Co-opting of nonATP-generating glycolytic enzymes for TBSV replication

Molho, Melissa; Chuang, Ching-Kai; Nagy, Peter D.

doi:10.1016/j.virol.2021.03.011

Cited by 7 publications

(12 citation statements)

References 61 publications

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“…The vir-condensates were also formed during full tombusvirus infections in N. benthamiana cells. The formation of special vir-condensate facilitated enrichment and sequestration of co-opted cytosolic enzymes, which were associated with the membranous VROs during the entire replication process [34][35][36][37]48]. We propose that the vir-condensate is likely associated with and surround the "core" clustered peroxisomes or mitochondria (Fig 11).…”

Section: Discussionmentioning

confidence: 89%

“…What makes these proteins trapped in the VROs? For example, the co-opted glycolytic and fermentation enzymes are soluble proteins, but stably and tightly associated with VROs during tombusvirus infections [34,48] Not only condensate formation, but other mechanism might also lead to substantial enrichment of proteins within substructures. For example, proteins may undergo low valency interactions with a large scaffold, such as segments of chromosomes or very long polymeric protein scaffolds [49].…”

Section: Resultsmentioning

confidence: 99%

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Co-opted cytosolic host proteins form unique condensate substructures within the membranous tombusviral replication organelles

Lin¹,

Nagy²

2023

Preprint

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Positive-strand RNA viruses co-opt intracellular organellar membranes for the biogenesis of viral replication organelles (VROs). The membranous VROs are the sites of viral replication. Tombusviruses co-opt numerous host proteins, many of them cytosolic, not membrane-bound, such as the glycolytic and fermentation enzymes. It is currently not known what type of molecular organization keeps these enzymes enriched and sequestered within the membranous VROs. Here, we show evidence that the tomato bushy stunt virus (TBSV) p33 and the closely-related carnation Italian ringspot virus (CIRV) p36 replication proteins sequester several co-opted cytosolic proteins in unique condensate substructures associated with the membranous VROs. We find that TBSV p33 and CIRV p36 replication proteins form dropletsin vitroorganized by their intrinsically disordered region (IDR). The replication proteins also organize the partitioning of several co-opted host proteins to p33/p36 droplets. We also show that the VRO-associated condensates containing co-opted glycolytic and fermentation enzymes are critical for local ATP production within the VRO to support the energy need of virus replication. A short segment with charged amino acids in p33/p36 IDR plays a critical role in droplet formationin vitro, in organizing the VRO-associated condensates and affects both ATP production in VROs and viral replication. We find that the co-opted ER membranes and actin filaments form meshworks within and around the condensate, likely contributing to the unique composition and structure of VROs. We propose that the p33/p36 replication proteins organize liquid-liquid phase separation of co-opted highly concentrated host proteins in condensate substructures within VROs to allow for the dynamic regulation of viral replication. Overall, we demonstrate that distinct substructures co-exist in tombusvirus VROs. These are the subverted membranes and condensate substructures, both of which are critical for VRO functions. The p33/p36 replication proteins induce and connect the two substructures within the VROs. Interfering with condensate formation in VROs might open up new antiviral approaches.

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Section: Discussionmentioning

confidence: 89%

Section: Resultsmentioning

confidence: 99%

Co-opted cytosolic host proteins form unique condensate substructures within the membranous tombusviral replication organelles

Lin¹,

Nagy²

2023

Preprint

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“…It has been demonstrated that host glycolytic and fermentation pathway enzymes are recruited to the virus replication sites due to both p36 and p33 activities [44,45]. Moreover, the co-opted glycolytic and fermentative enzymes allow the production of ATP locally, which serves tombusvirus replication [44].…”

Section: Discussionmentioning

confidence: 99%

Carnation Italian Ringspot Virus p36 Expression Induces Mitochondrial Fission and Respiratory Chain Complex Impairment in Yeast

Petrosillo,

De Stradis,

Marzulli

et al. 2023

IJMS

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Positive-strand RNA virus replication invariably occurs in association with host cell membranes, which are induced to proliferate and rearrange to form vesicular structures where the virus replication complex is assembled. In particular, carnation Italian ringspot virus (CIRV) replication takes place on the mitochondrial outer membrane in plant and yeast cells. In this work, the model host Saccharomyces cerevisiae was used to investigate the effects of CIRV p36 expression on the mitochondrial structure and function through the determination of mitochondrial morphology, mitochondrial respiratory parameters, and respiratory chain complex activities in p36-expressing cells. CIRV p36 ectopic expression was shown to induce alterations in the mitochondrial network associated with a decrease in mitochondrial respiration and the activities of NADH–cyt c, succinate–cyt c (C II-III), and cytochrome c oxidase (C IV) complexes. Our results suggest that the decrease in respiratory complex activity could be due, at least in part, to alterations in mitochondrial dynamics. This yeast-based model will be a valuable tool for identifying molecular targets to develop new anti-viral strategies.

