African horse sickness in equids, with a mortality rate of up to 95% in naive horses. African horse sickness has become a serious threat for countries outside Africa, since endemicCulicoidesspecies in moderate climates are supposed to be competent vectors. By using reverse genetics, viruses of all nine serotypes were constructed by the exchange of Seg-2 expressing the serotype-determining VP2 protein. Furthermore, we demonstrated that the nonstructural protein NS3/NS3a is not essential for virus replicationin vitro. However, the potential spread of the virus by biting midges is supposed to be blocked, since thein vitrorelease of the virus was strongly reduced due to this deletion. VP2 exchange and NS3/NS3a deletion in African horse sickness virus were combined in the concept of a disabled infectious single animal vaccine for all nine serotypes.

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

1999 ◽  
Vol 73 (4) ◽  
pp. 2963-2973 ◽  
Author(s):  
Judit Jané-Valbuena ◽  
Max L. Nibert ◽  
Stephan M. Spencer ◽  
Stephen B. Walker ◽  
Timothy S. Baker ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Virus Entry ◽  
Cysteine Proteinase ◽  
Reverse Genetic System ◽  
Lag Phase ◽  
Cysteine Proteinase Inhibitor ◽  
Outer Capsid Protein ◽  
Viral Mrnas ◽  
Outer Capsid

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.

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

2004 ◽  
Vol 78 (13) ◽  
pp. 6974-6981 ◽  
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.

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

2012 ◽  
Vol 37 (6) ◽  
pp. 659-664 ◽  
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

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

2011 ◽  
Vol 85 (16) ◽  
pp. 8141-8148 ◽  
Author(s):  
A. Fokine ◽  
M. Z. Islam ◽  
Z. Zhang ◽  
V. D. Bowman ◽  
V. B. Rao ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Outer Capsid Protein ◽  
Outer Capsid
Sign in / Sign up

Export Citation Format

Share Document