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

Abstract

Replication of (؉)RNA viruses depends on several co-opted host proteins but is also under the control of cell-intrinsic restriction factors (CIRFs). By using tombusviruses, small model viruses of plants, we dissect the mechanism of inhibition of viral replication by cellular WW-domain-containing proteins, which act as CIRFs. By using fusion proteins between the WW domain and the p33 replication protein, we show that the WW domain inhibits the ability of p33 to bind to the viral RNA and to other p33 and p92 replication proteins leading to inhibition of viral replication in yeast and in a cell extract. Overexpression of WWdomain protein in yeast also leads to reduction of several co-opted host factors in the viral replicase complex (VRC). These host proteins, such as eEF1A, Cdc34 E2 ubiquitin-conjugating enzyme, and ESCRT proteins (Bro1p and Vps4p), are known to be involved in VRC assembly. Simultaneous coexpression of proviral cellular factors with WW-domain protein partly neutralizes the inhibitory effect of the WW-domain protein. We propose that cellular WW-domain proteins act as CIRFs and also as regulators of tombusvirus replication by inhibiting the assembly of new membrane-bound VRCs at the late stage of infection. We suggest that tombusviruses could sense the status of the infected cells via the availability of cellular susceptibility factors versus WWdomain proteins for binding to p33 replication protein that ultimately controls the formation of new VRCs. This regulatory mechanism might explain how tombusviruses could adjust the efficiency of RNA replication to the limiting resources of the host cells during infections. IMPORTANCEReplication of positive-stranded RNA viruses, which are major pathogens of plants, animals, and humans, is inhibited by several cell-intrinsic restriction factors (CIRFs) in infected cells. We define here the inhibitory roles of the cellular Rsp5 ubiquitin ligase and its WW domain in plant-infecting tombusvirus replication in yeast cells and in vitro using purified components. The WW domain of Rsp5 binds to the viral RNA-binding sites of p33 and p92 replication proteins and blocks the ability of these viral proteins to use the viral RNA for replication. The WW domain also interferes with the interaction (oligomerization) of p33 and p92 that is needed for the assembly of the viral replicase. Moreover, WW domain also inhibits the subversion of several cellular proteins into the viral replicase, which otherwise play proviral roles in replication. Altogether, Rsp5 is a CIRF against a tombusvirus, and it possibly has a regulatory function during viral replication in infected cells. P lus-stranded (ϩ)RNA viruses, which are widespread and emerging pathogens, replicate in the cytosol of infected cells by assembling membrane-bound viral replicase complexes (VRCs). The VRCs consist of the viral RNA and viral proteins, as well as co-opted host-coded proteins (1-8). Rapid progress has recently been made in understanding the functions of the viral replication proteins, including the v...

Viruses go modular

“…Plants have developed innate and adaptive immune pathways, including cell-intrinsic restriction factors (CIRFs), that limit plant virus replication and disease (136). Indeed, the yeast E3 ubiqui-JBC REVIEWS: Viruses go modular tin ligase Rsp5 was identified as a CIRF, as its WW domains interact with the RNA-binding sites on two TBSV replication proteins (p33 and p92pol), thus blocking their ability to function in viral replication (38,137,138). Interestingly, the ability to block viral replication was independent of the catalytic HECT domain and was instead dependent on binding of the viral polymerase proteins by the WW domain of Rsp5 itself (38).…”

“…Although a canonical PPxY motif is not present in either p33 or p92pol, these two viral proteins do share structural similarity that likely renders them capable of interacting with the WW domains of Rsp5. The Rsp5 WW domains not only interfere with the formation and assembly of the viral RNA replicase but also appear to inhibit subversion of additional host proteins required by the vial replicase to complete viral replication (138). In sum, these studies not only have identified host WW-domain interactors that can regulate replication of TBSV and perhaps other related plant viruses, but also have laid the groundwork for future studies to determine whether these modular interactions and WW domains themselves could serve as the basis for new antiviral strategies.…”

Viral Sensing of the Subcellular Environment Regulates the Assembly of New Viral Replicase Complexes during the Course of Infection

“…Depletion of multiple WW domain proteins in yeast resulted in especially error-prone replicase at the late stage of replication (17). This is likely due to incorrect VRC assembly, possibly caused by (i) prior depletion of one or more proviral factors at the earlier rounds of VRC assembly and (ii) the fact that the depleted WW domain proteins could not block new VRC assembly.…”

“…1, regulation 1). Increasing WW domain protein amount by overexpression interfered with complex formation between p33 replication protein and several cellular host factors, such as eEF1A, Bro1p, Vps4p, and Cdc34p, involved in VRC assembly (17). Moreover, the WW domain inhibited the binding of p33 to the viral RNA and the oligomerization of p33, thus blocking tombusvirus replication.…”

“…However, the cellular WW domain proteins bind to the replication proteins with lower affinity than the proviral host factors (17), making them poor competitors with coopted proviral host factors when these subverted cellular proteins are abundant. It was proposed that the WW domain proteins bind to newly translated viral replication proteins, blocking their functions, when the proviral factors have been exhausted (Fig.…”