ABSTRACT

The innate immune restriction factor SAMHD1 can inhibit diverse viruses in myeloid cells. Mechanistically, SAMHD1 inhibits lentiviral replication including HIV-1 by depleting the nucleotide pool to interfere with their reverse transcription. Equine infectious anemia virus (EIAV) is an ancient lentivirus that preferentially attacks macrophages. However, the mechanism by which EIAV successfully establishes infection in macrophages with functional SAMHD1 remains unclear. Here, we demonstrate that while equine SAMDH1 can limit EIAV replication in equine macrophages at the reverse transcription stage, the antiviral effect is counteracted by the well-known transcriptional regulator Rev, which downregulates equine SAMHD1 through the lysosomal pathway. Remarkably, Rev hijacks BECN1 (beclin 1) and PIK3C3 to mediate SAMHD1 degradation in a canonical macroautophagy/autophagy-independent pathway. Our study illustrates that equine lentiviral Rev possesses important functions in evading cellular innate immunity in addition to its RNA regulatory function, and may provide new insights into the co-evolutionary arms race between SAMHD1 and lentiviruses.

Abbreviations:3-MA: 3-methyladenine; AA: amino acid; ACTB: actin beta; AD: activation domain; ATG: autophagy related; Baf A1: bafilomycin A₁; BD: binding domain; BECN1: beclin 1; BH3: BCL2-homology-3 domain; BiFC: bimolecular fluorescence complementation; CCD: coiled-coil domain; class III PtdIns3K: class III phosphatidylinositol 3-kinase; CQ: chloroquine; Co-IP: co-immunoprecipitation; dNTPase: dGTP-stimulated deoxynucleoside triphosphate triphosphohydrolase; ECD: evolutionarily conserved domain; EIAV: equine infectious anemia virus; eMDMs: equine monocyte-derived macrophages; GFP: green fluorescent protein; HD: histidine-aspartic; HIV-1: human immunodeficiency virus-1; hpi: hours post infection; hpt: hours post transfection; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LMB: leptomycin B; PMA: phorbol 12-myristate 13-acetate; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ND: unknown non-essential domain; NES: nuclear export signal; NLS: localization signal; NS: statistically non-significant; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; RBD: RNA binding domain; RT: reverse transcriptase; siRNAs: small interfering RNAs; SAMHD1: SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1; SIV: simian immunodeficiency virus; VN: C-terminal residues of Venus 174 to 238; VC: N-terminal residues 2 to 173 of Venus

摘要

先天免疫限制因子SAMHD1可以抑制骨髓细胞中的多种病毒。从机制上讲，SAMHD1 通过耗尽核苷酸库来干扰它们的逆转录，从而抑制包括 HIV-1 在内的慢病毒复制。马传染性贫血病毒 (EIAV) 是一种古老的慢病毒，它优先攻击巨噬细胞。然而，EIAV 成功地在具有功能性 SAMHD1 的巨噬细胞中建立感染的机制仍不清楚。在这里，我们证明虽然马 SAMDH1 可以在逆转录阶段限制马巨噬细胞中的 EIAV 复制，但抗病毒作用被众所周知的转录调节因子 Rev 抵消，它通过溶酶体途径下调马 SAMHD1。值得注意的是，Rev 劫持 BECN1 (beclin 1) 和 PIK3C3 以介导典型的巨自噬/自噬非依赖性途径中的 SAMHD1 降解。我们的研究表明，马慢病毒 Rev 除了其 RNA 调节功能外，还具有逃避细胞先天免疫的重要功能，并可能为 SAMHD1 和慢病毒之间的共同进化军备竞赛提供新的见解。

缩写：3-MA：3-甲基腺嘌呤；AA：氨基酸；ACTB：肌动蛋白β；AD：激活域；ATG：自噬相关；Baf A1：巴弗洛霉素 A ₁; BD：结合域；BECN1：beclin 1；BH3：BCL2-homology-3 结构域；BiFC：双分子荧光互补；CCD：盘绕线圈域；III类PtdIns3K：III类磷脂酰肌醇3-激酶；CQ：氯喹；Co-IP：免疫共沉淀；dNTPase：dGTP-刺激的脱氧核苷三磷酸三磷酸水解酶；ECD：进化保守域；EIAV：马传染性贫血病毒；eMDMs：马单核细胞衍生的巨噬细胞；GFP：绿色荧光蛋白；HD：组氨酸-天冬氨酸；HIV-1：人类免疫缺陷病毒-1；hpi：感染后数小时；hpt：转染后的小时数；KO：淘汰赛；LAMP2：溶酶体相关膜蛋白 2；LMB：细霉素B；PMA：佛波醇 12-肉豆蔻酸酯 13-乙酸酯；MAP1LC3/LC3：微管相关蛋白1轻链3；ND：未知的非必要域；NES：核输出信号；国家统计局：定位信号；NS：统计上不显着；PIK3C3：磷脂酰肌醇 3-激酶催化亚基 3 型；RBD：RNA结合域；RT：逆转录酶；siRNAs：小干扰RNA；SAMHD1：含有脱氧核苷三磷酸三磷酸水解酶 1 的 SAM 和 HD 结构域；SIV：猴免疫缺陷病毒；VN：Venus 174 至 238 的 C 端残基；VC：Venus 的 N 端残基 2 至 173