Mono and bi ubiquitination of two lysines namely
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 cycloheximide solubility 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.
Discussion High throughput genome-wide and proteome-wide screens in yeast model host for TBSV have helped tremendously the identification and characterization of host factors subverted for viral replication (Nagy et al., 2014). Follow up experiments using tombusviruses and plants (based on N. benthamiana host) have also showed that many of the identified host factors in yeast are relevant in the plant hosts, too. For example, co-opted host proteins, such as Hsp70, eEF1A, eEF1Bγ, GAPDH, DEAD-box helicases, cellular ion pumps, and ESCRT factors or the roles of sterols and phospholipids have all been confirmed to play important roles in the assembly of the tombusvirus VRCs or viral RNA synthesis in vitro, in yeast and plant cells (Barajas and Nagy, 2010, Jaag et al., 2010, Kovalev and Nagy, 2014, Li et al., 2009, Nagy et al., 2012, Nagy and Pogany, 2012, Sasvari et al., 2011, Sasvari et al., 2013, Wang and Nagy, 2008, Wang et al., 2009a, Xu and Nagy, 2015). In spite of systematic efforts, however, the previously identified and characterized yeast Cdc34p E2 ub-conjugating enzyme, which is a component of the tombusvirus replicase with pro-viral functions (Li et al., 2008), has not yet been confirmed in plants due to the lack of obvious ortholog(s). Therefore, we continued our search to find an E2 ubiquitin-conjugating enzyme from plants that is a component of the tombusvirus replicase with pro-viral functions. Our search was facilitated by the previous identification of a second E2 ubiquitin-conjugating enzyme from yeast, called Rad6p, which has an ortholog in plants, called Ubc2p (Qin, 2013, Strzalka et al., 2013, Xu et al., 2009). Accordingly, in this paper, we demonstrate similar characteristics for Rad6p/Ubc2p to Cdc34p, including (i) direct binding to the p33 replication protein in the MYTH assay; (ii) Co-purification of Rad6p/Ubc2p with the tombusvirus replicase from membrane fractions; (iii) Pro-viral function in tombusvirus replication, based on deletion of RAD6 in yeast or knockdown of UBC2 in N. benthamiana led to diminished tombusvirus RNA accumulation; (iv) Reduced level of p33 mono- and bi-ubiquitinylation when RAD6 has been deleted; (v) direct ubiquitination of recombinant p33 with purified Rad6p and AtUbc2p; (vi) Complementation of p33 mono- and bi-ubiquitinylation and TBSV repRNA accumulation in rad6Δ yeast expressing Rad6p or AtUbc2p from plasmids; (vii) Over-expression of AtUbc2p and Rad6p increased TBSV repRNA accumulation in wt yeast; (viii) Increased level of TBSV RNA synthesis with purified replicase from yeast expressing Rad6p/Ubc2p; and (ix) a ~3-fold increase in TBSV RNA replication in CFEs containing Rad6p/Ubc2p. All these data point at the important role of the plant Ubc2p and, similarly, the yeast Rad6p E2 ub-conjugating enzymes in tombusvirus replication in plants, yeast and in vitro.