The Nedd4-Type Rsp5p Ubiquitin Ligase Inhibits Tombusvirus Replication by Regulating Degradation of the p92 Replication Protein and Decreasing the Activity of the Tombusvirus Replicase

Abstract

pol replication proteins. We find that overexpression of Rsp5p inhibits TBSV replication in Saccharomyces cerevisiae yeast, while downregulation of Rsp5p leads to increased TBSV accumulation. The inhibition is caused by Rsp5p-guided degradation of p92 pol , while the negative effect on the p33 level is less pronounced. Interestingly, recombinant Rsp5p also inhibits TBSV RNA replication in a cell-free replication assay, likely due to its ability to bind to p33 and p92 pol . We show that the WW domain of Rsp5p, which is involved in protein interactions, is responsible for inhibition of TBSV replication, whereas the HECT domain, involved in protein ubiquitination, is not necessary for Rsp5p-mediated inhibition of viral replication. Overall, our data suggest that direct binding between Rsp5p and p92 pol reduces the stability of p92 pol , with consequent inhibition of TBSV replicase activity.Various interactions with their host cells are critical for plus-stranded (ϩ)RNA viruses as they attempt to utilize the host translation machinery to produce viral proteins, gain access to the resources of the host cells, co-opt host proteins, and subvert host membranes (1, 17). Additional levels of interaction between virus and host reflect antiviral responses which may involve innate immunity, as well as other antiviral processes and factors. On-going research with several model viruses is striving to map all the interactions between viruses and hosts and characterize the functions of the co-opted host factors. In this regard, recent research has led to the identification of a large number of host proteins which affect the replication of various (ϩ)RNA viruses and minus-stranded RNA viruses (4,5,9,11,22,35,39). The roles and functions of most of the host proteins identified as being involved in RNA virus replication, however, are currently unknown.Tombusviruses, such as Tomato bushy stunt virus (TBSV), are among the most advanced model systems in relation to the identification of host factors affecting (ϩ)RNA virus replication. The TBSV genome codes for only five proteins, two of which are the replication proteins translated directly from the genomic RNA (45). One of these replication proteins is the abundant p33 replication cofactor; the other is the RNA-dependent RNA polymerase (RdRp) p92pol . Due to the overlapping expression strategy, p33 is identical with the N-terminal portion of the larger p92 pol protein (Fig. 1A). Both replication proteins contain an RNA-binding motif (arginine-proline-rich motif), phosphorylation sites that affect RNA binding by the p33 protein, a p33-p33/p92 interaction domain, and two transmembrane domains (Fig. 1A) (18,19,32,36,37). Three short stretches of amino acids in p33 and p92 pol are involved in binding to the Pex19p host protein that facilitates the transportation of p33 and p92 pol from the cytosol to the cytosolic surface of the peroxisomes, the site of replicase complex formation and viral RNA replication (25). The essential nature of the above-named domains for obtaining functional rep...

Ubiquitin and Plant Viruses, Let’s Play Together!

“…Modifying the proper stoichiometry of replication complex subunits via selective degradation is thus likely to affect the outcome of viral replication. In some instances, proteasome subunits, E3 ligases, or cellular DUBs have been shown to affect the efficiency of viral replication, but such effects could not be linked directly to the degradation of viral proteins by the UPS and possibly could be due to indirect effects caused by cellular stress and/or perturbation of Ub homeostasis (Barajas et al, 2009b;Yamaji et al, 2010;Gancarz et al, 2011;Wang et al, 2011).…”

Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins

“…Plasmids pGBK-Hisp33-CUP1/DI72-GAL1, pGAD-His92-CUP1, pGAD-Flag92-CUP1, and pGBK-His33-CUP1 have been described before (47,64,68). To create plasmid pGAD-CFP-p92-CUP1, the 6ϫHis-CFP-p92 cassette was amplified by PCR with the primers 807 and 952 using plasmid pGAD-CFP-p92 as the template (34).…”

“…In addition to Rsp5p, several cellular WWdomain proteins, including Wwm1p, Prp40p, and plant AtDrh1, AtFCA, and AtPrp40c, bind to the tombusvirus replication proteins and inhibit their functions (64,65). Accordingly, binding of Rsp5p and other WW-domain proteins to the p92 pol replication protein leads to the degradation of p92 pol (64,65). The WW domain is a simple and highly conserved protein domain involved in protein-protein interactions (66,67).…”

“…The yeast Nedd4-type Rsp5p E3 ubiquitin ligase carrying WW domain was identified in several genome-wide screens previously (13,42,48). In addition to Rsp5p, several cellular WWdomain proteins, including Wwm1p, Prp40p, and plant AtDrh1, AtFCA, and AtPrp40c, bind to the tombusvirus replication proteins and inhibit their functions (64,65). Accordingly, binding of Rsp5p and other WW-domain proteins to the p92 pol replication protein leads to the degradation of p92 pol (64,65).…”

“…To test the possible regulatory functions of recruited cellular proteins, we chose the WW-domain-containing cellular proteins (64). The yeast Nedd4-type Rsp5p E3 ubiquitin ligase carrying WW domain was identified in several genome-wide screens previously (13,42,48).…”