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1.
Viruses ; 14(10)2022 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-36298748

RESUMO

African horse sickness is a deadly and highly infectious disease of equids, caused by African horse sickness virus (AHSV). AHSV is one of the most economically important members of the Orbivirus genus. AHSV is transmitted by the biting midge, Culicoides, and therefore replicates in both insect and mammalian cell types. Structural protein VP7 is a highly conserved major core protein of orbiviruses. Unlike any other orbivirus VP7, AHSV VP7 is highly insoluble and forms flat hexagonal crystalline particles of unknown function in AHSV-infected cells and when expressed in mammalian or insect cells. To examine the role of AHSV VP7 in virus replication, a plasmid-based reverse genetics system was used to generate a recombinant AHSV that does not form crystalline particles. We characterised the role of VP7 crystalline particle formation in AHSV replication in vitro and found that soluble VP7 interacted with viral proteins VP2 and NS2 similarly to wild-type VP7 during infection. Interestingly, soluble VP7 was found to form uncharacteristic tubule-like structures in infected cells which were confirmed to be as a result of unique VP7-NS1 colocalisation. Furthermore, it was found that VP7 crystalline particles play a role in AHSV release and yield. This work provides insight into the role of VP7 aggregation in AHSV cellular pathogenesis and contributes toward the understanding of the possible effects of viral protein aggregation in other human virus-borne diseases.


Assuntos
Vírus da Doença Equina Africana , Ceratopogonidae , Animais , Humanos , Vírus da Doença Equina Africana/genética , Agregados Proteicos , Replicação Viral , Proteínas do Core Viral/metabolismo , Ceratopogonidae/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Mamíferos
2.
Viruses ; 14(8)2022 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-35893692

RESUMO

A unique characteristic of the African horse sickness virus (AHSV) major core protein VP7 is that it is highly insoluble, and spontaneously forms crystalline particles in AHSV-infected cells and when expressed in vitro. The aggregation of AHSV VP7 into these crystals presents many problems in AHSV vaccine development, and it is unclear whether VP7 aggregation affects AHSV assembly or contributes to AHSV pathogenesis. Here, we set out to abolish VP7 self-assembly by targeting candidate amino acid regions on the surface of the VP7 trimer via site-directed mutagenesis. It was found that the substitution of seven amino acids resulted in the complete disruption of AHSV VP7 self-assembly, which abolished the formation of VP7 crystalline particles and converted VP7 to a fully soluble protein still capable of interacting with VP3 to form core-like particles. This work provides further insight into the formation of AHSV VP7 crystalline particles and the successful development of AHSV vaccines. It also paves the way for future research by drawing comparisons with similar viral phenomena observed in human virology.


Assuntos
Vírus da Doença Equina Africana , Proteínas do Core Viral , Vírus da Doença Equina Africana/genética , Animais , Antígenos Virais , Proteínas do Core Viral/metabolismo
3.
Virus Res ; 232: 80-95, 2017 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-28167267

RESUMO

VP7 is the major core protein of orbiviruses and is essential for virion assembly. African horse sickness virus (AHSV) VP7 self-assembles into highly insoluble crystalline particles - an attribute that may be related to the role of AHSV VP7 in virus assembly but also prevents crystallization. Given that this inherent insolubility is unique to AHSV VP7, we use amino acid sequence conservation analysis between AHSV VP7 and other orbiviruses to identify putative key residues that drive AHSV VP7 self-assembly. A homology model of the AHSV VP7 trimer was generated to analyze surface properties of the trimer and to identify surface residues as candidates for the AHSV VP7 trimer-trimer interactions that drive AHSV VP7 self-assembly. Nine regions were identified as candidate residues for future site-directed mutagenesis experiments that will likely result in a soluble AHSV VP7 protein. Additionally, we identified putative residues that function in the intermolecular interactions within the AHSV VP7 trimer as well as several epitopes. Given the many previous efforts of solubilizing AHSV VP7, we propose a useful strategy that will yield a soluble AHSV VP7 that can be used to study AHSV assembly and increase yield of recombinant vaccine preparations.


Assuntos
Vírus da Doença Equina Africana/química , Anticorpos Monoclonais/química , Antígenos Virais/química , Vírus Bluetongue/química , Proteínas do Core Viral/química , Vírus da Doença Equina Africana/imunologia , Vírus da Doença Equina Africana/metabolismo , Sequência de Aminoácidos , Anticorpos Monoclonais/metabolismo , Antígenos Virais/imunologia , Antígenos Virais/metabolismo , Sítios de Ligação , Vírus Bluetongue/imunologia , Vírus Bluetongue/metabolismo , Mapeamento de Epitopos , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Alinhamento de Sequência , Homologia Estrutural de Proteína , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/metabolismo , Montagem de Vírus
4.
Virology ; 456-457: 279-91, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24889247

RESUMO

African horse sickness virus (AHSV) VP7 is the major core protein of the virion. Apart from its role in virus assembly, VP7 forms crystalline-like particles during infection and when expressed in insect cells. The aim of this study was to investigate the process of VP7 crystalline-like particle formation. The intracellular distribution of VP7 was characterized in different systems and the association of VP7 with virus factories during AHSV infection was investigated. It was shown that the majority of VP7 is sequestered into these particles, and is therefore not available for new virion assembly. This is likely to have a negative impact on virus assembly and yield. By using specific markers and inhibitors of host trafficking pathways, VP7 localization was shown to be independent of host trafficking mechanisms and evaded host defenses against aggregation. Studying the process of VP7 crystalline-like particle formation will help us further understand AHSV replication and assembly.


Assuntos
Vírus da Doença Equina Africana/fisiologia , Antígenos Virais/metabolismo , Proteínas do Core Viral/metabolismo , Montagem de Vírus , Replicação Viral , Animais , Transporte Proteico , Células Sf9 , Spodoptera
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