Structural Insight into African Horsesickness Virus Infection

Manole, Violeta; Laurinmäki, Pasi; Wyngaardt, Wouter Van; Potgieter, Christiaan; Wright, Isabel M.; Venter, Gert J.; Dijk, Alberdina A. van; Sewell, B. Trevor; Butcher, Sarah J.

doi:10.1128/jvi.00517-12

Cited by 44 publications

(55 citation statements)

References 61 publications

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“…Mutant AHSV7-tVP2 forms RNA-filled, spherical, intact virus particles containing a smaller VP2 protein as shown by cryo-electron microscopy analysis (48). In our exper- iments, the protein band of tVP2 of serotype 4 is very weak as observed by Western blot analysis (Fig.…”

Section: Discussionsupporting

confidence: 61%

“…Regarding structural proteins VP1 to -7 of orbiviruses, it has been assumed that these are all essential for virus replication. Surprisingly, a region in VP2 of AHSV7, probably located at the outside of the virus particle, is not essential for virus replication in vitro (48). We show here that corresponding regions in VP2 of other serotypes are also dispensable.…”

Section: Discussionmentioning

confidence: 61%

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

Gennip

Water

Potgieter

et al. 2017

J Virol

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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, inframe 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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Section: Discussionsupporting

confidence: 61%

Section: Discussionmentioning

confidence: 61%

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

Gennip

Water

Potgieter

et al. 2017

J Virol

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“…3A). A marked structural resemblance exists in the overall architecture of the major core proteins and core particles of these two related orbiviruses (Burroughs et al, 1994;Basak et al, 1996Basak et al, , 1997Manole et al, 2012), as well as acceptable phylogenetic relationships between their VP3 or VP7 proteins. The amino acid sequence identity shared between VP3 of AHSV-9 and BTV-1 was 57.5%, while their VP7 proteins shared 42.8% identity, thus exceeding the 30% cut-off value for homology modeling (Xiang, 2006).…”

Section: Engineering and Assembly Of Mutant Ahsv Clpsmentioning

confidence: 99%

“…The AHSV genome is composed of 10 segments of linear double-stranded (ds) RNA, which encode four nonstructural proteins (NS1 to NS4) and seven structural proteins (Bremer, 1976;Grubman and Lewis, 1992;Zwart et al, 2015). The virion is a nonenveloped icosahedral particle (Oellermann et al, 1970;Burroughs et al, 1994) composed of three concentric protein layers (Manole et al, 2012). The core particle comprises of a VP7 surface layer and an underlying VP3 layer, which encloses the genomic dsRNA segments and three enzymatic minor proteins (VP1, VP3 and VP6).…”

Section: Introductionmentioning

confidence: 99%

Synthesis of empty african horse sickness virus particles

Maree

Putterill

et al. 2016

Virus Research

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As a means to develop African horse sickness (AHS) vaccines that are safe and DIVA compliant, we investigated the synthesis of empty African horse sickness virus (AHSV) particles. The emphasis of this study was on the assembly of the major viral core (VP3 and VP7) and outer capsid proteins (VP2 and VP5) into architecturally complex, heteromultimeric nanosized particles. The production of fully assembled corelike particles (CLPs) was accomplished in vivo by baculovirus-mediated co-synthesis of VP3 and VP7. The two different outer capsid proteins were capable of associating independently of each other with preformed cores to yield partial virus-like particles (VLPs). Complete VLPs were synthesized, albeit with a low yield. Crystalline formation of AHSV VP7 trimers is thought to impede high-level CLP production. Consequently, we engineered and co-synthesized VP3 with a more hydrophilic mutant VP7, resulting in an increase in the turnover of CLPs.

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“…In a second, alternate approach to identifying the presence of exposed loop regions in VP2, the similarity of BTV VP2 to the VP2 protein of African horse sickness virus (AHSV), a closely related orbivirus (38), was assessed. A database search of the available AHSV (4 of the 9 serotypes) and BTV (24 of the 26 serotypes) VP2 peptide sequences (data not shown) revealed that VP2 proteins from AHSV serotypes are generally longer (ranging from 1,051 to 1,060 residues) than those from BTV serotypes (ranging from 950 to 962 residues).…”

Section: Resultsmentioning

confidence: 99%

Trafficking of Bluetongue Virus Visualized by Recovery of Tetracysteine-Tagged Virion Particles

Bhattacharya

Ward

et al. 2014

J Virol

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Bluetongue virus (BTV), a member of the Orbivirus genus in the Reoviridae family, is a double-capsid insect-borne virus enclosing a genome of 10 double-stranded RNA segments. Like those of other members of the family, BTV virions are nonenveloped particles containing two architecturally complex capsids. The two proteins of the outer capsid, VP2 and VP5, are involved in BTV entry and in the delivery of the transcriptionally active core to the cell cytoplasm. Although the importance of the endocytic pathway in BTV entry has been reported, detailed analyses of entry and the role of each protein in virus trafficking have not been possible due to the lack of availability of a tagged virus. Here, for the first time, we report on the successful manipulation of a segmented genome of a nonenveloped capsid virus by the introduction of tags that were subsequently fluorescently visualized in infected cells. The genetically engineered fluorescent BTV particles were observed to enter live cells immediately after virus adsorption. Further, we showed the separation of VP2 from VP5 during virus entry and confirmed that while VP2 is shed from virions in early endosomes, virus particles still consisting of VP5 were trafficked sequentially from early to late endosomes. Since BTV infects both mammalian and insect cells, the generation of tagged viruses will allow visualization of the trafficking of BTV farther downstream in different host cells. In addition, the tagging technology has potential for transferable application to other nonenveloped complex viruses. IMPORTANCELive-virus trafficking in host cells has been highly informative on the interactions between virus and host cells. Although the insertion of fluorescent markers into viral genomes has made it possible to study the trafficking of enveloped viruses, the physical constraints of architecturally complex capsid viruses have imposed practical limitations. In this study, we have successfully genetically engineered the segmented RNA genome of bluetongue virus (BTV), a complex nonenveloped virus belonging to the Reoviridae family. The resulting fluorescent virus particles could be visualized in virus entry studies of both live and fixed cells. This is the first time a structurally complex capsid virus has been successfully genetically manipulated to generate virus particles that could be visualized in infected cells.

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Structural Insight into African Horsesickness Virus Infection

Cited by 44 publications

References 61 publications

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

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

Synthesis of empty african horse sickness virus particles

Trafficking of Bluetongue Virus Visualized by Recovery of Tetracysteine-Tagged Virion Particles

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