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

Yuyuan Yang; Runjiang Song; Shaobo Wang; Guangcheng Zu; Baoan Song

doi:10.1016/j.jare.2025.05.062

. 2025 Jun 1;81:43–56. doi: 10.1016/j.jare.2025.05.062

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

Yuyuan Yang ¹, Runjiang Song ¹, Shaobo Wang ¹, Guangcheng Zu ¹, Baoan Song ^1,^⁎

PMCID: PMC12957839 PMID: 40460937

Graphical abstract

Keywords: Purine nucleoside analogues, Sulfonamide moiety, Pepper mild mottle virus, Antiviral activities, Mechanisms of action

Highlights

•
Thirty-eight compounds of purine morpholine nucleoside analogues containing a sulfonamide moiety with favorable inhibitory activities against PMMoV were designed and synthesized.
•
ABPP technology, western blot, and MST demonstrated that PMMoV CP was the target protein of C1. Molecular docking, MD simulations, confocal, Western blot, and RT-qPCR revealed that Y13A in PMMoV CP could severely inhibit virus infection.
•
Co-IP MS, RNA-seq, LCA, and BiFC revealed that PMMoV CP could aggregate after interacting with I3QHX5, while PMMoV CP^Y13A could not under confocal. And the aggregates could be inhibited by applying of C1.
•
Fusion, fission and FRAP demonstrated that the condensed material formed by CP and I3QHX5 had the properties of LLPS both in vivo and in vitro. Thus, PMMoV CP could promote viral infection, while PMMoV CP^Y13A could not.
•
Further VIGS demonstrated that silencing of I3QHX5 leading to an attenuated accumulation of PMMoV CP.

Abstract

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, 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^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.

Introduction

Chili peppers (Capsicum spp.), also known as bell peppers, are one of the most significant agricultural crops in the world, with a total global production of 752,000 tonnes and an overall market revenue of $4.1 billion in 2018 (https://www.researchandmarkets.com/reports/4701016/world-pepper-market-analysis -forecast). Rich in vitamins C, A, and E, and they are also a vital source of carotenoids as well as other essential nutrients such as fiber, potassium, iron, calcium, and folic acid [1,2], with properties of antioxidant, anticancer, anti-inflammatory, antimicrobial, and insecticidal [3,4]. Among all the categories of plant pathogens, viruses are the most destructive biological agents found in chili peppers [[5], [6], [7]]. Currently, 68 viruses have been reported in chili peppers [8], among which pepper mild mottle virus (PMMoV) poses a severe threat to the production of chili peppers due to its high infectivity and soil persistence [7]. The incidence of PMMoV has been reported to be as high as 95 %, which in turn leads to yield losses of 75–95 %, thus taking a severe threat to chili peppers [2]. Recent studies have demonstrated that PMMoV is correlated with clinical symptoms such as fever, itching, and abdominal pain, indicating that it might render a substantial risk to human health [9,10]. Commercially available antiviral agents, ningnanmycin and ribavirin, have been restricted from widespread use because of their photosensitivity, water viscosity and unsatisfactory efficacy [11]. Therefore, developing an efficient and eco-friendly antiviral agent and elucidating its mechanism of action has become our top priority.

Purine nucleoside derivatives harbor extensive antiviral activities in medicine, such as for herpes simplex virus (HSV), varicella-zoster virus (VZV), human cytomegalovirus (HCMV), canine distemper virus (CDV), hepatitis B virus (HBV), human immunodeficiency virus (HIV) and so on [[12], [13], [14], [15], [16]]. However, fewer studies have been conducted as plant antiviral agents, most purine nucleoside analogs from pharmaceuticals are used directly to screen plant viruses, such as tobacco mosaic virus (TMV), potato virus X (PVX), cucumber mosaic virus (CMV), and it has been reported that they might target S-adenosyl homocysteine hydrolase (SAHH) [[17], [18], [19], [20], [21]]. The sulfonamide skeleton plays a crucial role in the development of antiviral agents and is regarded as a “molecular heterozygote” capable of forming hydrogen bonds and interacting with the unipolar environment in proteins [[22], [23], [24], [25], [26], [27], [28]]. Morpholine derivatives have a wide range of biological activities such as insecticides, fungicides, herbicides, and antivirals [29]. In this work, a series of purine morpholine nucleoside analogs containing a sulfonamide fragment were designed and synthesized by introducing sulfonamide and morpholine scaffolds into the purine ring and modifying both the sugar and purine rings for the first time (see Scheme 1 in Supporting Information for details).

In this study, thirty-seven purine morpholine nucleoside analogues C1, C3-C38 containing a sulfonamide fragment with favorable activities against PMMoV were designed and synthesized. Based on the preferable inactivating activity of compound C1, small molecule probe C2 was then synthesized. Its EC₅₀ of inactivating activities against PMMoV was 40.7 µg/mL, which was comparable to C1 (37.0 µg/mL) and superior to the control agent ningnanmycin (67.3 µg/mL), indicating that C2 could serve as a small molecule probe to capture target proteins. Activity-based protein profiling (ABPP) technology, Western blot and microscale thermophoresis (MST) manifested that PMMoV CP was the target protein of C1. The results of 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. Further studies of co-immunoprecipitation mass spectrometry (Co-IP MS), RNA sequencing (RNA-seq), luciferase complementation assay (LCA), bimolecular fluorescence complementation (BiFC), fluorescence recovery after photobleaching (FRAP), and virus-induced gene silencing (VIGS) revealed that the discrepancies in the interaction between I3QHX5 and PMMoV CP, as well as PMMoV CP^Y13A might be the main cause of the differences in infection. To the best of our knowledge, this work not only stated C1 could be employed as a novel antiviral agent but also interpreted its mechanism for the first time.

