Genome wide screens to identify host factors affecting TBSV

Genome-wide screens to identify host factors affecting TBSV RNA replication in yeast led to the identification of host genes known to be involved in various aspects of protein ubiquitination, such as BRE1, DOA4, RAD6, LGE1, UBP3 (Jiang et al., 2006, Panavas et al., 2005b, Serviene et al., 2005, Serviene et al., 2006). In addition, proteomics approach has revealed interaction between p33 replication protein and Uba1p ubiquitin- (Ub)-activating enzyme, Cdc34p E2 Ub-conjugating enzyme, Rsp5p E3 Ub-ligase, Ubp10p and Ubp15p Ub-specific proteases (Li et al., 2008). More detailed analysis with Cdc34p E2 Ub-conjugating enzyme showed that Cdc34p is present in the tombusvirus replicase complex and it can mono- and bi-ubiquitinate p33 in vitro in the absence of an E3 Ub-ligase (Li et al., 2008). 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). Altogether, the emerging idea from these studies on TBSV that ubiquitin and protein ubiquitination is a major Conessine mg in virus replication and evolution. Mono- and bi-ubiquitination of two lysines, namely K70 and K76, in a small fraction of p33 replication co-factor has been demonstrated in yeast (Barajas and Nagy, 2010, Li et al., 2008). Because mutations of these lysines reduced TBSV repRNA replication in yeast and affected the ability of p33 to interact with Vps23p ESCRT factor, we have proposed that one of the functions of p33 ubiquitination is to assist the recruitment of Vps23p ESCRT-I protein for TBSV replication (Barajas and Nagy, 2010). The recruitment of Vps23p, followed by subversion of additional ESCRT proteins could aid the formation of VRCs, which require membrane deformation to induce spherule-like structures (Barajas et al., 2009a, Barajas et al., 2014). Although the previous genome-wide and proteome-wide screens with TBSV in yeast have identified 10 yeast proteins involved in various aspects of the ubiquitin pathway (Nagy et al., 2014), we still do not know the functional roles of most of these cellular factors in virus replication. In the current paper, we have undertaken studies with Rad6p E2 ubiquitin-conjugating enzyme and its plant ortholog, Arabidopsis thaliana AtUbc2, in yeast and plants in combination with in vitro approaches. We find that both Rad6p and AtUbc2p interact with the p33 and p92pol replication proteins and they could be co-purified with the tombusvirus replicase. Rad6p/Ubc2p affects the ubiquitination level of p33 and deletion of RAD6 or knockdown of NbUBC2 reduces tombusvirus replication in yeast and plants, respectively. Both E2 ubiquitin-conjugating enzymes also facilitate TBSV replication in vitro, suggesting that they are directly involved in tombusvirus replication. We also provide evidence that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for VRC assembly.