RNA-dependent RNA polymerase from Atlantic Halibut Nodavirus contains two signals for localization to the mitochondria

Mézeth, Kjersti Bringsvor; Nylund, Stian; Henriksen, Håvard; Patel, Sonal; Nerland, Audun Helge; Szilvay, Anne Marie

doi:10.1016/j.virusres.2007.05.014

Cited by 25 publications

(19 citation statements)

References 33 publications

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“…Recently, nodavirus RdRp was shown to be targeted to mitochondria [41,42] and replicated on the mitochondrial membrane [42,43]. We asked if GRP78 played a role in regulating the function of protein A (RdRp) in mitochondria.…”

Section: Knockdown Of Ggrp78 Reduces Viral Titers and Increases Cell mentioning

confidence: 99%

Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death

Hong

2010

Apoptosis

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Whether viral pathogens that induce ER stress responses benefit the host or the virus remains controversial. In this study we show that betanodavirus induced ER stress responses up-regulate GRP78, which regulates the viral replication and host cellular mitochondrial-mediated cell death. Betanodavirus (redspotted grouper nervous necrosis virus, RGNNV) infection resulted in the following increased ER stress responses in fish GF-1 grouper fin cells: (1) IRE-1 and ATF-6 sensors at 48 h post-infection (p.i.) that up-regulated chaperone protein GRP78; (2) activation of caspase-12; and (3) PERK phosphorylation and down-regulation of Bcl-2. Analyses of GRP78 functions during viral replication using either loss-of-function or gain-of-function approaches showed that GRP78 over-expression also enhanced viral replication and induced cell death. Then, we found that zfGRP78 localization gradually increased in mitochondria after RGNNV infection by EGFP tagging approach. Furthermore, zfGRP78 can interact with viral RNA-dependent RNA polymerase (RdRp) by using immunofluorescent and immunoprecipitation assays. Finally, we found that blocking GRP78-mediated ER signals can reduce the viral death factors protein α and protein B2 expression and decrease the Bcl-2 down-regulation mediated mitochondria-dependent cell death, which also enhances host cellular viability. Taken together, our results suggest that RGNNV infection and expression can trigger ER stress responses, which up-regulate the chaperone GRP78 at early replication stage. Then, GRP78 can interact with RdRp that may enhance the viral replication for increasing viral death factors' expressions at middle-late replication stage, which can enhance mitochondrial-mediated cell death pathway and viral spreading. These results may provide new insights into the mechanism of ER stress-mediated cell death in RNA viruses.

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Section: Knockdown Of Ggrp78 Reduces Viral Titers and Increases Cell mentioning

confidence: 99%

Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death

Hong

2010

Apoptosis

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“…The MARs predicted for NoV do not share homology with those found within the FHV, GGNNV, or AHNV RdRp sequences (35,36,38), which also fail to overlap one another. This is not surprising, since the RdRps of alpha-and betanodaviruses share little (less than 30%) sequence identity at the amino acid level (39).…”

Section: Expression and Localization Kinetics Of The Nov Rdrp In Tranmentioning

confidence: 88%

“…The FHV RdRp and RCs colocalize to spherules within the outer mitochondrial membrane (32,36,37). The RdRp of a second betanodavirus, AHNV, localizes to mitochondria in infected fish cells and in transfected African green monkey kidney COS-7 cells when expressed from a plasmid in the absence of viral RNA replication, but the significance of this localization for the life cycle of the virus is unknown (38).…”

mentioning

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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“…The 276 bp PCR product was cloned into the pET200/ D-Topo vector (Invitrogen) creating a plasmid encoding a B2 protein with a 6xHis tag; the resulting plasmid was named pETB2-His. Using pCMV-RNA1-myc (Mézeth et al 2007) as the template and the primers 5'-GCG GCC GCA TAC TCT GTC TCC ATC GGC T-3' and 5'-AAA CTG ACA ACC ATG GAA CA-3', a plasmid expressing Myc-tagged B2 protein from a eukaryotic promoter was constructed. The B2 stop codon TAG was changed to TAT, followed by cloning of the 248 bp fragment with NcoI and Not I overhangs into the NcoI and Not I sites in the pCMV/myc/cyto vector (Clontech).…”

