Structure of Broadhaven Virus by Cryoelectron Microscopy: Correlation of Structural and Antigenic Properties of Broadhaven Virus and Bluetongue Virus Outer Capsid Proteins

ABSTRACTBluetongue virus (BTV), in the familyReoviridae, is an insect-borne, double-capsid virus causing hemorrhagic disease in livestock around the world. Here, we elucidate how outer capsid proteins VP2 and VP5 coordinate cell entry of BTV. To identify key functional residues, we used atomic-level structural data to guide mutagenesis of VP2 and VP5 and a series of biological and biochemical approaches, including site-directed mutagenesis, reverse genetics-based virus recovery, expression and characterization of individual recombinant mutant proteins, and variousin vitroandin vivoassays. We demonstrate the dynamic nature of the conformational change process, revealing that a unique zinc finger (CCCH) in VP2 acts as the major low pH sensor, coordinating VP2 detachment, subsequently allowing VP5 to sense low pH via specific histidine residues at key positions. We show that single substitution of only certain histidine residues has a lethal effect, indicating that the location of histidine in VP5 is critical to inducing changes in VP5 conformation that facilitates membrane penetration. Further, we show that the VP5 anchoring domain alone recapitulates sensing of low pH. Our data reveal a novel, multiconformational process that overcomes entry barriers faced by this multicapsid nonenveloped virus.IMPORTANCEVirus entry into a susceptible cell is the first step of infection and a significant point at which infection can be prevented. To enter effectively, viruses must sense the cellular environment and, when appropriate, initiate a series of changes that eventually jettison the protective shell and deposit virus genes into the cytoplasm. Many viruses sense pH, but how this happens and the events that follow are often poorly understood. Here, we address this question for a large multilayered bluetongue virus. We show key residues in outer capsid proteins, a pH-sensing histidine of a zinc finger within the receptor-binding VP2 protein, and certain histidine residues in the membrane-penetrating VP5 protein that detect cellular pH, leading to irreversible changes and propel the virus through the cell membrane. Our data reveal a novel mechanism of cell entry for a nonenveloped virus and highlight mechanisms which may also be used by other viruses.

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Bluetongue Virus Outer Capsid Proteins Are Sufficient To Trigger Apoptosis in Mammalian Cells

Journal of Virology ◽

10.1128/jvi.78.6.2875-2883.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 2875-2883 ◽

Cited By ~ 46

Author(s):

Eduardo Mortola ◽

Rob Noad ◽

Polly Roy

Keyword(s):

Cell Lines ◽

Bluetongue Virus ◽

Mammalian Cells ◽

Capsid Proteins ◽

Apoptotic Response ◽

Mammalian Cell Lines ◽

Endosomal Acidification ◽

Insect Cell Lines ◽

Outer Capsid ◽

Productive Virus

ABSTRACT Bluetongue virus (BTV) is transmitted by Culicoides sp. biting midges to livestock, causing severe hemorrhagic disease in sheep, but is asymptomatic in the insect host. Similarly, BTV causes rapid cell death in infected mammalian cells in culture, whereas infections of insect cells are long-term and unapparent, despite productive virus replication. To assess whether apoptosis plays any role in these two distinct cell responses, we have investigated apoptosis in cultured insect and mammalian cells. Three different mammalian cell lines and three different insect cell lines including Culicoides variipennis (KC) cells were infected with BTV serotype 10, and the key apoptosis indicators of cell morphology, chromosomal DNA fragmentation, and caspase-3 activation were monitored. BTV infection induced apoptosis with the activation of the transcription factor nuclear factor κB (NF-κB) in all three mammalian cell lines. In contrast, no evidence for apoptosis was detected in any of the three insect cell lines in response to BTV infection. Using inhibitors of endosomal acidification and UV-inactivated virus, we established that virus uncoating, but not productive virus replication, is necessary for BTV to trigger apoptosis in mammalian cells. Intracellular expression of the viral outer capsid proteins VP2 and VP5 or the two major nonstructural proteins NS1 and NS2 was not sufficient to trigger an apoptotic response. However, extracellular treatment with a combination of purified recombinant VP2 and VP5, but not with each protein used separately, resulted in an apoptotic response. Virus- and VP2-VP5-stimulated apoptotic responses were both inhibited by inhibitors of endosomal acidification. Thus, for BTV the viral outer capsid proteins alone are sufficient to trigger apoptosis.

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Analysis of the roles of bluetongue virus outer capsid proteins VP2 and VP5 in determination of virus serotype

Virology ◽

10.1016/0042-6822(89)90447-9 ◽

1989 ◽

Vol 170 (2) ◽

pp. 561-565 ◽

Cited By ~ 113

Author(s):

P.P.C. Mertens ◽

S. Pedley ◽

J. Cowley ◽

J.N. Burroughs ◽

A.H. Corteyn ◽

...

Keyword(s):

Bluetongue Virus ◽

Capsid Proteins ◽

Virus Serotype ◽

Outer Capsid

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High level expression of the two outer capsid proteins of bluetongue virus serotype 10: their relationship with the neutralization of virus infection

Virus Research ◽

10.1016/0168-1702(90)90027-9 ◽

1990 ◽

Vol 15 (3) ◽

pp. 189-195 ◽

Cited By ~ 29

Author(s):

Jonathon J.A. Marshall ◽

Polly Roy

Keyword(s):

Virus Infection ◽

Bluetongue Virus ◽

Capsid Proteins ◽

High Level Expression ◽

Virus Serotype ◽

High Level ◽

Outer Capsid

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In Vitro Recoating of Reovirus Cores with Baculovirus-Expressed Outer-Capsid Proteins μ1 and ς3

Journal of Virology ◽

10.1128/jvi.73.5.3941-3950.1999 ◽

1999 ◽

Vol 73 (5) ◽

pp. 3941-3950 ◽

Cited By ~ 84

Author(s):

Kartik Chandran ◽

Stephen B. Walker ◽

Ya Chen ◽

Carlo M. Contreras ◽

Leslie A. Schiff ◽

...

Keyword(s):

Buoyant Density ◽

Protein Composition ◽

Particle Morphology ◽

Capsid Proteins ◽

Productive Infection ◽

Cryoelectron Microscopy ◽

Mutant Form ◽

L Cells ◽

Outer Capsid

ABSTRACT Reovirus outer-capsid proteins μ1, ς3, and ς1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed μ1 and ς3 onto purified cores that lack μ1, ς3, and ς1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein ς1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of μ1 and ς3 onto cores had induced rearrangement of the pentameric λ2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the ς1 protein. To examine the utility of r-cores for genetic dissections of μ1 functions in reovirus entry, we generated r-cores containing a mutant form of μ1 that had been engineered to resist cleavage at the δ:φ junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in δ:φ cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection.

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