Targeting eEF1A reprograms cellular translation and uncovers alternative broad-spectrum antivirals against cap or m6A protein synthesis routes. Targeting eEF1A reprograms cellular translation and uncovers alternative broad-spectrum antivirals against cap or m6A protein synthesis routes

Plitidepsin is an antitumoral drug safe for treating COVID-19 infection that impairs SARS-CoV-2 replication by targeting the translation elongation factor eEF1A. Here we show that plitidepsin decreased all SARS-CoV-2 transcripts along the early translation of viral R1AB polyproteins and the late synthesis of structural viral proteins. It also reduced de novo cap-dependent translation of viral and non-viral RNAs but affected less than 13% of the host proteome, thus preserving cellular viability. In response to plitidepsin, cells upregulated EIF2AK3 and additional proteins that reduce translation. Other proteins supported proteostasis via ribosome synthesis and cap-independent translation routes, which relied on eIF4G2, PABPC1, and readers such as IGF2BP2. While cap- or internal ribosome entry sites (IRES)-mediated translation pathways were inhibited by plitidepsin, impact on N6-methyladenosine (m6A) translation was limited. The molecular landscape reprogrammed by cells treated with plitidepsin predicted a potential broad-spectrum activity against viruses translated via cap- or IRES-dependent pathways that were suppressed by m6A. In agreement, plitidepsin inhibited the replication of members from the Coronaviridae, Flaviviridae, Pneumoviridae and Herpesviridae families. Yet, it failed to block retroviruses that exploit m6A synthesis routes and are inhibited by drugs against specific m6A readers such as IGF2BP2. By deciphering the molecular fingerprint of cells treated with host-directed therapies targeting protein translation, we identified a rational approach to select for broad-spectrum antivirals with complementary activities and potential to counteract future pandemic viruses. Overall design: 1x10^6 Vero E6 cells per well seeded in a 24-well plate were infected with D614G at MOI 0.02 in the presence or absence of 50 nM plitidepsin for 4 h, 24 h and 48 h using 3 biological replicates per condition. At the indicated timepoints, cells were lysed with TRIzol™ (Invitrogen, 15596026 and 15596018). RNA was extracted with a Nucleospin RNA Magenerey-Nagel extraction kit and sequenced with Illumina TruSeq RNA w/ Ribo-Zero library, with coverage Novaseq 6000 150bp PE (150x2bp) 60M reads/spl (9Gb/spl) by Macrogen. Paired-end sequencing was performed according to standard TruSeq Stranded Total RNA Reference Guide (1000000040499 v00) protocol using TruSeq Stranded Total RNA Library Prep Gold Kit. All samples passed quality control checks and were aligned using bowtie255 version 2.5.1 to the SARS-CoV-2 variant EPI_ISL_510689 and the reference ASM985889v3. For host alignment, the reference genome GCA_023783515.1 and the human genome (GRCh38, ENSEMBL version 109) were used. RSEM56 v1.3.1 was used to create the indexes of these genomes and calculate the gene expression.

普利他滨（plitidepsin）是一种可安全用于治疗新型冠状病毒肺炎（COVID-19）感染的抗肿瘤药物，其通过靶向翻译延伸因子eEF1A来抑制严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的复制。本研究表明，普利他滨可降低SARS-CoV-2的所有转录本水平，涉及病毒R1AB多聚蛋白的早期翻译阶段以及结构性病毒蛋白的后期合成过程。同时，该药物可减少病毒与非病毒RNA的从头帽依赖型翻译过程，但仅影响不到13%的宿主蛋白质组，因此可维持细胞活力。
经普利他滨处理后，细胞会上调EIF2AK3及其他可抑制翻译过程的蛋白的表达。另有部分蛋白通过核糖体合成以及不依赖帽的翻译途径维持蛋白质稳态（proteostasis），此类途径依赖于eIF4G2、PABPC1以及IGF2BP2等阅读蛋白（readers）。尽管帽依赖型或内部核糖体进入位点（IRES）介导的翻译通路可被普利他滨抑制，但该药物对N6-甲基腺嘌呤（m6A）相关翻译的影响十分有限。
经普利他滨处理的细胞所重塑的分子谱，预示该药物具有潜在的广谱抗病毒活性，可靶向那些通过帽依赖型或IRES依赖型通路翻译、且可被m6A调控的病毒。与此一致，普利他滨可抑制冠状病毒科（Coronaviridae）、黄病毒科（Flaviviridae）、肺病毒科（Pneumoviridae）以及疱疹病毒科（Herpesviridae）成员的复制。但该药物无法阻断那些利用m6A合成通路、且可被针对特定m6A阅读蛋白（如IGF2BP2）的药物所抑制的逆转录病毒。
本研究通过解析以蛋白质翻译为靶点的宿主靶向疗法处理后的细胞分子特征，确立了一种可筛选具有互补活性、且有望对抗未来大流行病毒的广谱抗病毒药物的合理策略。

实验整体设计：将每孔1×10^6个Vero E6细胞接种于24孔板中，以感染复数（MOI, multiplicity of infection）0.02的D614G毒株感染细胞，分别在存在或不存在50 nM普利他滨的条件下培养4 h、24 h及48 h，每组设置3个生物学重复。在指定时间点，使用TRIzol™试剂（Invitrogen，货号15596026与15596018）裂解细胞。采用Nucleospin RNA试剂盒（Magenerey-Nagel）提取总RNA，并由Macrogen公司使用Illumina TruSeq RNA建库试剂盒（搭配Ribo-Zero磁珠）进行测序，测序平台为NovaSeq 6000，采用150 bp双端测序（150×2 bp），每个样本的测序读数约为60M（对应数据量9 Gb/样本）。双端测序流程严格遵循《TruSeq Stranded Total RNA参考指南》（编号1000000040499 v00），使用TruSeq Stranded Total RNA Library Prep Gold试剂盒完成建库。
所有样本均通过质量控制检测，使用bowtie255 2.5.1版本将测序数据比对至SARS-CoV-2变异株EPI_ISL_510689以及参考基因组ASM985889v3。宿主基因组比对则采用参考基因组GCA_023783515.1以及人类基因组（GRCh38，ENSEMBL版本109）。使用RSEM56 v1.3.1工具构建上述基因组的索引文件，并计算基因表达量。