Yuliandari Putu, Matsui Chieko, Deng Lin, Abe Takayuki, Mori Hiroyuki, Taguwa Shuhei, Ono Chikako, Fukuhara Takasuke, Matsuura Yoshiharu, Shoji Ikuo
Division of Infectious Disease Control, Center for Infectious Diseases, Kobe University Graduate School of Medicine, Kobe, Japan.
Medicine, Udayana University Department of Clinical Microbiology, Faculty of, Bali, Indonesia.
Autophagy Rep. 2022 Jul 22;1(1):264-285. doi: 10.1080/27694127.2022.2095591. eCollection 2022.
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. 3-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.
许多病毒常常利用蛋白质降解系统（如泛素 - 蛋白酶体途径或溶酶体途径）来调控病毒的增殖和发病机制。我们报道过，丙型肝炎病毒（HCV）感染通过伴侣介导的自噬（CMA）促进肝细胞核因子 -1α（HNF -1α）的溶酶体降解，这一过程是通过NS5A介导HNF -1α与细胞伴侣热休克同源70 kDa（HSC70）蛋白的结合来实现的。HSC70与HNF -1α蛋白上的五肽KFERQ基序（也称为CMA靶向基序）结合，并促进HNF -1α的溶酶体降解。KFERQ基序在CMA和内体微自噬（eMI）这两条溶酶体降解途径中起着关键作用。在此，我们通过检测携带KFERQ基序的NS5A相互作用蛋白，寻找HCV诱导的溶酶体降解的新底物。我们鉴定出二酰甘油O - 酰基转移酶1（DGAT1），它是HCV颗粒形成的关键因子，是HCV诱导的溶酶体降解途径的候选底物。DGAT1蛋白第149 - 153位氨基酸区域符合KFERQ基序的规则。DGAT1蛋白与HSC70共免疫沉淀，而DGAT1 Q149A突变体不与HSC70共免疫沉淀，这表明KFERQ基序负责DGAT1与HSC70之间的相互作用。在HCV J6/JFH1感染的细胞中敲低溶酶体相关膜蛋白2A（LAMP -2A）蛋白并不能恢复DGAT1蛋白水平，而敲低液泡蛋白分选相关蛋白4B（VPS4B）则能恢复DGAT1蛋白水平，这表明DGAT1是通过eMI降解的。这些发现使我们提出，HCV NS5A蛋白促进HSC70募集到DGAT1，从而通过eMI促进DGAT1的溶酶体降解。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：直接作用抗病毒药物；DGAT1：二酰甘油O - 酰基转移酶1；DMSO：二甲基亚砜；EL：细胞外腔；eMI：内体微自噬；ESCRT：运输所需的内体分选复合物；HA：血凝素；HCV：丙型肝炎病毒；HNF -1α：肝细胞核因子 -1α；HRP：辣根过氧化物酶；HSC70：热休克同源70 kDa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：单克隆抗体；MOI：感染复数；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：聚合酶链反应；PLA：邻近连接分析；PS：磷脂酰丝氨酸；RT：室温；TM：跨膜；TSG：肿瘤易感基因；VPS4A：液泡蛋白分选相关蛋白4A；VPS4B：液泡蛋白分选相关蛋白4B