Materials and methods

Materials

Reagents and solvents involved in the experiments were purchased from commercial suppliers without ulteriorly processing. Antibody of PMMoV CP and plasmid of pCB-GFP-PMMoV were graciously provided by Prof. Fei Yan (Ningbo University, Zhejiang, China).

General procedures for the preparation of target compoundsC1,C3-C38

Intermediates a–e and h were synthesized with reference to the methods depicted in literature [[30], [31], [32], [33], [34], [35]]. See Supporting Information for details.

To the mixture of morpholine (20.00 mg, 229.56 µmol) and triethylamine (69.69 mg, 688.69 µmol) in ethanol (10 mL), 2-(acetoxymethyl)-5-((substituted phenyl)sulfonamidyl)-6-(2-substituted-6-chloro-9H-purin-9-yl)tetrahydro-2H-pyran-3,4-dicarboxylic acid dihydrate h (183.65 µmol) dissolved in ethanol (5 mL) was slowly added, and the mixture was heated to reflux. The reaction was monitored by TLC (DCM/EA, 1:1 v/v) until full completion, and the solvent was removed by rotary evaporation. Then the residue was dissolved in DCM (20 mL) and washed with water (3 × 20 mL). The organic layer was dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. Finally, the products C1, C3-C35 were purified by column chromatography (DCM/EA, 1:1 v/v) [36,37].

Randomly selected compounds C1, C25, and C34 (146.82 mol) in anhydrous methanol (10 mL), and added methanol/sodium methoxide solution dropwise until the reaction mixture was basic (pH = 10). The mixture was agitated at rt until the reaction was complete. It was then concentrated under reduced pressure, and the residue was purified by column chromatography (DCM/MeOH, 10:1 v/v) to acquire compounds C36-C38 [34,38,39].

General procedures for the preparation of target compoundC2

Intermediate i was prepared following the method reported in the literature [36].

To the mixture of propargyl bromide (35.16 mg, 295.57 µmol) and potassium carbonate (30.64 mg, 221.68 µmol) in acetone (10 mL), 2-(acetoxymethyl)-5-((4-nitrophenyl)sulfonylamino)-6-(6-(piperazine-1-yl)-9H-purine-9-yl)tetrahydro-2H-pyrano-3,4-diacetyldiacetate (100 mg, 147.79 µmol) dissolved in acetone (5 mL) was dropwise added at rt. The mixture was concentrated under reduced pressure after the reaction was finished. And the residue was dissolved in DCM (20 mL) and washed with water (3 × 20 mL). Then the organic layer was dried over anhydrous MgSO₄, filtered, and concentrated under reduced pressure. Finally, the residue was purified by column chromatography (DCM/EA, 1:1 v/v) to yield the target compound C2.

Antiviral activity assay

Virions were isolated and extracted from leaves of N. benthamiana infected with PMMoV referring to the method recorded in the literature [40]. And the inhibiting activities of C1–C38 against PMMoV were assessed with reference to the literature as well [26,34].

Microscale thermophoresis (MST)

The binding affinities of compounds with PMMoV CP were analyzed by MST based on the procedures delineated in the literature [41,42].

Molecular modeling

Homology modeling

Homology modeling was performed by Discovery Studio 4.5 to predict the three-dimension (3D) structure of PMMoV CP with TMV CP (PDB code 1EI7) [43,44] as a template. The model of PMMoV CP was subjected to energy minimization, and the rationality of protein structure was analyzed through Protein Health [45] in Discovery Studio 4.5 and MolProbity Ramachandran analysis in MolProbity (https://altmolprobity.biochem.duke.edu/) [46,47].

Molecular docking

Molecular docking was performed with reference to the methods described in the literature [48]. The 3D structures of compounds were intended by SYBYL-X2.0 followed by 2000-steps steepest descent minimization and 2000-sreps conjugate gradient minimization, respectively. PMMoV CP was obtained by above mentioned homology modeling, and the hydrogens of the PMMoV CP were added by Discovery Studio 4.5. Compounds were docked into the binding pocket of PMMoV CP through AutoDock 4.2 [49]. The grid was set to be 60 × 60 × 60 with a default spacing 0.375 Å. The genetic algorithm (GA) was used to perform 256 runs. Conformation with top rank was chosen as the representative for further analysis.

Molecular dynamics (MD) simulations

In order to obtain the stable binding mode of compounds with PMMoV CP, MD simulations were performed based on the docking results of ligands and proteins, respectively. AM1-BCC charge model in Antechamber module of Amber22 program was used to assign charges for ligand atoms [50,51]. The topology and coordinate files of the complex were built with the Leap module in Amber22 program. AMBER ff14SB force field for amino acid residues, and general AMBER force field (GAFF) for ligands [[52], [53], [54]], Cl^- or Na⁺ for neutralizing the system. All molecules were dissolved in a rectangular TIP3P aqueous cassette extending at least 10 Å in each direction from the solute [55,56]. The Cpptraj module in Amber22 was used to perform root mean square deviation (RMSD), root mean square fluctuation (RMSF) and radius of gyration (Rg) analysis for measuring the stability of the complex during the MD process.

