The matrix protein of lyssavirus hijacks autophagosome for efficient egress by recruiting NEDD4 through its PPxY motif.

ABSTRACT

Lyssaviruses are well-known worldwide and often cause fatal encephalitis. Previous studies have shown that autophagy is beneficial for the replication of rabies virus (RABV), the representative lyssavirus, but the detailed mechanism remains obscure. In this study, we showed that the rabies virus matrix protein (RABV-M) used its PPxY motif to interact with the E3 ubiquitin-protein ligase NEDD4. NEDD4 then recruited MAP1LC3/LC3 via its LC3-interacting region (LIR). Interestingly, after binding to the ubiquitinated RABV-M, NEDD4 could bind more LC3 and enhance autophagosome accumulation, while NEDD4 knockdown significantly reduced M-induced autophagosome accumulation. Further study revealed that RABV-M prevented autophagosome-lysosome fusion and facilitated viral budding. Inhibition of RABV-M-induced autophagosome accumulation reduced the production of extracellular virus-like particles. We also found that M proteins of most lyssaviruses share the same mechanism to accumulate autophagosome by hijacking NEDD4. Collectively, this study revealed a novel strategy for lyssaviruses to achieve efficient viral replication by exploiting the host autophagy system.Abbreviations ABLV Australian bat lyssavirus; ATG5 autophagy related 5; Baf A1bafilomycin A1;co-IP co-immunoprecipitation; CQ chloroquine; DAPI4',6-diamidino-2'-phenylindole; DMSO dimethyl sulfoxide; EBLVEuropean bat lyssavirus; GFP green fluorescent protein; GSTglutathione S-transferase; hpi hours post-infection; hpt hourspost-transfection; LIR LC3-interactingregion;MAP1LC3/LC3 microtubule-associated protein 1 light chain 3; mCherryred fluorescent protein; MOI multiplicity of infection; NC negativecontrol; MVB multivesicular body; NEDD4 neural precursorcell-expressed developmentally down-regulated 4; RABV rabies virus;SQSTM1/p62 sequestosome 1; VLP virus-like particle; VPS4B vacuolarprotein sorting 4B; TEM transmission electron microscopy; WBwestern blotting; WT wild-type; µm micrometer; µM micromole.