The IFN-I response is one of the first lines of protection against DENV infection, and serves to curb viral replication and dissemination by generating an antiviral intracellular environment 
. The potency of the type I IFN pathway is exemplified by the fact that DENV antagonizes both IFN synthesis and IFN signaling in order to ensure its replication and survival 
. DENV NS5 inhibits IFN-I signaling by mediating proteasome-dependent STAT2 degradation, and STAT2 degradation promotes DENV replication 
. With this study, we report the discovery of a host factor, UBR4, that is essential for DENV-dependent STAT2 degradation. We describe the interaction of UBR4 with NS5 and show that this interaction is crucial for inhibiting type-I IFN signaling and promoting efficient DENV replication.
UBR4 associates with DENV NS5 but not with the closely related YFV NS5 or WNV NS5. UBR4 also binds preferentially to proteolytically-processed DENV NS5, which is the form of NS5 that efficiently mediates STAT2 degradation. Binding of UBR4 to DENV NS5 requires amino acids T2 and G3 of NS5, which are also critical for STAT2 degradation. These amino acids are conserved amongst the four DENV serotypes but are absent in other flaviviruses (). Though NS5 is the most highly conserved flavivirus protein, the high degree of specificity exhibited by UBR4 for DENV NS5 underscores the differences between the various flaviviral NS5 proteins.
In 293T cells and primary human dendritic cells, DENV replicates best when UBR4 levels are normal, but when UBR4 levels are reduced, DENV-mediated STAT2 degradation is reduced and DENV replication decreases as a consequence ( and ). In Vero cells, which do not produce IFN-I 
, UBR4 depletion does not affect DENV replication unless these cells are treated with exogenous IFN-I (). Furthermore, the DENV replication defect caused by UBR4 knockdown in 293T cells can be decreased by treating the cells with antibodies that block the IFN-I receptor and decrease IFN-I signaling (). The DENV replication defect seen in UBR4-knockdown 293Ts and MDDCs can be explained by an increase in ISG levels in DENV-infected UBR4-knockdown cells versus DENV-infected control cells ( and ). Thus, in the absence of IFN-I, there is no need for DENV to antagonize IFN-I signaling and cellular levels of UBR4 are irrelevant for DENV replication. However, upon activation of the IFN-I signaling pathway, UBR4 becomes necessary for DENV replication. Reducing STAT2 levels is essential for DENV to preempt the establishment of a cellular antiviral state, thus ensuring its efficient replication.
Antagonism of IFN signaling is one of the factors responsible for the limited host tropism of DENV to human and nonhuman primates. DENV does not replicate to high levels or induce disease in IFN-competent mice 
. Our previous results indicated that the cellular machinery needed for DENV replication in murine cells is in place but is limited by the inability of NS5 to associate with murine STAT2 and inhibit murine IFN-I signaling 
. Other blocks such as the type II IFN pathway also diminish DENV replication in mice, but the IFN-I signaling pathway restricts early replication 
. Here we show that DENV NS5 associates with murine UBR4 in murine cells. This is in keeping with our previous results 
, and suggests that the development of a genetically-modified mouse that expresses a functional human STAT2 in place of its murine counterpart should allow increased DENV replication. We predict, therefore, that DENV NS5 will mediate human STAT2 degradation in these mice by co-opting mouse UBR4. Such a mouse might provide the basis for the development of an immune-competent mouse model of DENV infections.
The 600 kDa large UBR4 is highly conserved and found in organisms as diverse as mammals, insects, plants and worms. It belongs to the N-recognin family, which contains proven and predicted E3 ligases that recognize and degrade proteins containing destabilizing N termini. The seven members of the UBR family, UBR1 to UBR7, encode a 70-amino-acid zinc finger motif known as the UBR box, which is necessary for substrate recognition 
. The better-characterized members of the UBR family are UBR1, UBR2 and UBR5. UBR1 and UBR2 are RING domain-containing N-recognins, which recognize N-end rule substrates and target them for degradation 
. UBR1 and UBR2 are also involved in N-end-rule-independent quality control protein degradation 
. UBR5 is a HECT-domain containing E3 ligase that binds N-end rule substrates 
, but can also target non-N-end rule substrates like E6AP for degradation 
. UBR4 contains neither a HECT nor a RING domain.
A dearth of UBR4 literature exists because of the difficulty that manipulating the UBR4 gene presents. The UBR4 gene contains 106 exons, and produces multiple splice variants that conceivably have different functions. UBR4 forms a chromatin scaffold when bound to retinoblastoma protein (Rb) in the nucleus, and it also influences cytoskeleton organization by binding clathrin in the cytoplasm 
. Both of these are structural roles for which no N-end rule or other E3 ligase activities have been detected. A second virus, human papilloma virus, is known to exploit UBR4's role in cellular morphology to initiate anchorage-independent growth and cellular transformation 
. Although UBR4 is part of a family of UBR E3 ligases involved in the N-end rule pathway, the involvement of the N-end rule in the NS5-dependent degradation of STAT2 seems unlikely. Our group has previously demonstrated that the identity of NS5's first residue is not relevant for STAT2 degradation as long as the precursor is correctly processed 
. In addition, we show that residues T2 and G3 of NS5 are critical for binding to UBR4 and for mediating STAT2 degradation, but they are considered to be stabilizing residues within the N-end rule. This does not exclude UBR4 from having E3 ligase activity that is independent of the N-end rule. Though it lacks an obvious catalytic domain such as the HECT or RING domains, UBR4 contains a cysteine-rich domain (CRD) that is unique to the UBR4 group. It is currently unknown if CRD functions as a ligase domain. Our experiments with the N-terminal region of UBR4 suggest that domains from the C terminus, which contain the CRD, are necessary for its function in DENV-mediated STAT2 degradation.
Finally, we propose two working models: one based on the hypothesized UBR4 E3 ligase catalytic activity, and another which postulates a scaffolding role for UBR4 based on its described interactions with clathrin and retinoblastoma protein (). Efforts are currently being made to clone and express the predicted UBR4 isoforms so as to further evaluate the function of UBR4 in DENV-mediated STAT2 degradation, and to explore its potential as a target for rationally-designed DENV therapeutics.
Model of DENV-mediated STAT2 degradation.