Transient expression of agrobacterium tumefaciens

Infectious cDNA clones of GFP-PMMoV CP^Y13A and GFP-PMMoV CP^Y140A were constructed following the approach depicted in the literature [34]. And leaves with significant characteristics were collected and preserved at −80 ℃ for further Western blot [34,57] and RT-qPCR [34,58]. The sequences of primers used in the construction of plasmids and RT-qPCR were listed in Table S3 for details.

Co-immunoprecipitation mass spectrometry (Co-IP MS) and RNA-seq

Vector construction and material preparation

The Flag-tagged vector was digested with KpnI and AscI, then fusion proteins of Flag-PMMoV CP and Flag-PMMoV CP^Y13A were constructed through homologous recombination. Western blot was performed to determine the expression of proteins at 24, 48 and 72 h of post inoculation (hpi), and the time point with the highest expression was selected for further Co-IP MS and RNA-seq.

Luciferase complementation assay (LCA) and bimolecular fluorescence complementation (BiFC)

Designed primers to construct vectors of LCA and BiFC via homologous recombination. Extracted plasmids and waited for sequencing confirmation before transforming them into GV3101 (pU19). A. tumefaciens transient transformation solution (200 μM acetosyringone, 10 mM MgCl₂, and 10 mM MES, pH = 5.6) was prepared to resuspend the organisms with a final OD₆₀₀ of 1.0, then mixed them with equal OD₆₀₀ and volume before being infiltrated into N. benthamiana of the same growth stage. Interactions of prey proteins of A0A248QEL2, B1PSM0, I3QHX5, LOC109221810, and A2IBL2 with bait proteins of PMMoV CP and PMMoV CP^Y13A were recorded by UV or confocal at 48–72 hpi. The primers involved in LCA and BiFC were listed in Table S3.

Virus-induced gene silencing (VIGS)

pTRV2-I3QHX5, pTRV2-PDS, and pTRV2 with pTRV1 in equal OD₆₀₀ (OD₆₀₀ = 0.2) and equal volume were co-infiltrated into N. benthamiana with 3–5 leaves. When the positive control (TRV:PDS) exhibited the symptom of bleaching at 10 dpi, RT-qPCR was used to detect the silencing efficiency of I3QHX5. Concurrently, GFP-PMMoV of OD₆₀₀ = 0.1 was infiltrated. The expression levels of PMMoV CP and CP mRNA in TRV:00 and TRV:I3QHX5 infiltrated with GFP-PMMoV at 8 dpi were detected via Western blot and RT-qPCR. The primers involved in VIGS were listed in Table S3.

Results

Design and synthesis of target compoundsC1,C3-C38

A series of purine morpholine nucleoside analogues C1, C3-C38 containing a sulfonamide moiety were designed and synthesized through modification of both sugar and purine rings. The target compounds underwent comprehensive structural characterization via ¹H NMR, ¹³C NMR, ¹⁹F NMR, and HRMS. Slow crystallization with EA and DCM at rt yielded a single crystal of C9, and X-ray diffraction was further used to confirm its fine structure. (See Fig. 1A and Supporting Information for more details).

Fig. 1 — Determination of the target protein for C1 action. (A) Design, synthesis of target compounds C1-**C38**, and single-crystal structure of C9. (B) Concentration-dependent experiments between C2 and PMMoV CP. (C) Competitive inhibition assays of C2 and C1 against PMMoV CP. (D) Binding affinities of C1, **C24**, **C25**, and ningnanmycin with PMMoV CP.

Inhibitory activities ofC1,C3-*C38**against PMMoV*

As demonstrated in Table 1, C1, C10, C12, C19, C22, C31, C32, and C34 all exhibited favorable inhibitory activities against PMMoV, with EC₅₀ values of curative, protective, and inactivating activities were 17.0, 86.9, 37.0 µg/mL; 22.2, 17.8, 16.3 µg/mL; 53.0, 134.4, 43.1 µg/mL; 36.7, 159.1, 25.5 µg/mL; 26.2, 38.0, 13.7 µg/mL; 29.6, 99.6, 50.0 µg/mL; 23.8, 28.7, 38.0 µg/mL; and 33.8, 53.8, 53.0 µg/mL; respectively, which were significantly superior to the control agent ningnanmycin (257.6, 285.4, 67.3 μg/mL). And the inhibitory activities of C1 against PMMoV in vivo were displayed in Fig. S129.

Table 1.

Antiviral activities of purine morpholine nucleoside analogues C1-C38 incorporating a sulfonamide fragment against PMMoV.

