Hepatitis C virus NS5A protein promotes the lysosomal degradation of diacylglycerol O-acyltransferase 1 (DGAT1) via endosomal microautophagy
Hepatitis C virus NS5A protein promotes the lysosomal degradation of diacylglycerol O-acyltransferase 1 (DGAT1) via endosomal microautophagy
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丙型肝炎病毒 NS5A 蛋白通过内体微自噬促进二酰基甘油 O-酰基转移酶 1 (DGAT1) 的溶酶体降解
DOI：
10.1080/27694127.2022.2095591
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Ikuo Shoji
中科院分区：
文献类型：
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作者：
Putu Yuliandari;Chieko Matsui;Lin Deng;Takayuki Abe;Hiroyuki Mori;Shuhei Taguwa;Chikako Ono;Takasuke Fukuhara;Yoshiharu Matsuura;Ikuo Shoji
许多病毒通常使用蛋白质降解系统(如泛素-蛋白酶体途径或溶酶体途径)来调节病毒的繁殖和病毒的致病。我们报道丙型肝炎病毒感染通过NS5A介导的肝细胞核因子-1α与细胞伴侣热休克相关蛋白70 kDa的结合促进肝细胞核因子-1α的溶酶体降解。HSC70与hnf-1α蛋白上的五肽KFERQ基序(也称为CmA靶向基序)结合，促进hnf-1α的溶酶体降解。KFERQ基序在CMA和EMI两条溶酶体降解途径中起着至关重要的作用。在这里，我们通过检测携带KFERQ基序的NS5A相互作用蛋白来寻找一种新的丙型肝炎病毒诱导溶酶体降解的底物。我们发现二酰基甘油O-酰基转移酶1(DGAT1)是丙型肝炎病毒颗粒形成的关键因子，是丙型肝炎病毒诱导的溶酶体降解途径的候选底物。DGAT1蛋白149-153个氨基酸组成的区域与KFERQ基序的规则相匹配。DGAT1蛋白与HSC70共沉淀，而DGAT1 Q149A突变体不与HSC70共沉淀，提示KFERQ基序参与了DGAT1与HSC70的相互作用。在HCVJ6/JFH1感染的细胞中，敲除LAMP-2A蛋白不能恢复DGAT1蛋白，而VPS4B基因敲除后恢复了DGAT1蛋白的水平，表明DGAT1通过EMI降解。简写3-MA：3-甲基腺嘌呤；AA：氨基酸；AH：两亲性螺旋；BSA：牛血清白蛋白；CMA：伴侣介导的自噬；DAAs：直接作用抗病毒；DGAT1：二酰甘油O-酰基转移酶1；DMSO：二甲基亚砜；EL：细胞外腔；EMI：微体自噬；ESCRT：转运所需的内体分选复合体；丙型肝炎病毒；HNF-α：肝细胞核因子-1α；HRP：辣根过氧化物酶；HSC70：热休克认知k70蛋白；IB：免疫印迹；IL：细胞内腔；IP：免疫沉淀；LAMP-2A：溶酶体相关膜蛋白2A型；LCS：低复杂性序列；mAb：单抗；MOI：感染的多样性；MVB：多泡体；NS：非结构蛋白；Pab：多克隆抗体；PBS：磷酸盐缓冲盐水；PCR：聚合酶链式反应；聚乳酸：邻近连接试验；PS：磷脂酰丝氨酸；RT：室温；TM：跨膜；TSG：肿瘤易感基因；VPS4A：空泡蛋白分类相关蛋白4A；VPS4B：空泡蛋白分类相关蛋白4B
Many viruses often use a protein degradation system (e.g., the ubiquitin-proteasome pathway or lysosome pathway) to modulate viral propagation and viral pathogenesis. We reported that hepatitis C virus (HCV) infection promotes the lysosomal degradation of hepatocyte nuclear factor-1α (HNF-1α) via chaperone-mediated autophagy (CMA) through an NS5A-mediated association of HNF-1α with cellular chaperone heat shock cognate 70 kDa (HSC70) protein. HSC70 binds to the pentapeptide KFERQ motif (also known as a CMA-targeting motif) on HNF-1α protein and promotes the lysosomal degradation of HNF-1α. The KFERQ motif plays a crucial role in the two lysosomal degradation pathways, CMA and endosomal microautophagy (eMI). Herein, we searched for a novel substrate of HCV-induced lysosomal degradation by examining the NS5A-interacting proteins that carry the KFERQ motif. We identified diacylglycerol O-acyltransferase 1 (DGAT1), which is a key factor for HCV particle formation, as a candidate substrate for HCV-induced lysosomal degradation pathway. The region spanning from amino acids 149–153 of DGAT1 protein matches the rule for the KFERQ motif. DGAT1 protein was co-immunoprecipitated with HSC70, whereas DGAT1 Q149A mutant was not co-immunoprecipitated with HSC70, suggesting that the KFERQ motif is responsible for the interaction between DGAT1 and HSC70. Knockdown of LAMP-2A protein in HCV J6/JFH1-infected cells did not recover DGAT1 protein, whereas knockdown of VPS4B recovered the level of DGAT1 protein, suggesting that DGAT1 is degraded via eMI. These findings lead us to propose that HCV NS5A protein facilitates the recruitment of HSC70 to DGAT1, thereby promoting the lysosomal degradation of DGAT1 via eMI.Abbreviations3-MA: 3-methyladenine; aa: amino acids; AH: amphipathic helix; BSA: bovine serum albumin; CMA: chaperone-mediated autophagy; DAAs: direct-acting antiviral; DGAT1: diacylglycerol O-acyltransferase 1; DMSO: dimethyl sulfoxide; EL: extracellular lumen; eMI: endosomal microautophagy; ESCRT: endosomal sorting complex required for transport; HA: hemagglutinin; HCV: hepatitis C virus; HNF-1α: hepatocyte nuclear factor-1α; HRP: horseradish peroxidase; HSC70: heat shock cognate 70 kDa protein; IB: immunoblotting; IL: intracellular lumen; IP: immunoprecipitation; LAMP-2A: lysosome-associated membrane protein type 2A; LCS: low-complexity sequences; mAb: monoclonal antibody; MOI: multiplicity of infection; MVB: multivesicular bodies; NS: nonstructural protein; pAb: polyclonal antibody; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PLA: proximity ligation assay; PS: phosphatidylserine; RT: room temperature; TM: transmembrane; TSG: tumor susceptibility gene; VPS4A: vacuolar protein sorting-associated protein 4A; VPS4B: vacuolar protein sorting-associated protein 4B