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

Abstract

Mono- and multi-ubiquitination alters the functions and subcellular localization of many cellular and viral proteins. Viruses can co-opt or actively manipulate the ubiquitin network to support viral processes or suppress innate immunity. Using yeast (Saccharomyces cerevisiae) model host, we show that the yeast Rad6p (radiation sensitive 6) E2 ubiquitin-conjugating enzyme and its plant ortholog, AtUbc2, interact with two tombusviral replication proteins and these E2 ubiquitin-conjugating enzymes could be co-purified with the tombusvirus replicase. We demonstrate that TBSV RNA replication and the mono- and bi-ubiquitination level of p33 is decreased in rad6Δ yeast. However, plasmid-based expression of AtUbc2p could complement both defects in rad6Δ yeast. Knockdown of UBC2 expression in plants also decreases tombusvirus accumulation and reduces symptom severity, suggesting that Ubc2p is critical for virus replication in plants. We provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly.

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

“…The most in-depth examination of the role for the UPS in virus replication has involved studies of tomato bushy stunt virus (TBSV, genus Tombusvirus ) and cymbidium ringspot virus (CRSV, genus Tombusvirus ). Yeast has been used as a model genetic system to uncover critical host factors that contribute to tombusvirus infection [29,30,31], enabling the discovery of novel interactions involving cellular membranes which could be confirmed through focused experimentation in plant cells. In this yeast experimental system, cells are transformed to express the tombusviral p33 and p92 replication proteins, which function to replicate short tombusvirus replicons derived from viral defective interfering RNAs.…”

“…Rad6/ubc2 and cdc34p are E2 Ub-conjugating enzymes that are components of the viral replication complexes identified in pull down assays [31,35]. Both Rad6 and cdc34p are involved in several cellular processes and, with regard to tombusvirus replication, have redundant capabilities of adding ubiquitin moieties to the tombusvirus p33 protein.…”

“…Rad6p/ubc2b is capable of mono-ubiquitination of most substrates in the absence of an E3 ligase, and, in the case of the tombusvirus p33 protein, data indicates that this factor attaches two ubiquitin molecules to the substrate. Both Rad6p/ubc2p and cdc34p are important for recruiting the endosomal sorting complexes required for transport (ESCRT) proteins Vps23p and Vps4p to the viral replication complex [30,31,37]. TBSV replicase forms spherule compartments along membranes, and these ESCRT proteins function in membrane bending needed to create spherular compartments.…”

“…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.…”

Role of Viral RNA and Co-opted Cellular ESCRT-I and ESCRT-III Factors in Formation of Tombusvirus Spherules Harboring the Tombusvirus Replicase

“…The model proposed for the functions of ESCRT proteins in TBSV replication predicts that the membrane-bound p33 replication protein binds directly to Vps23p ESCRT-I protein (Tsg101 in mammals), based on its late domain sequence and the mono-and biubiquitin moieties after becoming posttranslationally modified by Ubc2/Rad6 or Cdc34 E2 ubiquitin-conjugating enzymes (32,38,50,51). This is followed by the additional recruitment of ESCRT-III cellular factors, such as Snf7p, Vps20p, and Vps24p, whose single deletions reduced TBSV replication in yeast (26).…”