Compd.	Curative activity^a^,^b (%)	EC₅₀ of curative activity^a^,^b (µg/mL)	Protective activity^a^,^b (%)	EC₅₀ of protective activity^a^,^b (µg/mL)	Inactivating activity^a^,^b (%)	EC₅₀ of inactivating activity^a^,^b (µg/mL)
C1	73.7 ± 5.2	17.0 ± 3.9	64.3 ± 2.8	86.9 ± 8.8	76.4 ± 2.6	37.0 ± 4.4
C2	67.3 ± 4.3	66.6 ± 4.5	65.9 ± 3.0	64.5 ± 4.5	67.4 ± 3.9	40.7 ± 2.3
C3	67.0 ± 3.6	11.5 ± 4.4	57.5 ± 4.3	387.8 ± 16.6	68.9 ± 4.9	198.1 ± 16.1
C4	61.2 ± 3.7	219.1 ± 9.0	52.6 ± 2.0	474.9 ± 15.8	64.1 ± 3.5	141.3 ± 12.1
C5	60.3 ± 2.5	63.8 ± 5.3	43.3 ± 2.3	2285.0 ± 24.8	47.5 ± 1.4	622.1 ± 14.7
C6	67.0 ± 4.9	131.7 ± 9.4	52.0 ± 2.3	357.9 ± 12.3	60.8 ± 1.9	181.7 ± 7.8
C7	55.8 ± 1.3	280.7 ± 10.3	44.7 ± 1.4	793.2 ± 17.1	36.6 ± 3.2	2414.6 ± 17.2
C8	69.6 ± 1.3	64.6 ± 4.6	44.3 ± 1.8	1043.6 ± 19.4	56.6 ± 4.0	222.2 ± 4.8
C9	70.9 ± 4.9	33.3 ± 1.2	58.9 ± 4.0	267.3 ± 8.6	65.8 ± 3.9	103.4 ± 9.1
C10	62.1 ± 5.8	22.2 ± 3.4	67.0 ± 4.3	17.8 ± 2.1	60.5 ± 1.8	16.3 ± 1.9
C11	51.8 ± 1.0	371.9 ± 18.4	58.8 ± 3.1	224.7 ± 11.9	51.8 ± 2.1	497.1 ± 3.0
C12	63.5 ± 2.6	53.0 ± 2.1	65.6 ± 4.7	134.4 ± 7.4	71.5 ± 5.0	43.1 ± 1.1
C13	72.5 ± 2.6	20.9 ± 3.0	46.5 ± 3.4	596.2 ± 10.9	47.3 ± 2.7	458.1 ± 3.3
C14	53.6 ± 2.4	100.1 ± 8.9	38.3 ± 4.4	925.9 ± 11.4	38.5 ± 2.0	4507.1 ± 24.2
C15	65.6 ± 3.4	61.0 ± 5.4	62.5 ± 3.3	103.2 ± 6.4	47.7 ± 0.69	593.0 ± 13.7
C16	58.9 ± 4.2	48.5 ± 3.1	45.3 ± 2.5	663.8 ± 12.4	55.9 ± 1.5	171.4 ± 9.6
C17	70.5 ± 3.8	16.1 ± 2.7	61.0 ± 5.3	140.4 ± 9.5	59.9 ± 4.0	84.0 ± 2.8
C18	63.5 ± 2.5	24.7 ± 1.3	41.9 ± 2.9	994.5 ± 11.2	61.3 ± 2.8	134.1 ± 8.5
C19	69.5 ± 4.7	36.7 ± 3.7	59.8 ± 3.6	159.1 ± 11.8	69.7 ± 1.9	25.5 ± 4.0
C20	74.8 ± 4.8	30.1 ± 3.5	54.8 ± 2.8	292.3 ± 17.6	72.0 ± 4.2	28.5 ± 4.2
C21	66.9 ± 1.8	61.6 ± 5.3	60.7 ± 2.5	74.0 ± 5.1	68.7 ± 4.5	83.2 ± 3.7
C22	67.6 ± 1.9	26.2 ± 1.3	61.2 ± 1.6	38.0 ± 3.5	61.3 ± 4.5	13.7 ± 1.6
C23	69.0 ± 3.5	11.3 ± 3.3	57.4 ± 1.6	144.7 ± 7.7	55.4 ± 2.6	239.5 ± 17.8
C24	72.5 ± 2.2	30.7 ± 1.2	40.8 ± 3.5	918.6 ± 10.2	65.1 ± 4.2	144.8 ± 4.7
C25	59.0 ± 3.5	167.1 ± 5.2	52.3 ± 1.1	345.1 ± 11.3	46.9 ± 1.8	572.7 ± 10.9
C26	67.6 ± 2.5	45.1 ± 4.7	44.5 ± 1.6	787.5 ± 12.8	45.5 ± 2.6	632.0 ± 15.2
C27	69.9 ± 3.6	51.1 ± 3.6	45.0 ± 3.4	534.0 ± 14.9	62.4 ± 4.7	184.4 ± 10.0
C28	69.4 ± 4.9	45.4 ± 3.0	42.9 ± 0.7	633.4 ± 13.6	44.0 ± 4.3	575.9 ± 14.9
C29	69.8 ± 4.3	64.8 ± 3.1	64.9 ± 4.7	88.4 ± 6.3	53.4 ± 0.8	352.8 ± 18.7
C30	67.9 ± 3.3	60.2 ± 3.1	58.9 ± 3.8	261.7 ± 10.3	61.7 ± 4.6	20.4 ± 4.9
C31	61.9 ± 3.3	29.6 ± 1.1	56.0 ± 1.9	99.6 ± 3.8	68.5 ± 1.7	50.0 ± 2.0
C32	68.0 ± 2.7	23.8 ± 5.5	68.9 ± 2.0	28.7 ± 4.4	67.9 ± 5.1	38.0 ± 4.4
C33	71.5 ± 4.4	32.0 ± 1.0	53.0 ± 3.7	305.4 ± 12.2	54.4 ± 3.3	176.4 ± 11.3
C34	67.4 ± 2.0	33.8 ± 4.6	65.6 ± 4.5	53.8 ± 4.8	72.7 ± 2.0	53.0 ± 2.2
C35	67.9 ± 3.3	17.0 ± 1.4	68.9 ± 2.0	33.9 ± 0.5	49.1 ± 1.6	457.3 ± 10.2
C36	57.3 ± 1.1	283.1 ± 10.5	52.2 ± 0.8	561.7 ± 11.1	50.6 ± 3.6	313.0 ± 7.1
C37	36.2 ± 4.6	1998.2 ± 21.8	50.6 ± 2.1	343.7 ± 15.8	57.8 ± 3.4	279.7 ± 13.9
C38	51.54 ± 0.3	441.9 ± 12.6	46.5 ± 1.0	473.8 ± 27.7	51.6 ± 0.5	420.9 ± 7.4
Ningnanmycin^c	54.5 ± 3.3	257.6 ± 7.6	60.8 ± 3.1	285.4 ± 5.3	71.2 ± 6.1	67.3 ± 3.5

