EV-D68 cleaves LARP1 and PABPC1 by 3Cpro to redirect host mRNA translation machinery toward its genomic RNA

Fig 1. Viral Cleavage of LARP1 and PABPC1 Inhibits Host Eukaryotic Translation. (A) RD cells were infected with EV-D68 at MOIs of 0.01, 0.1, and 1, and harvested at 6 h, 12 h, and 24 h. Cells were labelled with 10 µg/mL puromycin and treated with puromycin for 0.5 h. Cells were collected for western blot. (B) A model of the translation initiation complex, including eIF4E, eIF4G, LARP1, and PABPC1. Created in BioRender. Ruyang, T. (2025) https://BioRender.com/q79y826. (C) RD cells were infected with EV-D68 (MOI = 0.1) for 6 h, 12 h, and 24 h, and then collected for western blot. (D) RD cells were transfected with negative control (NC) or pooled PABPC1 siRNA for 48 h. Cells were labelled with 10 µg/mL puromycin for 0.5 h, and then collected for western blot. (E) RD cells were transfected with NC or pooled LARP1 siRNA for 48 h. Cells were labelled with 10 µg/mL puromycin for 0.5 h and then collected for western blot.

Fig 2. EV-D68 Cleaves PABPC1 and LARP1, Inhibiting eIF4E-Cap-Dependent mRNA Translation. (A) Empty Vector, 2A-HA, and 3C-HA plasmids were transfected into RD cells for 24 h, and then EV-D68 was infected for 24 h. Cells were then collected for western blot. (B) RD cells were infected with EV-D68, and Rupintrivir (10 µ M) was added at 6 h post-infection for a total of 18 h. Cells were harvested at 24 h post-infection for western blot analysis. (C) Empty Vector, 2A-HA, and 3C-HA plasmids were transfected into RD cells for 24 h, and then EV-D68 was infected for 24 h. Cells were then collected for western blot. (D) RD cells were infected with EV-D68, and treated with Rupintrivir (5 µ M or 10 µ M) starting at 6 h post-infection for 18 h. Cells were collected at 24 h post-infection for western blot analysis. (E) Empty Vector, 3C-HA, 3C-H40D-HA, 3C-E71A-HA, 3C-C147A-HA plasmids were transfected into RD cells for 24 h, then EV-D68 infected control cells for 12 h. Cells were harvested for western blot. (F) HEK293T cells were transfected with 4EBP1-HA plasmid for 24 h, infected with EV-D68 (MOI = 0.1) for 12 h, and cells were collected for Co-IP. (G) HEK293T cells were transfected with NC or…

Fig 3. PABPC1 and LARP1 Disrupt EV-D68 Replication. (A) PABPC1-Flag plasmid was transfected in RD cells for 24 h, then infected with EV-D68, and the lysates of the cells were collected after 24 h for western blot (i), and viral titer analysis (ii). (B) LARP1-Flag plasmid was transfected in RD cells for 24 h, then infected with EV-D68, and the lysates of the cells were collected after 24 h for western blot (i), and viral titer analysis (ii). (C) NC or pooled PABPC1 siRNA was transfected in RD cells for 24 h, then transfected with LARP1-Flag plasmid for 24 h, followed by infection with EV-D68 virus for 24 h. Finally, cells were collected for western blot. (D) PABPC1-Flag plasmid was transfected in RD cells for 24 h, then infected with EV-D68, and 12 h later the cells were labelled with 10 µg/mL puromycin for 0.5 h. Cells were then collected for western blot. (E) LARP1-Flag plasmid was transfected in RD cells for 24 h, then infected with EV-D68, and 12 h later the cells were labelled with 10 µg/mL puromycin for 0.5 h. Cells were then collected for western blot. (F) Schematic representation of EV-D68 minireplicon (i); Schematic representation of EV-D68 minireplicon Δ3′UTR (ii); Schematic…

