The NS3 Protein of Bluetongue Virus Exhibits Viroporin-like Properties

Abstract

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...

Bluetongue virus: virology, pathogenesis and immunity

“…Interestingly, NS3 and NS3A appear to be associated with smooth intracellular membranes, although they are also present at the plasma membrane. NS3 functions as a viroporin, facilitating virus release by inducing membrane permeabilization [33]. In addition, NS3 binds to the cellular protein Tsg101 [104], allowing BTV particles to also leave host cells by a budding mechanism, similarly to retroviruses.…”

“…Mature progeny virus particles are transported within the cytoplasm on microtubules involving VP2/vimentin interactions [8]. Release of virions from the infected cell occurs via cell membrane destabilization mediated by the NS3 viroporin activity [33], in some cases via budding, or as a result of cell death and lysis. Production of mature particles is exponential during the 8th and 24th hours post infection.…”

Bluetongue virus genetic and phenotypic diversity: Towards identifying the molecular determinants that influence virulence and transmission potential

“…Cell lysis with an associated cytopathic effect is mainly observed in mammalian cells, whereas budding of virions without cell death occurs in insect cells (Han and Harty, 2004;Hyatt et al, 1993). Cell lysis is likely mediated by two viroporin-like hydrophobic domains that are able to interact with the mammalian cell membrane to induce membrane pore formation (Han and Harty, 2004). The budding of progeny virus particles in contrast appears to be facilitated through the simultaneous interaction of VP2 and NS3/A with components of the host cell export machinery.…”

“…The proteins are associated with smooth intracellular vesicles as well as the plasma membrane (Wu et al, 1992) and co-localizes at sites of viral release at the cell surface (Hyatt et al, 1991). Cell lysis with an associated cytopathic effect is mainly observed in mammalian cells, whereas budding of virions without cell death occurs in insect cells (Han and Harty, 2004;Hyatt et al, 1993). Cell lysis is likely mediated by two viroporin-like hydrophobic domains that are able to interact with the mammalian cell membrane to induce membrane pore formation (Han and Harty, 2004).…”

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

“…1c) (Bansal et al, 1998;French et al, 1989;Hyatt et al, 1991;Wu et al, 1992). NS3 is a viroporin-like protein, facilitating virus release by membrane permeabilization (Han & Harty, 2004). It interacts with the calpactin light chain p11 of the cellular annexin II complex and with cellular Tsg101 at its N terminus (Beaton et al, 2002;Celma & Roy, 2011), which are involved in membrane-related events, secretion and intracellular trafficking (Raynal & Pollard, 1994;Wirblich et al, 2006).…”