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Average of three replicates.

The ± values represent standard deviation.

Ningnanmycin was used as a positive control.

Subsequently, C1, C25, and C34 were randomly selected for deacetylation, and the bioassay results demonstrated that the deprotected compounds of C36-C38 exhibited poor antiviral activities compared to those of C1, C25, and C34, which were consistent with our previous results [26,34].

In addition, the structure–activity relationship (SAR) of C1, C3-C38 against PMMoV was analyzed based on the data presented in Table 1. (1) When substituent R was identical, the antiviral activities of purine morpholine nucleoside analogues with R¹ = H were superior to those of the corresponding R¹ = F, R¹ = Cl, and R¹ = NH₂. e.g., C1 (R = 4-NO₂, R¹ = H, 37.0 µg/mL) < C4 (R = 4-NO₂, R¹ = Cl, 141.3 µg/mL) < C3 (R = 4-NO₂, R¹ = F, 198.1 µg/mL), C12 (R = 4-OCH₃, R¹ = H, 43.1 µg/mL) < C13 (R = 4-OCH₃, R¹ = F, 458.1 µg/mL) < C14 (R = 4-OCH₃, R¹ = NH₂, 4507.1 µg/mL), C21 (R = 2,4,6-triCH₃, R¹ = H, 83.2 µg/mL) < C23 (R = 2,4,6-triCH₃, R¹ = Cl, 239.5 µg/mL), C24 (R = 4-CH₃, R¹ = H, 83.2 µg/mL) < C25 (R = 4-CH₃, R¹ = Cl, 239.5 µg/mL), C30 (R = 4-F, R¹ = H, 20.4 µg/mL) < C32 (R = 4-F, R¹ = Cl, 38.0 µg/mL) < C31 (R = 4-F, R¹ = F, 50.0 µg/mL); (2) The antiviral activities of purine morpholine nucleoside analogues with electron-withdrawing groups were better than those of the corresponding electron-donating groups, when R¹ remained constant and R was a different substituent. For example, C1 (R = 4-NO₂, R¹ = H, 37.0 µg/mL) < C9 (R = 4-Cl, R¹ = H, 103.4 µg/mL) < C18 (R = 4-H, R¹ = H, 134.1 µg/mL) < C24 (R = 4-CH₃, R¹ = H, 144.8 µg/mL), C30 (R = 4-F, R¹ = H, 20.4 µg/mL) < C9 (R = 4-Cl, R¹ = H, 103.4 µg/mL) < C15 (R = 4-Br, R¹ = H, 593.0 µg/mL); (3) When the substituents of both R¹ and R were identical, the antiviral activities of the para-substituted purine morpholine nucleoside analogues were superior to those of the corresponding ortho- and meta-substituents. e.g., C1 (R = 4-NO₂, R¹ = H, 37.0 µg/mL) < C6 (R = 2-NO₂, R¹ = H, 181.7 µg/mL) < C5 (R = 3-NO₂, R¹ = H, 622.1 µg/mL), C33 (R = 4-OCH₃, R¹ = H, 176.4 µg/mL) < C35 (R = 3-OCH₃, R¹ = H, 457.3 µg/mL); (4) When the substituents at R¹ and R were identical, the antiviral activities of the purine morpholine nucleoside analogues were superior to those of the corresponding deacetylated analogues. For example, C1 (R = 4-NO₂, R¹ = H, 37.0 µg/mL) < C37 (R = 4-NO₂, R¹ = H, 279.7 µg/mL), C34 (R = 4-OCF₃, R¹ = F, 53.0 µg/mL) < C38 (R = 4-OCF₃, R¹ = F, 420.9 µg/mL). In summary, introducing electron-withdrawing groups at the para-position of the benzene ring while maintaining an unmodified purine ring was favorable for enhancing antiviral activities. Additionally, preserving the acetylated structure was crucial, as full exposure of the hydroxyl group might reduce activity due to increased polarity.

Searching for the target protein ofC1acted on PMMoV

As illustrated in Table 1, C1, C10, C12, C19, C22, C31, C32, and C34 all exhibited pleasant inhibitory activities against PMMoV, and the EC₅₀ of the inactivating activity of C1 (37.0 µg/mL) against PMMoV was superior to those of C12 (43.1 µg/mL), C31 (50.0 µg/mL), C32 (38.0 µg/mL), and C34 (53.0 µg/mL). Although the EC₅₀ values for the inactivating activities against PMMoV of C10 (16.3 µg/mL) and C22 (13.7 µg/mL) were better than that of C1, the inactivating activity of C1 (76.4 %) was better than those of C10 (60.5 %) and C22 (61.3) at 500 µg/mL. Based on the dual evaluation system of EC₅₀ value and inactivating efficiency, C1 was chosen as a representative to investigate the inactivating mechanism of series C acting on PMMoV.

