Protein disulfide isomerases (PDIs) negatively regulate ebolavirus structural glycoprotein expression in the endoplasmic reticulum (ER) via the autophagy-lysosomal pathway

Zaire ebolavirus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates with high morbidity and mortality. EBOV infection is dependent on its structural glycoprotein (GP), but high levels of GP expression also trigger cell rounding, detachment, and downregulation of many surface molecules that is thought to contribute to its high pathogenicity. Thus, EBOV has evolved an RNA editing mechanism to reduce its GP expression and increase its fitness. We now report that the GP expression is also suppressed at the protein level in cells by protein disulfide isomerases (PDIs). Although PDIs promote oxidative protein folding by catalyzing correct disulfide formation in the endoplasmic reticulum (ER), PDIA3/ERp57 adversely triggered the GP misfolding by targeting GP cysteine residues and activated the unfolded protein response (UPR). Abnormally folded GP was targeted by ER-associated protein degradation (ERAD) machinery and, unexpectedly, was degraded via the macroautophagy/autophagy-lysosomal pathway, but not the proteasomal pathway. PDIA3 also decreased the GP expression from other ebolavirus species but increased the GP expression from Marburg virus (MARV), which is consistent with the observation that MARV-GP does not cause cell rounding and detachment, and MARV does not regulate its GP expression via RNA editing during infection. Furthermore, five other PDIs also had a similar inhibitory activity to EBOV-GP. Thus, PDIs negatively regulate ebolavirus glycoprotein expression, which balances the viral life cycle by maximizing their infection but minimizing their cellular effect. We suggest that ebolaviruses hijack the host protein folding and ERAD machinery to increase their fitness via reticulophagy during infection. <b>Abbreviations:</b> 3-MA: 3-methyladenine; 4-PBA: 4-phenylbutyrate; ACTB: β-actin; ATF: activating transcription factor; ATG: autophagy-related; BafA1: bafilomycin A₁; BDBV: <i>Bundibugyo</i> ebolavirus; CALR: calreticulin; CANX: calnexin; CHX: cycloheximide; CMA: chaperone-mediated autophagy; ConA: concanamycin A; CRISPR: clusters of regularly interspaced short palindromic repeats; Cas9: CRISPR-associated protein 9; dsRNA: double-stranded RNA; EBOV: <i>Zaire</i> ebolavirus; EDEM: ER degradation enhancing alpha-mannosidase like protein; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; Env: envelope glycoprotein; ER: endoplasmic reticulum; ERAD: ER-associated protein degradation; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; GP: glycoprotein; HA: hemagglutinin; HDAC6: histone deacetylase 6; HMM: high-molecular-mass; HIV-1: human immunodeficiency virus type 1; HSPA5/BiP: heat shock protein family A (Hsp70) member 5; IAV: influenza A virus; IP: immunoprecipitation; KIF: kifenesine; Lac: lactacystin; LAMP: lysosomal associated membrane protein; MAN1B1/ERManI: mannosidase alpha class 1B member 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MARV: Marburg virus; MLD: mucin-like domain; NHK/SERPINA1: alpha1-antitrypsin variant null (Hong Kong); NTZ: nitazoxanide; PDI: protein disulfide isomerase; RAVV: Ravn virus; RESTV: Reston ebolavirus; SARS-CoV: severe acute respiratory syndrome coronavirus; SBOV: <i>Sudan</i> ebolavirus; sGP: soluble GP; SQSTM1/p62: sequestosome 1; ssGP: small soluble GP; TAFV: Taï Forest ebolavirus; TIZ: tizoxanide; TGN: thapsigargin; TLD: TXN (thioredoxin)-like domain; Ub: ubiquitin; UPR: unfolded protein response; VLP: virus-like particle; VSV: vesicular stomatitis virus; WB: Western blotting; WT: wild-type; XBP1: X-box binding protein 1.

