Cu cpt 4a

IPEC cells were seeded in 6-well plates at a density of 1 ​× ​10⁶ ​cells/well and grew for 24 ​h. Non-adherent cells and media were removed and replaced with fresh DMEM, and adherent IPEC cells were then treated with commercially available selective TLR3 inhibitor CU CPT 4a (#4883, R&D, Minneapolis MN, USA) at IC₅₀ ​= ​3.44 ​μM for a period of 1 ​h. Later, treated cells were mock-infected or infected with GDS01 or HX strain (MOI ​= ​1) for a period of 3, 6, and 12 ​h. Subsequently, samples were collected and processed for further experiments.

RNA40 [5′-GCCCGUCUGUUGUGUGACUC-3′; at U5 region 108–127 nt of the HIV genome (accession no. NC_001802.1)] [14 (link)], RNA41 (a derivative of RNA40 in which adenosine replaces all uracil nucleotides), RNA649 [5′-GUCAGAGUGUGUACUUG-3′; position 24 649–24 665 nt in the SARS-CoV-2 genome (S2 spike protein region) (accession no. NC_045512.2)] [16 (link)], RNA649A (a derivative of RNA649 in which adenosine replaces all uracil nucleotides), IRS 661 (5′-TGCTTGCAAGCTTGCAAGCA-3′) [45 (link)] and ODN (5′-TCCTGCAGGTTAAGT-3′) [45 (link)] were synthesised by Integrated DNA Technologies. LyoVec (InvivoGen), a cationic lipid-based transfection reagent, was used to complex GU-rich ssRNA in a 2 : 1 (LyoVec:RNA) ratio as previously described [16 (link)]. RNA40 and RNA41 precomplexed with LyoVec were purchased from InvivoGen. Recombinant human ubiquitin specific peptidase 2 (USP2) and CU-CPT4a were purchased from R&D Systems. CU-CPT9a, JH-X-119-01, maraviroc, pacritinib (SB1518), plerixafor, and zimlovisertib were purchased from Selleck Chemicals. Lambda protein phosphatase (λ-PPase) and MG132 were purchased from Sigma-Aldrich.