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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Notes/Citation Information

Published in PLOS Pathogens, v. 17, issue 6, e1009680.

© 2021 Molho et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Funding Information

PDN was supported by the National Science Foundation (MCB-1122039 and IOS-1922895) and a USDA hatch grant (KY012042) and USDA-NIFA grant (2020-70410-32901). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

ppat.1009680.s001.doc (166 kB)
S1 Text. Supplementary materials and methods. https://doi.org/10.1371/journal.ppat.1009680.s001

ppat.1009680.s002.tif (834 kB)
S1 Fig. Gene silencing of Rpn11 in N. benthamiana. https://doi.org/10.1371/journal.ppat.1009680.s002

ppat.1009680.s003.tif (1503 kB)
S2 Fig. Demonstration of sequestration of Rpn11 from the cytosol to the nucleus. https://doi.org/10.1371/journal.ppat.1009680.s003

ppat.1009680.s004.tif (507 kB)
S3 Fig. Temperature-sensitive mutation in Rpn11 reduces the co-purification of Tdh3 and Tdh2 glycolytic enzymes with the viral replicase. https://doi.org/10.1371/journal.ppat.1009680.s004

ppat.1009680.s005.tif (3818 kB)
S4 Fig. Transient expression of Legionella VipA effector affects the architecture of the actin network and TBSV VROs in GFP-mTalin N. benthamiana transgenic plants. https://doi.org/10.1371/journal.ppat.1009680.s005

ppat.1009680.s006.tif (1304 kB)
S5 Fig. Transient expression of RavK in N. benthamiana. https://doi.org/10.1371/journal.ppat.1009680.s006

ppat.1009680.s007.tif (1239 kB)
S6 Fig. Dependence of ATP generation within tombusvirus VROs on actin and cofilin in yeast. https://doi.org/10.1371/journal.ppat.1009680.s007