ABPP technology utilized its unique chemical probe strategy to selectively capture target proteins and was regarded as a well-established approach for discovering target proteins. According to the superior inactivating activity of C1 against PMMoV, the small molecule probe C2 was designed and synthesized by introducing an alkyne group into purine nucleoside with piperazine as a bridging bond. As can be seen from Table 1, C2 exhibited excellent inactivating activity against PMMoV, with an EC₅₀ value of 40.7 µg/mL, which was comparable to C1 (37.0 µg/mL) and superior to ningnanmycin (67.3 µg/mL). The aforementioned results indicated that C2 not only retains the antiviral properties of the parent compound, but also exhibits promising potential as a capture probe for target protein.

From the results of Western blot (Fig. S132), it could be seen that at the identical concentration, C2 was capable of capturing the protein with a molecular weight of 17 kDa, whereas neither C1 nor DMSO exhibited such a capacity. Of the four open reading frames of PMMoV, only PMMoV CP corresponded to a size of 17.5 kDa, indicating that the target protein of C1 acting on PMMoV was PMMoV CP.

Concentration dependent experiment ofC2with PMMoV CP and competitive inhibition betweenC2andC1with PMMoV CP

As illustrated in Fig. 1B, C2 could capture PMMoV CP, while C1 could not at the same concentration of 100 µM. Additionally, C2 successfully labeled protein at concentrations of 10–50 µM, but failed to do so when the concentrations were reduced to 5–1 µM. These results collectively established that the interaction of C2 with PMMoV CP occurred in a concentration-dependent manner.

As manifested in Fig. 1C, C2 formed a distinct protein band with PMMoV CP in the absence of C1, while no corresponding protein band was observed in DMSO. The intensity of this protein band was gradually weakened with increasing concentrations of C1. These findings collectively demonstrated that C1 competitively inhibited the interaction of C2 with PMMoV CP.

Detection of binding affinities of compounds with PMMoV CP via MST

C1, C24, and C25 with good, moderate, and poor inactivating activities against PMMoV were chosen to evaluate their binding affinities with PMMoV CP via MST using ningnanmycin as a control agent. According to Fig. 1D, compound C1 exhibited strong binding affinity to PMMoV CP, with a dissociation constant of 1.50 µM, which was better than ningnanmycin (K_d = 13.59 µM), C24 (K_d = 109.12 µM), and C25 (K_d = 267.24 µM).

Searching for the pivotal amino acid residues ofC1acting on PMMoV CP via molecular modeling

As shown in Fig. 2D, RMSD of the system remained in an upward phase until 35,000 ps, indicating that C1 and PMMoV CP were relatively unstable in the initial stage of MD simulations. However, as the simulation progressed, small molecule penetrated deeper into the protein pocket and formed additional hydrogen bonds, thus reaching a stable state for both. According to the binding mode obtained from equilibrated MD simulations (Fig. 2A), C1 mainly interacted with the Tyr13 and Tyr140 of PMMoV CP through hydrogen bonding.

In order to investigate which of these two amino acid residues was more critical, PMMoV CP^Y13A and PMMoV CP^Y140A were subsequently constructed using Modeller and the MD simulations of their binding modes with C1 were conducted via Amber22. From Fig. 2E-F, it could be seen that compared to PMMoV CP, PMMoV CP^Y13A and PMMoV CP^Y140A had larger fluctuations and they were basically in a dynamically stable state after 35000 ps. The fluctuation ranges of RMSF and Rg values of the proteins before and after the mutation were relatively minimal, indicating that the protein structures before and after the mutation were stabilized in the dynamic process. After reaching a relative equilibrium state, the binding energies (Table S2) of C1 to PMMoV CP^Y13A and PMMoV CP^Y140A were −8.53 and −10.71 kcal/mol, respectively, both lower than that of C1 to PMMoV CP (−24.08 kcal/mol), revealing that the binding stabilities of C1 to PMMoV CP^Y13A and PMMoV CP^Y140A was not as stable as that of C1 to PMMoV CP. And the binding energy of C1 to PMMoV CP^Y13A was the weakest, indicating that Tyr13 plays a more critical role in stabilizing the interaction. The aforementioned findings implied that Tyr13 and Tyr140 of PMMoV CP might be the key binding sites for C1, with Tyr13 being potentially more critical. However, further experimental validation was required to confirm this hypothesis.

Difference in symptoms among GFP-PMMoV CP^Y13A, GFP-PMMoV CP^Y140A, and GFP-PMMoV in N. benthamiana

As exhibited in Fig. 3B, the green fluorescence of the inoculated leaves infiltrated with GFP-PMMoV changed from stronger to weaker at 7 dpi. Concurrently, widespread green fluorescence appeared in the systemic leaves. Compared with GFP-PMMoV, no green fluorescence was observed both in the systemic and infiltrated leaves inoculated with GFP-PMMoV CP^Y13A and GFP-PMMoV CP^Y140A.

