Multiple Routes of Bluetongue Virus Egress

Bluetongue virus (BTV) is an arthropod-borne virus infecting livestock. Its frequent emergence in Europe and North America had caused significant agricultural and economic loss. BTV is also of scientific interest as a model to understand the mechanisms underlying non-enveloped virus release from mammalian and insect cells. The BTV particle, which is formed of a complex double-layered capsid, was first considered as a lytic virus that needs to lyse the infected cells for cell to cell transmission. In the last decade, however, a more in-depth focus on the role of the non-structural proteins has led to several examples where BTV particles are also released through different budding mechanisms at the plasma membrane. It is now clear that the non-structural protein NS3 is the main driver of BTV release, via different interactions with both viral and cellular proteins of the cell sorting and exocytosis pathway. In this review, we discuss the most recent advances in the molecular biology of BTV egress and compare the mechanisms that lead to lytic or non-lytic BTV release.

Download Full-text

Human IFITM3 restricts Chikungunya virus and Mayaro virus infection and is susceptible to virus-mediated counteraction

10.1101/2020.09.11.292946 ◽

2020 ◽

Author(s):

Sergej Franz ◽

Thomas Zillinger ◽

Fabian Pott ◽

Christiane Schüler ◽

Sandra Dapa ◽

...

Keyword(s):

Virus Infection ◽

Influenza A ◽

Chikungunya Virus ◽

Protein S ◽

Human Cells ◽

Structural Protein ◽

Infected Cells ◽

Mayaro Virus ◽

The Impact

AbstractInterferon-induced transmembrane (IFITM) proteins restrict infection by enveloped viruses through interfering with membrane fusion and virion internalisation. The role of IFITM proteins during alphaviral infection of human cells and viral counteraction strategies remain largely unexplored. Here, we characterized the impact of IFITM proteins and variants on entry and spread of Chikungunya virus (CHIKV) and Mayaro virus (MAYV) in human cells, and provide first evidence for a CHIKV-mediated antagonism of IFITM proteins. IFITM1, 2 and 3 restricted infection at the level of alphavirus glycoprotein-mediated entry, both in the context of direct infection and during cell-to-cell transmission. Relocalization of normally endosomal IFITM3 to the plasma membrane resulted in the loss of its antiviral activity. rs12252-C, a naturally occurring variant of IFITM3 that has been proposed to associate with severe influenza in humans, restricted CHIKV, MAYV and influenza A virus infection as efficiently as wild-type IFITM3. Finally, all antivirally active IFITM variants displayed reduced cell surface levels in CHIKV-infected cells involving a posttranscriptional process mediated by one or several non-structural protein(s) of CHIKV.

Download Full-text

Dengue activates mTORC2 signaling to counteract apoptosis and maximize viral replication

10.1101/2020.09.22.308890 ◽

2020 ◽

Author(s):

Christoph C. Carter ◽

Jean Paul Olivier ◽

Alexis Kaushansky ◽

Fred D. Mast ◽

John D. Aitchison

Keyword(s):

Viral Replication ◽

Cell Survival ◽

Structural Protein ◽

Protein Protein Interaction ◽

Mechanistic Target Of Rapamycin ◽

Promote Cell Survival ◽

Important Regulator ◽

Infected Cells ◽

Induced Apoptosis

ABSTRACTThe mechanistic target of rapamycin (mTOR) functions in at least two distinct complexes: mTORC1, which regulates cellular anabolic-catabolic homeostasis, and mTORC2, which is an important regulator of cell survival and cytoskeletal maintenance. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. In contrast, the role of mTORC2 in viral pathogenesis is unknown. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.

Download Full-text

Insights into the role of the non-structural protein 2 (NS2) in Bluetongue virus morphogenesis

Virus Research ◽

10.1016/j.virusres.2010.05.014 ◽

2010 ◽

Vol 151 (2) ◽

pp. 109-117 ◽

Cited By ~ 14

Author(s):

Carmen Butan ◽

Paul Tucker

Keyword(s):

Bluetongue Virus ◽

Structural Protein ◽

Virus Morphogenesis

Download Full-text

The VP3 Protein of Bluetongue Virus Associates with the MAVS Complex and Interferes with the RIG-I-Signaling Pathway

Viruses ◽

10.3390/v13020230 ◽

2021 ◽

Vol 13 (2) ◽

pp. 230

Author(s):

Marie Pourcelot ◽

Rayane Amaral Moraes ◽

Aurore Fablet ◽

Emmanuel Bréard ◽

Corinne Sailleau ◽

...

Keyword(s):

Bluetongue Virus ◽

Type I Interferon ◽

Type I ◽

Adapter Protein ◽

Structural Protein ◽

Innate Response ◽

Biting Midges ◽

Domestic Ruminants ◽

Infected Cells ◽

Culicoides Biting Midges

Bluetongue virus (BTV), an arbovirus transmitted by Culicoides biting midges, is a major concern of wild and domestic ruminants. While BTV induces type I interferon (alpha/beta interferon [IFN-α/β]) production in infected cells, several reports have described evasion strategies elaborated by this virus to dampen this intrinsic, innate response. In the present study, we suggest that BTV VP3 is a new viral antagonist of the IFN-β synthesis. Indeed, using split luciferase and coprecipitation assays, we report an interaction between VP3 and both the mitochondrial adapter protein MAVS and the IRF3-kinase IKKε. Overall, this study describes a putative role for the BTV structural protein VP3 in the control of the antiviral response.

