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

Abstract

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.

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

“…11). Indeed, BiFC experiments showed that a population of Pgk1 and Adh1 was bound to p33 within VROs (Fig S16) [38]. This model may explain the partial fluorescent recovery of the co-opted host proteins in VROs.…”

“…To test if additional co-opted host proteins are also present in the vir-condensate portion of VROs, we selected Rpn11, which is proteasomal key protein interaction 'hub', a cytosolic protein [38,51]. Rpn11 is a pro-viral host factor and it is efficiently co-opted into TBSV and CIRV VROs [38,51,52]. FRAP experiments revealed that Rpn11 was present in vir-condensates induced by TBSV p33 or CIRV p36 replication proteins (Fig 3A -B).…”

“…Interestingly, TBSV p33 replication protein targets the proteasomal Rpn11 key protein interaction 'hub', which is co-opted to facilitate the recruitment of glycolytic and fermentation enzymes into VROs [38]. It is currently not known how all these co-opted cytosolic proteins are sequestered and enriched within VROs.…”

Manipulation of the Cellular Membrane-Cytoskeleton Network for RNA Virus Replication and Movement in Plants

“…The stabilized actin filaments function as “trafficking highways” to ensure the efficient and rapid delivery of viral proteins and proviral host components required for VRO assembly [ 121 , 123 , 142 ]. For instance, through the subverted AFs, TBSV recruits Rpn11, a cytosolic protein interaction hub, to deliver cytosolic proteins, such as glycolytic and fermentation enzymes, to generate energy essential for VRO growth and viral replication [ 143 ]. Moreover, TBSV may also take advantage of the actin-assisted rapid biogenesis of VROs to limit the recruitment of cellular restriction factors (CIRFs) [ 144 ].…”

Recombination in Positive-Strand RNA Viruses

“…Host factors, combined with environmental factors that affect the Met22/Xrn1 pathway, seem to also play a role in viral RNA recombination ( Jaag and Nagy, 2010 ). Host cellular RNA helicase RH20 and proteasomal Rpn11 protein can affect RNA recombination and viral replication ( Prasanth et al, 2015 ; Wu and Nagy, 2020 ; Molho et al, 2021 ; Figure 3D ).…”