Membrane Association of Greasy Grouper Nervous Necrosis Virus Protein A and Characterization of Its Mitochondrial Localization Targeting Signal

Guo, Yan; Chan, Ssc; Kwang, Jimmy

doi:10.1128/jvi.78.12.6498-6508.2004

Cited by 43 publications

(36 citation statements)

References 47 publications

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“…Although the NoV RdRp was shown here to be an integral membrane protein with MAR structures that play critical roles in RC formation, it is not yet clear how this interaction is mediated. The RdRps of the related nodaviruses FHV and GGNNV are integral membrane proteins that interact with mitochondrial membranes via transmembrane ␣-helices (35,36). However, these RdRps are markedly different from that of NoV, with the NoV and FHV RdRps sharing 44% amino acid sequence identity (39) and those of NoV and GGNNV sharing only 26% amino acid sequence identity, as we calculated using ClustalW2 software (data not shown).…”

Section: Discussionmentioning

confidence: 99%

“…This is not surprising, since the RdRps of alpha-and betanodaviruses share little (less than 30%) sequence identity at the amino acid level (39). While the RdRps of FHV and GGNNV are both predicted to contain helices that span the membrane (35,36), the MARs in the NoV RdRp are unlikely to be membrane-spanning ␣-helices, due to the presence of charged residues within the predicted helical regions (Fig. 5A) not found for FHV and GGNNV.…”

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

confidence: 93%

“…Transfected BSR-T7/5 cells were washed twice with ice-cold PBS, harvested by scraping into PBS, and collected by centrifugation. Cell pellets were gently resuspended in hypotonic lysis buffer (1 mM Tris-HCl [pH 7.4], 0.1 mM EDTA, 15 mM NaCl) (35) supplemented with 1 mM each benzamidine (Sigma-Aldrich) and phenylmethylsulfonyl fluoride (PMSF; Sigma-Adrich). Cells were homogenized with a glass Dounce instrument and unbroken cells, large cell debris, and nuclei were removed by centrifugation.…”

Section: Methodsmentioning

confidence: 99%

“…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: 99%

“…Two other members of the Nodaviridae replicate their genomes in association with mitochondrial membranes, namely, FHV (in yeast and cultured Drosophila melanogaster cells) and the betanodavirus GGNNV (in cultured sea bass cells) (32,35,36). The FHV RdRp and RCs colocalize to spherules within the outer mitochondrial membrane (32,36,37).…”

mentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%