Abstract
BackgroundCarnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined.ResultsHere we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM).ConclusionOur results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.
Highlights
Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication
We show using a combination of p36reporter fusion proteins and p36-p33 hybrid proteins, wherein specific regions of p36 were replaced with those that constitute the peroxisomal targeting information in p33, that the mitochondrial sorting of p36 is mediated by an internal targeting signal consisting of its two moderately hydrophobic transmembrane domain (TMD) and a positively-charged face of an amphipathic helix located within the intervening loop sequence
Close inspection of these p36-containing globular structures at higher magnification revealed that they consisted of numerous torus or donut-shaped structures that contained myc-p36 or p36-myc and that enclosed spherical fluorescent structures containing matrix-localized E1β. These toroidal structures contained the endogenous mitochondrial outer membrane protein porin [24], as evidenced by the colocalization of p36-myc and porin (Figure 1). These results indicate that p36 sorts to mitochondrial outer membranes in Bright Yellow-2 (BY-2) cells and that the expression of this viral protein causes mitochondria to coalesce in perinuclear regions, in contrast to mitochondria in non-transformed cells that are distributed throughout the
Summary
Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. The hallmark of positive-strand RNA viruses is their ability to recruit distinct host-cell organelle membranes in order to create unique compartments at which viral RNA replication takes place [1,2,3] This process is exemplified during tombusvirus infections of plant cells where, depending on the virus and host, peroxisomes or mitochondria undergo a series of remarkable structural rearrangements that results in their transformation into so-called multivesicular bodies (MVBs) [reviewed in [4,5]]. These novel structures form initially by the proliferation and progressive invagination of the organelle's boundary (outer) membrane, resulting in the matrix or intermembrane space containing hundreds of small (~80–150 nm diameter) vesicles and/or spherules which serve as the sites of viral RNA replication. The events involved in the specific intracellular targeting and membrane integration/ assembly of the viral replication proteins, as well as the host-cell factors that facilitate these processes and/or mediate membrane remodelling are, in most cases, poorly studied and unclear
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