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. 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.