In spite of systematic efforts however the previously identified

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 Anti-diabetic Compound Library 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 Anti-diabetic Compound Library 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.
Based on the similar activities of Rad6p and Cdc34p (i.e., both are E2 ub-conjugating enzymes) on ubiquitinylation of p33 replication protein, we propose that Rad6p and the plant Ubc2p mono- and bi-ubiquitinate the viral replication proteins in order to facilitate the recruitment of the cellular ESCRT proteins via binding to Vps23p (Tsg101 in mammals) ESCRT-I protein and Bro1p (ALIX in mammals) ESCRT-accessory protein (Barajas and Nagy, 2010). These events then lead to subversion of the cellular ESCRT machinery consisting of additional ESCRT-I and ESCRT-III components and Vps4p AAA+ ATPase (Barajas et al., 2009a, 2014; Barajas and Nagy, 2010). These cellular ESCRT proteins are likely involved in membrane bending/invagination and viral spherule formation during the assembly of the membrane-bound tombusvirus replicase complexes (Barajas et al., 2009a, 2014; Barajas and Nagy, 2010). In support of the above model, co-purification experiments showed that Rad6p is involved in promoting the subversion of Vps23p and Vps4p ESCRT proteins for viral replicase complex assembly.
Protein ubiquitination and the ubiquitin proteosome system (UPS) play a role in many viral infections by targeting viral proteins for degradation or for modification that leads to altered viral protein functions (Alcaide-Loridan and Jupin, 2012; Okumura et al., 2006; Shackelford and Pagano, 2005). Viruses also usurp UPS to target host antiviral proteins for degradation to facilitate viral infections. Viral proteins could also reverse protein ubiquitination that likely regulate viral infections (Alcaide-Loridan and Jupin, 2012; Chenon et al., 2012; Lindner, 2007; Lombardi et al., 2013).

Materials and methods

Acknowledgments
The authors thank to Dr. C. Boone (U. Toronto) for Cdc34 yeast. This work was supported by a grant for Overseas Researcher from Nihon University to Y. I. and by NSF (MCB 1122039) to PDN.

Background
While current antiretroviral therapies prolong the lifespan of people living with HIV/AIDS, they fail to completely clear the virus reservoir, resulting in a chronic infection (Ketseoglou et al., 2014; Kumar et al., 2014; Li et al., 2012). These antiretroviral regimens also have significant side effects, including lipodystrophy, hyperglycemia, renal dysfunction, osteoporosis, and cardiovascular disease (Carr and Cooper, 2000; Kalayjian et al., 2012; Max and Sherer, 2000; Towner et al., 2012). Due to side effects as well as the common problem of drug resistance, novel antiretrovirals are needed to offer new treatment options. With exception of CCR5 antagonists and other entry inhibitors, current HIV-1 drug regimens only target the viral enzymes.