Reverse Genetics of Nodaviruses

Ball, L. Andrew; Johnson, Kyle L.

doi:10.1016/s0065-3527(08)60350-4

Cited by 67 publications

(68 citation statements)

References 36 publications

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“…Alphanodavirus predominantly infects insects, whereas Betanodavirus predominantly infects fish (3)(4)(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.…”

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

Hong

2010

Journal of Biological Chemistry

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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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“…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).…”

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confidence: 99%

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

Hong

2010

Journal of Biological Chemistry

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“…Alphanodavirus predominantly infects insects, while Betanodavirus predominantly infects fish [3][4][5][6][7] . Nodaviruses are small, nonenveloped, spherical viruses with bipartite positive-sense RNA genomes (RNA1 and RNA2) that are capped but not polyadenylated [3] . RNA1 encodes a non-structural protein of approximately 110 kDa that has been designated RNAdependent RNA polymerase or protein A.…”

Section: Betanodavirusmentioning

confidence: 99%

“…Nodaviruses also synthesize RNA3, a sub-genomic RNA species from the 3' terminus of RNA1. RNA3 contains two putative open reading frames that potentially encode a 111 amino-acid protein B1 and a 75 amino-acid protein B2 [3,10,11] . Recently, the betanodavirus B1 protein has been shown to have an anti-necrotic death function during the early replication stages [10] .…”

Section: Betanodavirusmentioning

confidence: 99%

Betanodavirus: Mitochondrial disruption and necrotic cell death

Hong¹

2013

WJV

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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. This disease can cause mass mortality in larval and juvenile populations of several teleost species and is of global economic importance. The mechanism of brain and retina damage during betanodavirus infection is poorly understood. In this review, we will focus recent results that highlight betanodavirus infection-induced molecular death mechanisms in vitro . Betanodavirus can induce host cellular death and post-apoptotic necrosis in fish cells. Betanodavirus-induced necrotic cell death is also correlated with loss of mitochondrial membrane potential in fish cells, as this necrotic cell death is blocked by the mitochondrial membrane permeability transition pore inhibitor bongkrekic acid and the expression of the antiapoptotic Bcl-2 family member zfBcl-xL. Moreover, this mitochondria-mediated necrotic cell death may require a caspase-independent pathway. A possible cellular death pathway involving mitochondrial function and the modulator zfBcl-xs is discussed which may provide new insights into the necrotic pathogenesis of betanodavirus.

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“…Originally isolated from the mosquito Culex tritaeniorhynchus, NoV lethally infects insects (mosquitoes, wax moth larvae, and honey bees) and mammals (suckling mice and suckling hamsters), causing hind segment or hind limb paralysis, respectively (1-7). The family Nodaviridae consists of two genera: members of the Alphanodavirus genus, which includes NoV, Flock House virus (FHV), and Black beetle virus (BBV), primarily infect insects, while the betanodaviruses, including Greasy grouper nervous necrosis virus (GGNNV) and Atlantic halibut nodavirus (AHNV), have been isolated only from fish (8,9). NoV provides an excellent model for deciphering the basic mechanism of viral RNA replication, due to its genetic simplicity, tremendous levels of RNA amplification, and ability to replicate in a wide range of host cells.…”

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confidence: 99%

Two Membrane-Associated Regions within the Nodamura Virus RNA-Dependent RNA Polymerase Are Critical for both Mitochondrial Localization and RNA Replication

Gant

Moreno

Varela-Ramı́rez

et al. 2014

J Virol

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Viruses with positive-strand RNA genomes amplify their genomes in replication complexes associated with cellular membranes. Little is known about the mechanism of replication complex formation in cells infected with Nodamura virus. This virus is unique in its ability to lethally infect both mammals and insects. In mice and in larvae of the greater wax moth (Galleria mellonella), Nodamura virus-infected muscle cells exhibit mitochondrial aggregation and membrane rearrangement, leading to disorganization of the muscle fibrils on the tissue level and ultimately in hind limb/segment paralysis. However, the molecular basis for this pathogenesis and the role of mitochondria in Nodamura virus infection remains unclear. Here, we tested the hypothesis that Nodamura virus establishes RNA replication complexes that associate with mitochondria in mammalian cells. Our results showed that Nodamura virus replication complexes are targeted to mitochondria, as evidenced in biochemical, molecular, and confocal microscopy studies. More specifically, we show that the Nodamura virus RNA-dependent RNA polymerase interacts with the outer mitochondrial membranes as an integral membrane protein and ultimately becomes associated with functional replication complexes. These studies will help us to understand the mechanism of replication complex formation and the pathogenesis of Nodamura virus for mammals. IMPORTANCEThis study will further our understanding of Nodamura virus (NoV) genome replication and its pathogenesis for mice. NoV is unique among the Nodaviridae in its ability to infect mammals. Here we show that NoV establishes RNA replication complexes (RCs) in association with mitochondria in mammalian cells. These RCs contain newly synthesized viral RNA and feature a physical interaction between mitochondrial membranes and the viral RNA-dependent RNA polymerase (RdRp), which is mediated by two membrane-associated regions. While the nature of the interaction needs to be explored further, it appears to occur by a mode distinct from that described for the insect nodavirus Flock House virus (FHV). The interaction of the NoV RdRp with mitochondrial membranes is essential for clustering of mitochondria into networks that resemble those described for infected mouse muscle and that are associated with fatal hind limb paralysis. This work therefore provides the first link between NoV RNA replication complex formation and the pathogenesis of this virus for mice.

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Reverse Genetics of Nodaviruses

Cited by 67 publications

References 36 publications

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

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

Betanodavirus: Mitochondrial disruption and necrotic cell death

Two Membrane-Associated Regions within the Nodamura Virus RNA-Dependent RNA Polymerase Are Critical for both Mitochondrial Localization and RNA Replication

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