Antiviral activity and mechanism of purine morpholine nucleoside analogues incorporating a sulfonamide fragment

Introduction Pepper mild mottle virus (PMMoV) poses an enormous threat to pepper production because of its high infectivity and soil persistence. Commercially available antiviral agents, ningnanmycin and ribavirin, have been restricted from widespread use due to their photosensitivity, water viscosity, and unsatisfactory efficacy. Objectives In order to synthesize compounds with superior antiviral activities against PMMoV and clarify their antiviral mechanisms. Methods A series of purine morpholine nucleoside analogues incorporating a sulfonamide moiety were successfully synthesized from D-glucosamine hydrochloride through a systematic multistage reaction involving condensation, acetylation, hydrolysis, sulfonation, bromination, and substitution. And activity-based protein profiling (ABPP) technology, Western blot, and microscale thermophoresis (MST) were combined to validate the target protein of C1 acting on PMMoV. Subsequently, molecular docking, MD simulations, Western blot, and RT-qPCR were employed to identify the key amino acid sites for C1 action. Next, co-immunoprecipitation mass spectrometry (Co-IP MS), RNA sequencing (RNA-seq), luciferase complementation assay (LCA), bimolecular fluorescence complementation (BiFC), and fluorescence recovery after photobleaching (FRAP) were applied to investigate the underlying mechanisms of infection diversity between GFP-PMMoV CP and GFP-PMMoV CP Y13A . Finally, the function of I3QHX5 in viral infection was further validated by virus-induced gene silencing (VIGS). Results Thirty-eight purine morpholine nucleoside analogues incorporating a sulfonamide fragment with preferable inhibiting activities against PMMoV were designed and synthesized for the first time. ABPP technology, Western blot, and MST manifested that PMMoV CP was the target protein of C1 . Molecular docking, RMSD, RT-qPCR, and Western blot revealed that the tyrosine at position 13 (Tyr13) of PMMoV CP might be the key amino site for C1 action. Co-IP MS, RNA-seq, LCA, BiFC, and FRAP indicated that the discrepancy in the liquid–liquid phase separation (LLPS) between PMMoV CP and PMMoV CP Y13A with I3QHX5 might be the main cause of infection differences. Further VIGS demonstrated that silencing of I3QHX5 led to an attenuated accumulation of PMMoV. Conclusion Our research revealed that purine morpholine nucleoside analogues containing a sulfonamide fragment possessed preferable inhibitory activities against PMMoV. Correlated mechanism studies indicated that C1 could inhibit the formation of LLPS by specifically targeting Tyr13 of PMMoV CP, thus achieving the purpose of antiviral effects.