Download Full-text

Effects of intrabodies specific for rotavirus NSP5 during the virus replicative cycle

Journal of General Virology ◽

10.1099/vir.0.80075-0 ◽

2004 ◽

Vol 85 (11) ◽

pp. 3285-3290 ◽

Cited By ~ 42

Author(s):

Fulvia Vascotto ◽

Michela Campagna ◽

Michela Visintin ◽

Antonino Cattaneo ◽

Oscar R. Burrone

Keyword(s):

Structural Protein ◽

Wild Type ◽

Single Chain ◽

Single Chain Fv ◽

Infected Cells ◽

Antibody Capture ◽

Virus Replication Cycle ◽

First Time ◽

Replicative Cycle

Intracellular antibodies or intrabodies (ICAbs) have great potential in protein knockout strategies for intracellular antigens. In this study, they have been used to investigate the role of the rotavirus non-structural protein NSP5 in the virus replication cycle. Intracellular antibody-capture technology was used to select single-chain Fv format (scFv) ICAbs against an NSP5 mutant. Five different specific ICAbs were selected and expressed in MA104 cells, in the scFv format, as cytoplasmic- and nuclear-tagged forms. By confocal microscopy, it was found that three of these ICAbs recognized the full-length wild-type NSP5 specifically, forming antigen-specific aggresomes in the cytoplasm of cotransfected cells. Expression of the ICAbs in rotavirus-infected cells largely reduced the assembly of viroplasms and cellular cytopathic effect. Replication of dsRNA was partially inhibited, despite there being no reduction in virus titre. These results demonstrate for the first time a key role for NSP5 during the virus replicative cycle.

Download Full-text

A Viral Nonstructural Protein Regulates Bluetongue Virus Trafficking and Release

Journal of Virology ◽

10.1128/jvi.00263-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6806-6816 ◽

Cited By ~ 71

Author(s):

Cristina C. P. Celma ◽

Polly Roy

Keyword(s):

Membrane Protein ◽

Bluetongue Virus ◽

Reverse Genetics ◽

Nonstructural Protein ◽

Genetic System ◽

Cellular Membrane ◽

Binding Motif ◽

Virus Particles ◽

Infected Cells ◽

Virus Egress

ABSTRACT Bluetongue virus (BTV), a nonenveloped insect-borne virus, is released from infected cells by multiple pathways. Unlike other nonenveloped viruses, in addition to cell lysis the newly synthesized virus particles also appear to use a unique “budding” process. The nonstructural protein NS3, the only membrane protein encoded by BTV in infected cells, has been implicated in this process, since it appears to interact not only with the outermost viral capsid protein VP2 but also with a component of the cellular ESCRT pathway. However, to date it had not been possible to obtain direct evidence for the involvement of NS3 in BTV morphogenesis due to the lack of a genetic system that would allow introducing the targeted mutation in NS3 gene. In this study, we have used the recently developed T7 transcript-based reverse genetics system for BTV to introduce mutations in the sequence of NS3 into the viral genome and have investigated the effect of these mutations in the context of a replicating virus. While certain NS3 mutations exhibited drastic effects on newly synthesized virus release, others had less pronounced effects. In particular, mutations of two residues in the Tsg101 binding motif, the putative L domain of NS3, altered normal virus egress patterns and left nascent particles tethered to the cellular membrane, apparently arrested in the process of budding. In cells infected with a mutant virus that was incapable of an NS3-VP2 interaction, no budding particles were visualized. These data suggest that NS3 may act like the membrane protein of enveloped viruses and is responsible for intracellular trafficking and budding of virus particles. NS3 is thus a bridge between the maturing virion particles and cellular proteins during virus egress.

Download Full-text

Functional Dissection of the Major Structural Protein of Bluetongue Virus: Identification of Key Residues within VP7 Essential for Capsid Assembly

Journal of Virology ◽

10.1128/jvi.74.18.8658-8669.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8658-8669 ◽

Cited By ~ 27

Author(s):

Chang-Kwang Limn ◽

Norbert Staeuber ◽

Katherine Monastyrskaya ◽

Patrice Gouet ◽

Polly Roy

Keyword(s):

Bluetongue Virus ◽

Cdna Clones ◽

Structural Protein ◽

Apparent Effect ◽

Dynamic Nature ◽

Virus Identification ◽

Substitution Mutations ◽

The Stability ◽

Key Residues

ABSTRACT A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.

Download Full-text

NTP binding and phosphohydrolase activity associated with purified bluetongue virus non-structural protein NS2

Journal of General Virology ◽

10.1099/0022-1317-81-8-1961 ◽

2000 ◽

Vol 81 (8) ◽

pp. 1961-1965 ◽

Cited By ~ 16

Author(s):

Nigel J. Horscroft ◽

Polly Roy

Keyword(s):

Bluetongue Virus ◽

Inclusion Bodies ◽

Insect Cells ◽

Expression System ◽

Baculovirus Expression System ◽

Structural Protein ◽

Baculovirus Expression ◽

Infected Cells ◽

Viral Inclusion ◽

Hydrolysis Of

The bluetongue virus ssRNA-binding protein, NS2, is a phosphoprotein that forms viral inclusion bodies in infected cells. Recombinant NS2 was expressed in the baculovirus expression system and purified to homogeneity from insect cells. Purified NS2 bound nucleosides. Further investigation revealed that the protein bound ATP and GTP and could hydrolyse both nucleosides to their corresponding NMPs, with a higher efficiency for the hydrolysis of ATP. The increased efficiency of hydrolysis of ATP correlated with a higher binding affinity of NS2 for ATP than GTP. Ca2+, Mg2+ and Mn2+ were able to function as the required divalent cation in the reactions. The phosphohydrolase activity was not sensitive to ouabain, an inhibitor of cellular ATPases, suggesting that this activity was not the result of a cellular contaminant.

Download Full-text