Nonstructural Protein 3 of Bluetongue Virus Assists Virus Release by Recruiting ESCRT-I Protein Tsg101

Abstract

The release of Bluetongue virus (BTV) and other members of the Orbivirus genus from infected host cells occurs predominantly by cell lysis, and in some cases, by budding from the plasma membrane. Two nonstructural proteins, NS3 and NS3A, have been implicated in this process. Here we show that both proteins bind to human Tsg101 and its ortholog from Drosophila melanogaster with similar strengths in vitro. This interaction is mediated by a conserved PSAP motif in NS3 and appears to play a role in virus release. The depletion of Tsg101 with small interfering RNA inhibits the release of BTV and African horse sickness virus, a related orbivirus, from HeLa cells up to fivefold and threefold, respectively. Like most other viral proteins which recruit Tsg101, NS3 also harbors a PPXY late-domain motif that allows NS3 to bind NEDD4-like ubiquitin ligases in vitro. However, the late-domain motifs in NS3 do not function as effectively in facilitating the release of mini Gag virus-like particles from 293T cells as the late domains from human immunodeficiency virus type 1, human T-cell leukemia virus, and Ebola virus. A mutagenesis study showed that the arginine residue in the PPRY motif is responsible for the low activity of the NS3 late-domain motifs. Our data suggest that the BTV late-domain motifs either recruit an antagonist that interferes with budding or fail to recruit an agonist which is different from NEDD4.

A PSAP motif in the ORF3 protein of hepatitis E virus is necessary for virion release from infected cells

“…Recent studies have revealed that viral matrix proteins play critical roles during the later stages of virus budding in many enveloped RNA viruses, including retroviruses, rhabdoviruses, filoviruses and orthomyxoviruses; these viral proteins possess a so-called late (L)-domain containing PT/SAP, PPXY and YXXL, which are critical motifs for efficient budding (Bouamr et al, 2003;Ciancanelli & Basler, 2006;Göttlinger et al, 1991;Harty et al, 1999Harty et al, , 2000Wirblich et al, 2006). The PTAP motif was first identified in HIV Gag and has been reported to bind to Tsg101, which functions in vacuolar protein sorting (Garrus et al, 2001).…”

Bluetongue virus: virology, pathogenesis and immunity

“…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. This budding mechanism might be involved in BTV egress from insect cells in which BTV does not induce significant cytopathic effect, whereas the viroporin mechanism would be more prominent in mammalian cells.…”

“…However, some cell types that support the replication of the virus do not demonstrate a cytopathic effect to BTV, such as insect cells [73], T cell lines [96] and activated blood lymphocytes [5]. The budding versus the viroporin-mediated viral exit mechanism might partially explain this difference [104].…”

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

“…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). BTV NS3 also contains highly conserved PSAP and PPXY late domain motifs common in Tsg101-recruiting proteins (Wirblich et al, 2006).…”

“…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). BTV NS3 also contains highly conserved PSAP and PPXY late domain motifs common in Tsg101-recruiting proteins (Wirblich et al, 2006). The C-terminal cytoplasmic domain interacts with the outer capsid protein VP2 (Beaton et al, 2002), suggesting that NS3 supports virus release by connecting virus to cellular transport mechanisms and by disruption of the cell membrane.…”