The Proteasomal Rpn11 Metalloprotease Suppresses Tombusvirus RNA Recombination and Promotes Viral Replication via Facilitating Assembly of the Viral Replicase Complex

Abstract

RNA viruses co-opt a large number of cellular proteins that affect virus replication and, in some cases, viral genetic recombination. RNA recombination helps viruses in an evolutionary arms race with the host's antiviral responses and adaptation of viruses to new hosts. Tombusviruses and a yeast model host are used to identify cellular factors affecting RNA virus replication and RNA recombination. In this study, we have examined the role of the conserved Rpn11p metalloprotease subunit of the proteasome, which couples deubiquitination and degradation of proteasome substrates, in tombusvirus replication and recombination in Saccharomyces cerevisiae and plants. Depletion or mutations of Rpn11p lead to the rapid formation of viral RNA recombinants in combination with reduced levels of viral RNA replication in yeast or in vitro based on cell extracts. Rpn11p interacts with the viral replication proteins and is recruited to the viral replicase complex (VRC). Analysis of the multifunctional Rpn11p has revealed that the primary role of Rpn11p is to act as a "matchmaker" that brings the viral p92 pol replication protein and the DDX3-like Ded1p/RH20 DEAD box helicases into VRCs. Overexpression of Ded1p can complement the defect observed in rpn11 mutant yeast by reducing TBSV recombination. This suggests that Rpn11p can suppress tombusvirus recombination via facilitating the recruitment of the cellular Ded1p helicase, which is a strong suppressor of viral recombination, into VRCs. Overall, this work demonstrates that the co-opted Rpn11p, which is involved in the assembly of the functional proteasome, also functions in the proper assembly of the tombusvirus VRCs. IMPORTANCERNA viruses evolve rapidly due to genetic changes based on mutations and RNA recombination. Viral genetic recombination helps viruses in an evolutionary arms race with the host's antiviral responses and facilitates adaptation of viruses to new hosts. Cellular factors affect viral RNA recombination, although the role of the host in virus evolution is still understudied. In this study, we used a plant RNA virus, tombusvirus, to examine the role of a cellular proteasomal protein, called Rpn11, in tombusvirus recombination in a yeast model host, in plants, and in vitro. We found that the cellular Rpn11 is subverted for tombusvirus replication and Rpn11 has a proteasome-independent function in facilitating viral replication. When the Rpn11 level is knocked down or a mutated Rpn11 is expressed, then tombusvirus RNA goes through rapid viral recombination and evolution. Taken together, the results show that the co-opted cellular Rpn11 is a critical host factor for tombusviruses by regulating viral replication and genetic recombination. V iruses and their hosts go through continuous evolution, including a genetic arms race between viral and host factors that decides if the given virus could accomplish successful infection in that host. RNA virus adaptation to the host is facilitated by the ability of RNA viruses to evolve rapidly due to high-frequency muta...

Plant Virus Infection and the Ubiquitin Proteasome Machinery: Arms Race along the Endoplasmic Reticulum

“…Normally, Rpn11p functions to couple de-ubiquitination of substrates with proteasomal degradation, and also contributes to tombusvirus replication [31,38,39]. Rpn11p gained the attention of researchers working on TBSV replication because it was initially identified to function in peroxisome division, and TBSV replication occurs inside spherules along peroxisomal membranes.…”

Cellular Ubc2/Rad6 E2 ubiquitin-conjugating enzyme facilitates tombusvirus replication in yeast and plants

“…The Nedd4-type Rsp5p E3 Ub-ligase has also been shown to bind to and ubiquitinylate p33 replication protein in vitro (Barajas et al, 2009b). Studies with the proteasomal Rpn11p metalloprotease, which acts as a deubiquitination (DUB) enzyme, has shown the role of Rpn11p in the assembly of TBSV VRCs, and the recruitment of the cellular DDX3-like Ded1p DEAD-box helicase into the viral replicase (Prasanth et al, 2014). Data also support the role of Rpn11p and the free ubiquitin pool in TBSV replication and viral RNA recombination (Prasanth et al, 2014).…”

“…Studies with the proteasomal Rpn11p metalloprotease, which acts as a deubiquitination (DUB) enzyme, has shown the role of Rpn11p in the assembly of TBSV VRCs, and the recruitment of the cellular DDX3-like Ded1p DEAD-box helicase into the viral replicase (Prasanth et al, 2014). Data also support the role of Rpn11p and the free ubiquitin pool in TBSV replication and viral RNA recombination (Prasanth et al, 2014). Altogether, the emerging idea from these studies on TBSV that ubiquitin and protein ubiquitination is a major element in virus replication and evolution.…”

Viral Sensing of the Subcellular Environment Regulates the Assembly of New Viral Replicase Complexes during the Course of Infection

“…Multiple genome-wide screens with TBSV in yeast indicated that depletion of a proviral host factor does not necessarily prevent the assembly of the VRCs (4) but frequently leads to incorrect VRC assembly that changes some of the activities of the viral replicase. For example, depletion of multifunctional proteasomal Rpn11p metalloprotease in yeast results in inefficient recruitment of Ded1p DEAD box helicase into the tombusvirus VRCs (14). The low level of Ded1p leads to reduced replication and the production of high levels of truncated viral RNAs and recombinant viral RNAs (14).…”

“…For example, depletion of multifunctional proteasomal Rpn11p metalloprotease in yeast results in inefficient recruitment of Ded1p DEAD box helicase into the tombusvirus VRCs (14). The low level of Ded1p leads to reduced replication and the production of high levels of truncated viral RNAs and recombinant viral RNAs (14). Also, depletion of ESCRT proteins (Vps24 or Vps4p) results in incorrect assembly of tombusvirus VRCs in yeast, which gives rise to spherules with wide openings, instead of the narrow "neck-like" opening in wild-type (wt) yeast (8).…”