With the extension of time, both the systemic leaves and veins of N. benthamiana inoculated with GFP-PMMoV exhibited significant green fluorescence at 14 dpi. Compared with GFP-PMMoV, only trace green fluorescence was discovered in the systemic leaves inoculated with GFP-PMMoV CP^Y140A, while there was no green fluorescence were detected in systemic leaves inoculated with GFP-PMMoV CP^Y13A.

Eventually, more obvious green fluorescence was appeared in the whole strain of N. benthamiana inoculated with GFP-PMMoV at 21 dpi. And a large amount of green fluorescence was found both in the systemic leaves and veins after inoculation of GFP-PMMoV CP^Y140A, whereas the green fluorescence of N. benthamiana injected with GFP-PMMoV CP^Y13A was hardly perceived.

Expression of CP and GFP by Western blot

The results of Western blot (Fig. 3C) revealed that the expression levels of CP and GFP were higher in the systemic leaves of N. benthamiana inoculated with GFP-PMMoV at 7, 14 and 21 dpi. In comparison with GFP-PMMoV, neither CP nor GFP was detectable in the systemic leaves inoculated with GFP-PMMoV CP^Y13A and GFP-PMMoV CP^Y140A at 7 dpi. And the systemic leaves of N. benthamiana inoculated with GFP-PMMoV CP^Y13A and GFP-PMMoV CP^Y140A displayed almost no expression of CP, while GFP was slightly expressed with inoculation of GFP-PMMoV CP^Y140A at 14 dpi (Fig. 3C). Finally, both CP and GFP were highly expressed in systemic leaves of N. benthamiana inoculated with GFP-PMMoV^Y140A, whereas neither CP nor GFP was expressed in systemic leaves inoculated with GFP-PMMoV^Y13A at 21 dpi (Fig. 3C).

Expression of CP gene via RT-qPCR

RT-qPCR (Fig. 3D) demonstrated that the CP gene was unexpressed in the systemic leaves of N. benthamiana inoculated with GFP-PMMoV CP^Y13A and GFP-PMMoV CP^Y140A, compared with those inoculated with GFP-PMMoV at 7 and 14 dpi. And the expression level of CP gene with inoculation of GFP-PMMoV CP^Y140A was approximately 26.0 % of GFP-PMMoV, whereas there was almost no expression of CP gene inoculated with GFP-PMMoV CP^Y13A at 21 dpi.

Looking for differential proteins that interacted with PMMoV CP and PMMoV CP^Y13Athrough Co-IP MS and RNA-Seq

The plant samples of the third day with highest expression level of Flag-CP (Fig. 4A) were selected for Co-IP MS and RNA-seq. As can be seen from Fig. 4B, the enrichment of magnetic beads was qualified, and then mass spectrometry could be further performed. According to the mapping result of Co-IP MS and RNA-seq (Fig. S136), the differentially expressed proteins (DEPs) between CP and CP^Y13A were LOC109206267, LOC109221810 and AFB70993.1. Combining the top ranked DEPs in Co-IP MS with the analysis of Co-IP MS and RNA-seq, 10 DEPs (Table 2) were chosen and analyzed by RT-qPCR to validate the omics data. According to the results of RT-qPCR (Fig. 4D), the up-regulated proteins were mainly A0A248QEL2, B1PSM0, I3QHX5, LOC109221810, and A2IBL2, which were in agreement with the data of omics. Therefore, these 5 proteins were chosen for further study.

Table 2.

List of differentially expressed proteins in Co-IP MS and RNA-seq.

No.	Protein ID	Protein Name	Organism	Length	Sig
1	A0A5B9Y6Q5	MLP-like protein 43	N. benthamiana	146 AA	up
2	A0A248QEL2	S-adenosylmethionine synthase	N. benthamiana	390 AA	up
3	A0A481NUV9	Ferredoxin-NADP(+) reductase (Fragment)	N. benthamiana	322 AA	up
4	B1PSM0	Methyltransferase	N. benthamiana	617 AA	up
5	I3QHX5	Adenosylhomocysteinase	N. benthamiana	485 AA	up
6	Q8H0B4	Mitogen-activated protein kinase	N. benthamiana	376 AA	up
7	A2IBL2	Histone H₂B	N. benthamiana	147 AA	up
8	LOC109206267	Putrescine N-methyltransferase 2	N. attenuata	681 AA	up
9	LOC109221810	Putrescine N-methyltransferase 2	N. attenuata	566 AA	up
10	AFB70993.1	Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit	N. attenuata	477 AA	down

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Enrichment analysis of the GO terms and KEGG pathways revealed that most of DEPs were mainly involved in primary and secondary metabolic processes. These findings further suggested that the infection of PMMoV significantly activated multiple proteins participating in metabolic pathways, thereby endowing plants with the ability to resist viruses.

Validation of differential proteins interacting with PMMoV CP and PMMoV CP^Y13Avia LCA

From the results of LCA in Fig. 5A, it can be seen that both PMMoV CP and PMMoV CP^Y13A interacted with A0A248QEL2, B1PSM0, I3QHX5, LOC109221810, and A2IBL2. (The LCA results of PMMoV CP and PMMoV CP^Y13A with LOC109221810 and A2IBL2 were shown in Fig. S137).

