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

introductionPepper 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.

objectivesIn order to synthesize compounds with superior antiviral activities against PMMoV and clarify their antiviral mechanisms.

methodsA 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

resultsThirty-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, MD simulations, 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

conclusionOur 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.