Betanodavirus B2 Causes ATP Depletion-induced Cell Death via Mitochondrial Targeting and Complex II Inhibition in Vitro and in Vivo

Abstract

The betanodavirus non-structural protein B2 is a newly discovered necrotic death factor with a still unknown role in regulation of mitochondrial function. In the present study, we examined protein B2-mediated inhibition of mitochondrial complex II activity, which results in ATP depletion and thereby in a bioenergetic crisis in vitro and in vivo. Expression of protein B2 was detected early at 24 h postinfection with red-spotted grouper nervous necrosis virus in the cytoplasm. Later B2 was found in mitochondria using enhanced yellow fluorescent protein (EYFP) and immuno-EM analysis. Furthermore, the B2 mitochondrial targeting signal peptide was analyzed by serial deletion and specific point mutation. The sequence of the B2 targeting signal peptide ( 41 RTFVISAHAA 50 ) was identified and its presence correlated with loss of mitochondrial membrane potential in fish cells. Protein B2 also was found to dramatically inhibit complex II (succinate dehydrogenase) activity, which impairs ATP synthesis in fish GF-1 cells as well as human embryonic kidney 293T cells. Furthermore, when B2 was injected into zebrafish embryos at the one-cell stage to determine its cytotoxicity and ability to inhibit ATP synthesis, we found that B2 caused massive embryonic cell death and depleted ATP resulting in further embryonic death at 10 and 24 h postfertilization. Taken together, our results indicate that betanodavirus protein B2-induced cell death is due to direct targeting of the mitochondrial matrix by a specific signal peptide that targets mitochondria and inhibits mitochondrial complex II activity thereby reducing ATP synthesis.Betanodaviruses cause viral nervous necrosis, an infectious neuropathological condition in fish that is characterized by necrosis of the central nervous system, including the brain and retina and by clinical signs (e.g. abnormal swimming behavior and development of a darker body color) (1). This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance (2).The family Nodaviridae is comprised of the genera Alphanodavirus and Betanodavirus. Alphanodavirus predominantly infects insects, whereas Betanodavirus predominantly infects fish (3-5). Nodaviruses are small, non-enveloped, spherical viruses with bipartite positive-sense RNA genomes (RNA1 and RNA2) that are capped but not polyadenylated (3). RNA1 encodes an ϳ110-kDa non-structural protein that has been designated RNA-dependent RNA polymerase or protein A. This protein is vital for replication of the viral genome. RNA2 encodes a 42-kDa capsid protein (6, 7), which may also function in the induction of cell death (8, 9). Nodaviruses also synthesize RNA3, a sub-genomic RNA species from the 3Ј terminus of RNA1. RNA3 contains two putative open reading frames that potentially encode an 111-amino acid protein B1 and a 75-amino acid protein B2 (3, 10, 11). Recently, betanodavirus B1 was found to play an anti-necrotic death function in the early replication stages (10). In contrast, betanodaviru...

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“…Betanodavirus virus [59] infection-induced viral protein a [60] and B2 protein have been shown to target the mitochondria [61,62]. Aquatic birnavirus infection induces mitochondria-mediated death in fish cells [63].…”

Viral susceptibility, transfection and growth of SPB – a fish neural progenitor cell line from the brain of snubnose pompano, Trachinotus blochii (Lacépède)

“…; Chen, Su & Hong ; Su, Wu & Hong ). Although B2 contains a mitochondrial targeting signal (Su & Hong ), the localization of B2 is uncertain and depends on host cells. Fenner et al .…”

Betanodavirus: Mitochondrial disruption and necrotic cell death

“…Does induction of autophagy affect necrotic cell death during viral replication? What parameters, in addition to mitochondria-shaping proteins, control mitochondrial fusion and fission [15,55] ? Hopefully, future studies will increase our understanding of the mechanisms underlying mitochondria-mediated necrosis, its functions in multiple biological processes, and the regulatory signaling pathways that control its activation.…”

“…Recently, the betanodavirus B1 protein has been shown to have an anti-necrotic death function during the early replication stages [10] . In contrast, the betanodavirus B2 protein appears to function as a suppressor of host siRNA silencing [12,13] or as a EDITORIAL necrotic death factor [14,15] . In addition, red-spotted grouper nervous necrosis virus (RGNNV) infection and expression can trigger the ER stress response, which results in the upregulation of the 78 kDa glucose regulated protein at the early replication stage [16] .…”