扎伊尔埃博拉病毒（Zaire ebolavirus, EBOV）可引发人类与非人灵长类动物罹患重症出血热，具有极高的发病率与死亡率。埃博拉病毒的感染依赖其结构糖蛋白（glycoprotein, GP），但GP的高表达同样会引发细胞变圆、脱落，并下调多种表面分子，这被认为是其高致病性的关键诱因之一。因此，埃博拉病毒演化出RNA编辑机制以降低GP的表达量，提升自身适配性。
本研究证实，细胞内的蛋白质二硫键异构酶（protein disulfide isomerases, PDIs）亦可在蛋白质层面抑制GP的表达。尽管PDIs可通过催化内质网（endoplasmic reticulum, ER）内正确二硫键的形成，促进氧化型蛋白质折叠，但PDIA3/ERp57却会靶向GP的半胱氨酸残基，诱导GP错误折叠并激活未折叠蛋白反应（unfolded protein response, UPR）。
异常折叠的GP会被内质网相关蛋白降解（ER-associated protein degradation, ERAD）系统识别，且出乎意料的是，其降解途径为巨自噬/自噬-溶酶体通路，而非蛋白酶体通路。
PDIA3同样可降低其他埃博拉病毒物种的GP表达，但却会提升马尔堡病毒（Marburg virus, MARV）的GP表达，这与下述观察结果一致：MARV-GP不会引发细胞变圆与脱落，且马尔堡病毒在感染过程中不会通过RNA编辑机制调控GP的表达。此外，其余五种PDI同样对埃博拉病毒GP具有类似的抑制活性。
综上，PDIs负向调控埃博拉病毒糖蛋白的表达，通过最大化病毒感染能力同时削弱其细胞毒性，平衡病毒的生命周期。我们推测，埃博拉病毒在感染过程中可劫持宿主的蛋白质折叠与ERAD系统，经由网状自噬提升自身适配性。
<b>缩略语：</b>3-MA：3-甲基腺嘌呤；4-PBA：4-苯基丁酸；ACTB：β-肌动蛋白；ATF：激活转录因子；ATG：自噬相关；BafA1：巴弗洛霉素A₁；BDBV：本迪布焦埃博拉病毒（Bundibugyo ebolavirus）；CALR：钙网蛋白；CANX：钙连蛋白；CHX：环己酰亚胺；CMA：分子伴侣介导的自噬；ConA：伴刀豆球蛋白A；CRISPR：成簇规律间隔短回文重复序列；Cas9：CRISPR相关蛋白9；dsRNA：双链RNA；EBOV：扎伊尔埃博拉病毒（Zaire ebolavirus）；EDEM：内质网降解增强α-甘露糖苷酶样蛋白；EIF2AK3/PERK：真核翻译起始因子2α激酶3；Env：包膜糖蛋白；ER：内质网；ERAD：内质网相关蛋白降解；ERN1/IRE1：内质网至细胞核信号转导蛋白1；GP：糖蛋白；HA：血凝素；HDAC6：组蛋白脱乙酰酶6；HMM：高分子量；HIV-1：人类免疫缺陷病毒1型；HSPA5/BiP：热休克蛋白家族A（Hsp70）成员5；IAV：甲型流感病毒；IP：免疫沉淀；KIF： kifenesine；Lac：乳胞素；LAMP：溶酶体相关膜蛋白；MAN1B1/ERManI：α-甘露糖苷酶1B家族成员1；MAP1LC3/LC3：微管相关蛋白1轻链3；MARV：马尔堡病毒；MLD：黏蛋白样结构域；NHK/SERPINA1：α1-抗胰蛋白酶缺失突变体（香港型）；NTZ：硝唑尼特；PDI：蛋白质二硫键异构酶；RAVV：拉文病毒；RESTV：莱斯顿埃博拉病毒；SARS-CoV：严重急性呼吸综合征冠状病毒；SBOV：苏丹埃博拉病毒（Sudan ebolavirus）；sGP：可溶性GP；SQSTM1/p62：自噬受体p62/隔离体1；ssGP：小型可溶性GP；TAFV：塔伊森林埃博拉病毒；TIZ：替哨唑；TGN：毒胡萝卜素；TLD：硫氧还蛋白样结构域；Ub：泛素；UPR：未折叠蛋白反应；VLP：病毒样颗粒；VSV：水疱性口炎病毒；WB：蛋白质免疫印迹；WT：野生型；XBP1：X盒结合蛋白1。