Expression of nonstructural protein NS3 of African horsesickness virus (AHSV): evidence for a cytotoxic effect of NS3 in insect cells, and characterization of the gene products in AHSV infected Vero cells

Staden, Vida van; Stoltz, Malin; Huismans, H.

doi:10.1007/bf01309863

Cited by 21 publications

(24 citation statements)

References 34 publications

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“…For example, electron microscopic studies suggested that NS3 was associated with areas of plasma membrane perturbation (3). Similar findings have been reported for the NS3 protein encoded by the closely related African horse sickness virus (5,8,9). More recently, an elegant study by Beaton et al (10) demonstrated that NS3 of BTV facilitates virus release by interacting with specific host-trafficking proteins at the plasma membrane.…”

supporting

confidence: 54%

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The NS3 Protein of Bluetongue Virus Exhibits Viroporin-like Properties

Han

Harty

2004

Journal of Biological Chemistry

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Bluetongue virus (BTV) 1 is a member of the Orbivirus genus within the Reoviridae family of segmented double-stranded RNA viruses. BTV remains an agriculturally important veterinary pathogen transmitted primarily to sheep by biting midges. BTV encodes seven structural (VP1-7) and four nonstructural (NS1, NS2, NS3, and NS3A) proteins. NS3 is a 229-amino acid protein encoded by the viral S10 RNA segment, and NS3A is a 216-amino acid protein in which synthesis is initiated from an in-frame downstream AUG codon within the Ser-10 RNA segment (1, 2). Both proteins have been shown to be N-linked glycosylated and associated with lipid membranes in infected cells (2-5). Hyatt et al. (3,6,7) were the first to provide evidence that NS3/NS3A may be involved in the late stages of BTV assembly and that NS3/NS3A were required for efficient release of virus-like particles from BTV-infected cells. For example, electron microscopic studies suggested that NS3 was associated with areas of plasma membrane perturbation (3). Similar findings have been reported for the NS3 protein encoded by the closely related African horse sickness virus (5, 8, 9). More recently, an elegant study by Beaton et al. (10) demonstrated that NS3 of BTV facilitates virus release by interacting with specific host-trafficking proteins at the plasma membrane. The authors suggest that NS3 functions as a bridge, linking progeny virions with cellular export machinery to promote virus release (10). Thus, NS3 appears to play a key role in virus assembly and release. We were initially intrigued by the presence of conserved PPXY and PSAP motifs within NS3 that are identical in sequence to late budding domains (L-domains) that we and others have identified in matrix proteins of RNA viruses (11). These L-domains have been shown to interact with specific host proteins, and the resultant virus-host interactions are thought to facilitate virus budding by an, as yet, undetermined mechanism. Results from our initial experiments indicated that the PPXY and PSAP motifs of NS3 did not appear to function as typical L-domains, in that these motifs did not promote the release of NS3-containing virus-like particles from mammalian cells. Interestingly, we did find that cells expressing NS3 were permeable to the translational inhibitor hygromycin B in a dose-dependent manner. This finding suggested that NS3 may be capable of permeabilizing or destabilizing lipid membranes; a property shared by a group of viral proteins termed viroporins (12). We went on to demonstrate that NS3 possesses additional characteristics commonly associated with viroporins. For example, NS3 was able to form homo-oligomers when expressed in mammalian cells. Furthermore, like other viroporins, NS3 is a transmembrane protein containing two transmembrane domains (TM1 and TM2). Mutations introduced into TM1 to disrupt the hydrophobic nature of this region abolished the ability of NS3 to permeabilize the plasma membrane. In contrast, the deletion of amino acids comprising TM2 did not result in disruption of NS3-i...

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confidence: 54%

“…Indeed, expression of the NS3 protein of African horse sickness virus was shown to destabilize the plasma membrane (8,9). The ability of NS3 to perturb the plasma membrane, together with its proposed role as a bridging molecule between virions and host proteins, may ensure efficient release of mature virus particles from infected cells (10).…”

Section: Discussionmentioning

confidence: 99%

The NS3 Protein of Bluetongue Virus Exhibits Viroporin-like Properties

Han

Harty

2004

Journal of Biological Chemistry

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“…Analysis of all the AHSV NS3 sequences showed a well conserved proline-rich region between residues 20 and 28, as described by van Staden et al (1995). This region was surrounded by hydrophilic, variable residues under positiveselective pressure (Fig.…”