Section: Methodsmentioning

confidence: 99%

“…The fibroblast cell line (SSN-1) from whole fry tissue of striped snakehead Channa striatus was infected with AHNV (Frerichs et al 1996). The cells were grown on poly-Llysine coated coverslips in a 24-well plate and infected as described previously (Mézeth et al 2007). COS-7 cells were transfected with Lipofectamine 2000 (Gibco) using 0.25 µg plasmid DNA and 1.25 µl Lipofectamine 2000 per coverslip.…”

Section: Methodsmentioning

confidence: 99%

B2 protein from betanodavirus is expressed in recently infected but not in chronically infected fish

Mézeth¹,

Patel²,

Henriksen³

et al. 2009

Dis. Aquat. Org.

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Betanodavirus infects both larvae and juvenile fish and can cause the disease viral encephalopathy and retinopathy (VER). During an acute outbreak of VER, infected individuals display several clinical signs of infection, i.e. abnormal swimming pattern and loss of appetite. Betanodaviruses can also cause chronic or persistent infection where the infected individuals show no clinical signs of infection. During infection the viral sub-genomic RNA3 and the RNA3-encoded B2 protein are expressed. Antibodies against the B2 protein from Atlantic halibut nodavirus were raised and used together with antibodies against the capsid protein to detect the presence of these 2 viral proteins in infected cells in culture and at different stages of infection in Atlantic halibut Hippoglossus hippoglossus and Atlantic cod Gadus morhua. The B2 protein was detected in recently infected, but not in chronically infected fish. Results suggest that the detection of B2 may be used to discriminate a recent and presumably active infection from a chronic and presumably persistent infection. KEY WORDS: Fish nodavirus · Atlantic halibut · Atlantic cod · B2 protein · Viral nervous necrosis Resale or republication not permitted without written consent of the publisherDis Aquat Org 83: [97][98][99][100][101][102][103] 2009 for both alpha-and betanodavirus replication (Johnson et al. 2004, Fenner et al. 2006b).RNA interference (RNAi) is a conserved evolutionary mechanism widely found in nature and is considered to be a cellular defense mechanism against foreign genes and viral infection (Cullen 2002, Plasterk 2002. The B2 proteins from the alphanodaviruses Flock House virus (FHV) and Nodamura virus (NoV) and the betanodavirus greasy grouper nervous necrosis virus (GGNNV) have been shown to suppress RNAi by binding to double stranded RNA, thus preventing cleavage, leading to enhanced accumulation of viral RNAs in plant, insect and mammalian cells (Johnson et al. 2004, Lingel et al. 2005, Sullivan & Ganem 2005, Fenner et al. 2006a.Atlantic halibut nodavirus (AHNV) was first isolated from hatchery-reared larvae of Atlantic halibut in 1995 (Grotmol et al. 1995). The clinical signs of AHNV infection are loss of appetite, abnormal swimming pattern, and changes in pigmentation (Munday et al. 2002). Mass mortality in farmed Atlantic cod and diseased juveniles with similar clinical signs to AHNV-infected Atlantic halibut led to the identification of Atlantic cod nodavirus (ACNV) , Nylund et al. 2008. The RdRp and capsid protein of AHNV and ACNV are more than 97% identical (GenBank accession nos. AJ401165, AJ245641, EF617332, EF617326), and both strains belong to the barfin flounder genotype of nodavirus (Nishizawa et al. 1997). Diagnosis of VER is performed by histopathology with identification of necrosis and vacuolation in the brain and retina, often combined with immunohistochemistry (IHC) using antibodies against the capsid protein (Grotmol et al. 1997). Other diagnostic tests, including enzyme-linked immunosorbant assays (ELISA) and rever...

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RNA-dependent RNA polymerase from Atlantic Halibut Nodavirus contains two signals for localization to the mitochondria

Cited by 25 publications

References 33 publications

Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death

Betanodavirus up-regulates chaperone GRP78 via ER stress: roles of GRP78 in viral replication and host mitochondria-mediated cell death

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

B2 protein from betanodavirus is expressed in recently infected but not in chronically infected fish

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