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“…We predict that the most abundant host proteins, which interact with Rpn11, will have the best chances to be recruited by p33 into VROs. The productions of many host proteins, including glycolytic and fermentation enzymes, are greatly induced by TBSV infection [19][20][21]55]. The increased amounts of the induced host proteins would likely favor their associations with Rpn11.…”

Section: Discussionmentioning

confidence: 99%

“…Rpn11 is an essential protein, and therefore, we applied different approaches to manipulate Rpn11 availability for pro-viral functions. First, we knocked down Rpn11 mRNA level via VIGS in N. benthamiana (S1A Fig) followed by transient expression of p33 replication protein and three glycolytic enzymes and two fermentation enzymes, which are known pro-viral host factors [19][20][21]55]. The glycolytic enzymes included the ATP generating Pgk1 (phosphoglycerate kinase 1) and PK (pyruvate kinase, Cdc19 in yeast and PKM2/PKLR in humans) as well as Fba2 (fructose 1,6-bisphosphate aldolase), whereas the fermentation enzymes included Pdc1 (pyruvate decarboxylase 1) and Adh1 (alcohol dehydrogenase 1).…”

Section: Critical Role Of the Cytosolic Rpn11 In Assisting Tombusviruses During Recruitment Of Pro-viral Glycolytic And Fermentation Enzymentioning

confidence: 99%

A novel viral strategy for host factor recruitment: The co-opted proteasomal Rpn11 protein interaction hub in cooperation with subverted actin filaments are targeted to deliver cytosolic host factors for viral replication

2021

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Positive-strand (+)RNA viruses take advantage of the host cells by subverting a long list of host protein factors and transport vesicles and cellular organelles to build membranous viral replication organelles (VROs) that support robust RNA replication. How RNA viruses accomplish major recruitment tasks of a large number of cellular proteins are intensively studied. In case of tomato bushy stunt virus (TBSV), a single viral replication protein, named p33, carries out most of the recruitment duties. Yet, it is currently unknown how the viral p33 replication protein, which is membrane associated, is capable of the rapid and efficient recruitment of numerous cytosolic host proteins to facilitate the formation of large VROs. In this paper, we show that, TBSV p33 molecules do not recruit each cytosolic host factor one-by-one into VROs, but p33 targets a cytosolic protein interaction hub, namely Rpn11, which interacts with numerous other cytosolic proteins. The highly conserved Rpn11, called POH1 in humans, is the metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates. However, TBSV takes advantage of a noncanonical function of Rpn11 by exploiting Rpn11’s interaction with highly abundant cytosolic proteins and the actin network. We provide supporting evidence that the co-opted Rpn11 in coordination with the subverted actin network is used for delivering cytosolic proteins, such as glycolytic and fermentation enzymes, which are readily subverted into VROs to produce ATP locally in support of VRO formation, viral replicase complex assembly and viral RNA replication. Using several approaches, including knockdown of Rpn11 level, sequestering Rpn11 from the cytosol into the nucleus in plants or temperature-sensitive mutation in Rpn11 in yeast, we show the inhibition of recruitment of glycolytic and fermentation enzymes into VROs. The Rpn11-assisted recruitment of the cytosolic enzymes by p33, however, also requires the combined and coordinated role of the subverted actin network. Accordingly, stabilization of the actin filaments by expression of the Legionella VipA effector in yeast and plant, or via a mutation of ACT1 in yeast resulted in more efficient and rapid recruitment of Rpn11 and the selected glycolytic and fermentation enzymes into VROs. On the contrary, destruction of the actin filaments via expression of the Legionella RavK effector led to poor recruitment of Rpn11 and glycolytic and fermentation enzymes. Finally, we confirmed the key roles of Rpn11 and the actin filaments in situ ATP production within TBSV VROs via using a FRET-based ATP-biosensor. The novel emerging theme is that TBSV targets Rpn11 cytosolic protein interaction hub driven by the p33 replication protein and aided by the subverted actin filaments to deliver several co-opted cytosolic pro-viral factors for robust replication within VROs.

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Co-opting of nonATP-generating glycolytic enzymes for TBSV replication

Cited by 7 publications

References 61 publications

Co-opted cytosolic host proteins form unique condensate substructures within the membranous tombusviral replication organelles

Co-opted cytosolic host proteins form unique condensate substructures within the membranous tombusviral replication organelles

Carnation Italian Ringspot Virus p36 Expression Induces Mitochondrial Fission and Respiratory Chain Complex Impairment in Yeast

A novel viral strategy for host factor recruitment: The co-opted proteasomal Rpn11 protein interaction hub in cooperation with subverted actin filaments are targeted to deliver cytosolic host factors for viral replication

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