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 A1:bafilomycin A₁;co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI:4’,6-diamidino-2’-phenylindole; DMSO: dimethyl sulfoxide; EBLV:European bat lyssavirus; GFP: green fluorescent protein; GST:glutathione S-transferase; hpi: hours post-infection; hpt: hourspost-transfection; LIR: LC3-interactingregion;MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry:red 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; WB:western blotting; WT: wild-type; μm: micrometer; μM: micromole.

KEYWORDS:

• Autophagosome
• lyssavirus
• matrix protein
• Rabies virus (RABV)
• viral budding
• virus-like particle (VLP)

Acknowledgements

This study was supported by the National Key Research and Development Program of China (No. 2022YFD1800100) and Fundamental Research Funds for the Central Universities (No. 2662023PY005). The funders had no conflicts of interest in the study design, data collection and analysis, publication decision, or manuscript preparation.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethics statement

All the animals used in this study were maintained in the animal facility of Huazhong Agricultural University and in compliance with the Regulations for the Administration of Affairs Concerning Experimental Animals made by the Ministry of Science and Technology of China. The experiments were carried out in accordance with the protocols (permit number HZAU-MO-2021–0063) approved by the Scientific Ethics Committee of Huazhong Agricultural University.

Data availability statement

We authors declare all data and materials are available on request.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2024.2338575

Additional information

Funding

The work was supported by the National Key Research and Development Program of China [2022YFD1800100].