Section: Ahsv Ns3 Proteinmentioning

confidence: 98%

“…The AHSV NS3 is not as well conserved as the BTV NS3, which varies by only 7-10 % (van Niekerk et al, 2001b) and the equine encephalosis virus (EEV) NS3, which varies by 17 % (van Niekerk et al, 2003). Conserved regions within AHSV NS3 have been reported to be (i) the initiation codon for NS3a, (ii) a proline-rich area between residues 22 and 34, (iii) a sequence of highly conserved amino acids from residues 46 to 90 and (iv) two hydrophobic regions (residues 116-137 and 154-170, respectively) predicted to form transmembrane helices (van Staden et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Molecular epidemiology of the African horse sickness virus S10 gene

Quan

Vuuren

Howell

et al. 2008

Journal of General Virology

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Between 2004 and 2006, 145 African horse sickness viruses (AHSV) were isolated from blood and organ samples submitted from South Africa to the Faculty of Veterinary Science, University of Pretoria. All nine serotypes were represented, with a range of 3-60 isolates per serotype. The RNA small segment 10 (S10) nucleotide sequences of these isolates were determined and the phylogeny investigated. AHSV, bluetongue virus (BTV) and equine encephalosis virus (EEV) all formed monophyletic groups and BTV was genetically closer to AHSV than EEV. This study confirmed the presence of three distinct S10 phylogenetic clades (a, b and c). Some serotypes (6, 8 and 9 in a; 3 and 7 in b; 2 in c) were restricted to a single clade, while other serotypes (1, 4 and 5) clustered into both the a and c clades. Strong purifying selection was evident and a constant molecular clock was inappropriate. The S10 gene is the second most variable gene of the AHSV genome and the use of S10 in molecular epidemiology was illustrated by an AHS outbreak in the Western Cape in 2004. It was shown that two separate AHSV were circulating in the area, even though AHSV serotype 1 was the only isolate from the outbreak. The small size of the gene (755-764 bp) and conserved terminal regions facilitate easy and quick sequencing. The establishment of an S10 sequence database is important for characterizing outbreaks of AHS. It will be an essential resource for elucidating the epidemiology of AHS.

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“…NS3/NS3a proteins are conserved among orbivirus species (Huismans et al, 2004;van Niekerk et al, 2003;van Staden & Huismans, 1991) and their function is likely similar (Jensen et al, 1994;Meiring et al, 2009;van de Water et al, 2015;van Staden et al, 1995). However, reassortants consisting of genome segments of different orbivirus species have never been isolated and virus rescue of such Seg-10 mono-reassortants using reverse genetics has failed.…”

Section: Discussionmentioning

confidence: 99%

Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses

Feenstra

Gennip²,

Schreuder³

et al. 2016

Journal of General Virology

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Orbiviruses are insect-transmitted, non-enveloped viruses with a ten-segmented dsRNA genome of which the bluetongue virus (BTV) is the prototype. Viral non-structural protein NS3/NS3a is encoded by genome segment 10 (Seg-10), and is involved in different virus release mechanisms. This protein induces specific release via membrane disruptions and budding in both insect and mammalian cells, but also the cytopathogenic release that is only seen in mammalian cells. NS3/NS3a is not essential for virus replication in vitro with BTV Seg-10 containing RNA elements essential for virus replication, even if protein is not expressed. Recently, new BTV serotypes with distinct NS3/NS3a sequence and cell tropism have been identified. Multiple studies have hinted at the importance of Seg-10 in orbivirus replication, but the exact prerequisites are still unknown. Here, more insight is obtained with regard to the needs for orbivirus Seg-10 and the balance between protein expression and RNA elements. Multiple silent mutations in the BTV NS3a ORF destabilized Seg-10, resulting in deletions and sequences originating from other viral segments being inserted, indicating strong selection at the level of RNA during replication in mammalian cells in vitro. The NS3a ORFs of other orbiviruses were successfully exchanged in BTV1 Seg-10, resulting in viable chimeric viruses. NS3/NS3a proteins in these chimeric viruses were generally functional in mammalian cells, but not in insect cells. NS3/NS3a of the novel BTV serotypes 25 and 26 affected virus release from Culicoides cells, which might be one of the reasons for their distinct cell tropism.

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Expression of nonstructural protein NS3 of African horsesickness virus (AHSV): evidence for a cytotoxic effect of NS3 in insect cells, and characterization of the gene products in AHSV infected Vero cells

Cited by 21 publications

References 34 publications

The NS3 Protein of Bluetongue Virus Exhibits Viroporin-like Properties

The NS3 Protein of Bluetongue Virus Exhibits Viroporin-like Properties

Molecular epidemiology of the African horse sickness virus S10 gene

Balance of RNA sequence requirement and NS3/NS3a expression of segment 10 of orbiviruses

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