Molecular basis for Ebolavirus VP35 suppression of human dendritic cell maturation

Zaire ebolavirus (EBOV) VP35 is a double-stranded RNA (dsRNA)-binding protein that inhibits RIG-I signaling and alpha/beta interferon (IFN-α/β) responses by both dsRNA-binding-dependent and -independent mechanisms. VP35 also suppresses dendritic cell (DC) maturation. Here, we define the pathways and mechanisms through which VP35 impairs DC maturation. Wildtype VP35 (VP35-WT) and two well-characterized VP35 mutants (F239A and R322A) that independently ablate dsRNA binding and RIG-I inhibition were delivered to primary human monocyte-derived DCs (MDDCs) using a lentivirus-based expression system. VP35-WT suppressed not only IFN-α/β but also proinflammatory responses following stimulation of MDDCs with activators of RIG-I-like receptor (RLR) signaling, including RIG-I activators such as Sendai virus (SeV) or 5'-triphosphate RNA, or MDA5 activators such as encephalomyocarditis virus (EMCV) or poly(I·C). The F239A and R322A mutants exhibited greatly reduced suppression of IFN-α/β and proinflammatory cytokine production following treatment of DCs with RLR agonists. VP35-WT also blocked the upregulation of DC maturation markers and the stimulation of allogeneic T cell responses upon SeV infection, whereas the mutants did not. In contrast to the RLR activators, VP35-WT and the VP35 mutants impaired IFN-β production induced by Toll-like receptor 3 (TLR3) or TLR4 agonists but failed to inhibit proinflammatory cytokine production induced by TLR2, TLR3, or TLR4 agonists. Furthermore, VP35 did not prevent lipopolysaccharide (LPS)-induced upregulation of surface markers of MDDC maturation and did not prevent LPS-triggered allogeneic T cell stimulation. Therefore, VP35 is a general antagonist of DC responses to RLR activation. However, TLR agonists can circumvent many of the inhibitory effects of VP35. Therefore, it may be possible to counteract EBOV immune evasion by using treatments that bypass the VP35-imposed block to DC maturation.