Fig 4. EV-D68 3Cpro cleaves LARP1 at Q371 disrupting its LAM domain and weakening its interaction with the viral 5’UTR. (A) LARP1-Flag plasmid was transfected into HEK293T cells for 24 h. Then HEK293T cells were infected with EV-D68 (MOI = 0.1) for 12 h. Cells were then collected for RIP, followed by qPCR (i, ii) and western blot(iii). (B - D) 293T cells were co-transfected with LARP1-Flag plasmid with EV-D68 minireplicon (B), EV-D68 minireplicon Δ3′UTR (C), EV-D68 minireplicon Δ5′UTR (D) for 36 h, respectively, and then the cells were collected for RIP, followed by qPCR(i) and western blot(ii). (E) HEK293T cells were co-transfected with various LARP1 deletion mutants (ΔN1-68, ΔN1-279, ΔLAM, ΔRRM-L5 and ΔDM15) along with the EV-D68 minireplicon for 36h. Cells were collected for RIP, followed by qPCR (i) and western blot analysis (ii). (F) Diagram of the functional domains and identified cleavage site of LARP1. (G) RD cells were co-transfected with LARP1-FLAG wild-type (WT), LARP1-Q371A-FLAG, or LARP1-Q790A-FLAG plasmids along with 3C-HA for 24 h. Cells were collected and subjected to western blot analysis. (H) HEK293T cells were transfected with full-length LARP1-FLAG or the…

Fig 5. EV-D68 Infection Impairs mTOR and CDK1 signaling pathways, Leading to Enhanced LARP1 Binding to Viral RNA. (A, B) RD cells were infected with EV-D68 for 24 h and then cells were collected for western blot. (C) LARP1-Flag plasmid was transfected in RD cells for 24 h, then RAPA (10 µ M) was added and treated for 12 h. Cells were labelled with 10 µg/mL puromycin for 0.5 h, and then collected for western blot. (D) NC or pooled CDK1 siRNA was transfected in RD cells for 24 h, followed by transfection of the LARP1-Flag plasmid for 24 h. Cells were labelled with 10 µg/mL puromycin for 0.5 h, and then collected for western blot. (E) LARP1-Flag plasmid was transfected in HEK293T cells for 24 h, then RAPA (10 µ M) was added and treated for 12 h. Cell samples were collected for RIP, followed by qPCR(i) and western blot(ii). (F) HEK293T cells were transfected with the LARP1-Flag plasmid for 24 h, then infected with EV-D68. RAPA (10 µ M) was added 12 h post-infection, followed by cell collection for RIP analysis. Viral RNA levels were quantified by qPCR (i), and associated protein expression were validated by western blot (ii). (G) LARP1-Flag plasmid was transfected in HEK293T cells for 24 h,…

Fig 6. Potential Universal Inhibitory Role of PABPC1 and LARP1 in Enteroviruses. (A, B) RD cells were infected with EV-A71 (MOI = 0.1) for 6 h, 12 h and 24 h, Cells were collected for western blot. (C, D) RD cells were infected with CV-A16 (MOI = 0.5), and cells infected for 6 h, 12 h and 24 h were collected for western blot. (E) PABPC1-Flag plasmid was transfected in RD cells for 24 h, then infected with EV-A71 (MOI = 0.1), and the lysates of the cells were collected after 24 h for western blot (i), qPCR (ii). (F) LARP1-Flag plasmid was transfected in RD cells for 24 h, then infected with EV-A71 (MOI = 0.1), and the lysates of the cells were collected after 24 h for western blot (i), qPCR (ii). (G) PABPC1-Flag plasmid was transfected in RD cells for 24 h, then infected with CV-A16 (MOI = 0.5), and the lysates of the cells were collected after 24 h for western blot (i), qPCR (ii). (H) LARP1-Flag plasmid was transfected in RD cells for 24 h, then infected with CV-A16 (MOI = 0.5), and the lysates of the cells were collected after 24 h for western blot (i), qPCR (ii).

Fig 7. Proposed model for EV-D68 cleavage of LARP1 and PABPC1 to inhibit host cell translation and redirect translation initiation factors for viral RNA translation. Under normal nutrient available conditions, mTOR and CDK1 regulate the dissociation of downstream LARP1 from bound mRNA through phosphorylation, allowing the eIF4E protein to bind to the 5′ end of the mRNA, thereby recruiting the translation initiation complex and initiating translation. EV-D68 infection reduces the protein levels of mTOR and CDK1 in the cell, and the corresponding phosphorylation levels are also reduced, resulting in the inability of LARP1 to dissociate from the binding RNA, and EV-D68 translation is inhibited. To this end, EV-D68 cleaves LARP1 and PABPC1 so that the translation initiation site of viral RNA is not occupied by LARP1 and is able to bind to translation initiation factors and recruit the corresponding translation initiation proteins for its own use. Created in BioRender. Ruyang, T. (2025) https://BioRender.com/c23t611.