Abstract：
The Lassa virus (LASV) is a widely recognized virulent pathogen that frequently results in lethal viral hemorrhagic fever (VHF). Earlier research has indicated that macroautophagy/autophagy plays a role in LASV replication, but, the precise mechanism is unknown. In this present study, we show that LASV matrix protein (LASV-Z) is essential for blocking intracellular autophagic flux. LASV-Z hinders actin and tubulin folding by interacting with CCT2, a component of the chaperonin-containing T-complexes (TRiC). When the cytoskeleton is disrupted, lysosomal enzyme transit is hampered. In addition, cytoskeleton disruption inhibits the merge of autophagosomes with lysosomes, resulting in autophagosome accumulation that promotes the budding of LASV virus-like particles (VLPs). Inhibition of LASV-Z-induced autophagosome accumulation blocks the LASV VLP budding process. Furthermore, it is found that glutamine at position 29 and tyrosine at position 48 on LASV-Z are important in interacting with CCT2. When these two sites are mutated, LASV-mut interacts with CCT2 less efficiently and can no longer inhibit the autophagic flux. These findings demonstrate a novel strategy for LASV-Z to hijack the host autophagy machinery to accomplish effective transportation.Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; ATG7: autophagy related 7; Baf-A1: bafilomycin A1; CCT2: chaperonin containing TCP1 subunit 2; co-IP: co-immunoprecipitation; CTSD: cathepsin D; DAPI: 4\',6-diamidino-2\'-phenylindole; DMSO: dimethyl sulfoxide; EGFR: epidermal growth factor receptor; GFP: green fluorescent protein; hpi: hours post-infection; hpt: hours post-transfection; LAMP1: lysosomal-associated membrane protein 1; LASV: lassa virus; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry: red fluorescent protein; PM: plasma membrane; SQSTM1/p62: sequestosome 1; STX6: syntaxin 6; VLP: virus-like particle; TEM: transmission electron microscopy; TRiC: chaperonin-containing T-complex; WB: western blotting; μm: micrometer; μM: micromole.
摘要：
拉沙病毒（LASV）是一种公认的毒力病原体，通常会导致致命的病毒性出血热（VHF）。早期的研究表明，巨自噬/自噬在LASV复制中起作用，但是,确切的机制是未知的。在本研究中,我们表明LASV基质蛋白（LASV-Z）对于阻断细胞内自噬通量至关重要。LASV-Z通过与CCT2相互作用而阻碍肌动蛋白和微管蛋白折叠,CCT2是含伴侣蛋白的T复合物(TRiC)的组分。当细胞骨架被破坏时，溶酶体酶转运受阻。此外,细胞骨架破坏抑制自噬体与溶酶体的合并，导致自噬体积累，促进LASV病毒样颗粒（VLP）的出芽。抑制LASV-Z诱导的自噬体积累阻断LASVVLP出芽过程。此外，发现LASV-Z上29位的谷氨酰胺和48位的酪氨酸在与CCT2的相互作用中是重要的。当这两个位点发生突变时,LASV-mut与CCT2的相互作用效率较低，并且不再抑制自噬通量。这些发现证明了LASV-Z劫持宿主自噬机制以实现有效运输的新策略。
