Mutation of Ebola virus VP35 Ser129 uncouples interferon antagonist and replication functions

Ebolaviruses are non-segmented, negative-sense RNA viruses (NNSVs) within the order that possess the multifunctional virion protein 35 (VP35), a major determinant of virulence and pathogenesis that is indispensable for viral replication and host innate immune evasion. VP35 is functionally equivalent to the phosphoprotein (P) of other mononegaviruses such as rhabdoviruses and paramyxoviruses. Phosphorylation of the P protein is universally regarded as functionally important however, a regulatory role(s) of phosphorylation on VP35 function remains unexplored. Here, we identified a highly conserved Ser129 residue near the homo-oligomerization coiled coil motif, which is essential for VP35 functions. Affinity-purification MS followed by post-translational modification (PTM) analysis predicted phosphorylation of Ser129. Co-immunoprecipitation, cross-linking, and biochemical characterization studies revealed a moderately decreased capacity of VP35-S129A to oligomerize. Functional analysis showed that Ser-to-Ala substitution of Ebola virus (EBOV) VP35 did not affect IFN inhibitory activity but nearly abolished EBOV minigenome activity. Further coimmunoprecipitation studies demonstrated a lost interaction between VP35-S129A and the amino terminus of the viral polymerase but not between viral nucleoprotein (NP) or VP35-WT. Taken together, our findings provide evidence that phosphorylation modulates VP35 function, supporting VP35 as a NNSV P protein and providing a potentially valuable therapeutic target.