Confirmation of differential proteins interacting with PMMoV CP and PMMoV CP^Y13Avia BiFC

As can be seen from Fig. 5D-E, PMMoV CP and PMMoV CP^Y13A interacted with A0A248QEL2, B1PSM0, and I3QHX5, but not with A0A248QEL2 and LOC109221810 (The BiFC results of PMMoV CP and PMMoV CP^Y13A with LOC109221810 and A2IBL2 were exhibited in Fig. S138). Notably, there were differences in the interaction between PMMoV CP and PMMoV CP^Y13A with I3QHX5. Furthermore, the addition of C1 also affected the interaction between PMMoV CP and PMMoV CP^Y13A with I3QHX5 at 30, 36 and 42 hpi (Fig. 6A).

Fig. 6 — C1 attenuated viral replication by mediating the liquid–liquid phase separation (LLPS) formed by PMMoV CP and I3QHX5. (A) Differences in the interaction of PMMoV CP and PMMoV CP^Y13A with I3QHX5 at 30, 36 and 42 hpi before and after application of C1. (B) Phase separation and fluorescence recovery after photobleaching (FRAP) of PMMoV CP with I3QHX5 *in vivo* and *in vitro*.

Interaction of PMMoV CP with I3QHX5 in vitro

The result of Octet (Fig. 5B) demonstrated that PMMoV CP had a nanomolar binding affinity to I3QHX5, with a binding constant of 1.3 × 10^-8 M, which suggested that there was a strong interaction between PMMoV CP and I3QHX5 in vitro.

Liquid-liquid phase separation (LLPS) of PMMoV CP with I3QHX5

BiFC results (Fig. 5D) illustrated that there were obvious fluorescent aggregation and droplet formation after the interaction of PMMoV CP with I3QHX5, and these droplets would undergo fusion and fission, after which they would relax into spherical shapes (Fig. 6). And with fluorescence bleaching, the fluorescence intensity of the aggregates decreased to less than 50 %, and after stopping fluorescence bleaching, the visible fluorescence intensity gradually recovered. These results indicated that the condensed material formed by CP and I3QHX5 has the properties of LLPS in vivo. Subsequently, PMMoV CP-GFP and I3QHX5-GFP fusion expression vectors were constructed and the proteins were purified, and the results of phase separation and FRAP were basically in accord with those in vivo (Fig. 6B).

Based on the above results, it was further demonstrated that CP had the properties of LLPS both in vitro and in vivo after interacting with I3QHX5.

Silencing of I3QHX5 attenuated the infection of GFP-PMMoV in N. benthamiana

As displayed in Fig. 7, compared with the N. benthamiana inoculated with TRV:00, the expression of I3QHX5 with TRV:I3QHX5 decreased to approximately 60 % of normal levels, while the plant did not show any obvious phenotype at 10 dpi. And N. benthamiana with TRV:00 showed more severe crumpling symptoms and a broader range of green fluorescence than silenced plants of TRV:I3QHX5 inoculated with GFP-PMMoV at 8 dpi. Western blot and RT-qPCR demonstrated that silencing of I3QHX5 leading to an attenuated accumulation of PMMoV, revealing that PMMoV CP could regulate the protein of I3QHX5 to facilitate viral transmission (see Fig. 8).

Fig. 8 — Proposed antiviral mechanism of C1 through modulating phase separation in PMMoV infection. Overexpression of Flag-PMMoV CP triggered rapid upregulation of I3QHX5, and silencing of I3QHX5 significantly attenuated systemic invasion of PMMoV. PMMoV CP promoted LLPS by interacting with I3QHX5 to facilitate viral replication, while PMMoV CP^Y13A could not. And C1 specifically targeted PMMoV CP^Y13A to inhibit LLPS, thereby suppressing viral replication.

As shown in Fig. 7F, the accumulation of PMMoV RNAs in protoplasts of TRV:00 and TRV:I3QHX5 were lower after transfection of pCB-GFP-PMMoV for 12 h, while the accumulation level of PMMoV RNAs in TRV:I3QHX5 was less than that in TRV:00. And the accumulation level of PMMoV RNAs in TRV:00 was significantly higher than that of TRV:I3QHX5 after transfection of pCB-GFP-PMMoV for 24 h. The above results further indicated that silencing of I3QHX5 impaired the level of viral replication.

Discussion

In this study, a series of purine morpholine nucleoside analogues containing a sulfonamide fragment, including C1, C10, C12, C19, C22, C31, C32, and C34 with preferable inhibitory activities towards PMMoV, were designed and synthesized by modifying and reforming both the purine and sugar rings. And the EC₅₀ value for the inactivating activity of C1 against PMMoV was superior to those of C12, C31, C32, and C34. Although the EC₅₀ values for inactivating activities against PMMoV of C10, C19, and C22 were better than that of C1, the inactivating activity of C1 was superior to those of C10, C19, and C22 at 500 µg/mL. Based on the dual evaluation system of EC₅₀ value and inactivating efficiency, C1 was chosen as a representative to investigate the inactivating mechanism of series C acting on PMMoV.

Firstly, the small molecule probe C2 was synthesized by introducing an alkyne group into the purine ring through a modification on C1, and the inactivating activity of C2 against PMMoV was comparable to that of C1, suggesting that C2 could be used as a small molecule probe to capture target protein. It could be observed from the results of ABPP that, at the identical concentration, C2 was capable of capturing the protein with a molecular weight of 17 kDa, whereas neither C1 nor DMSO exhibited such a capacity. Of the four open reading frames of PMMoV, only PMMoV CP corresponded to a size of 17.5 kDa, indicating that the target protein of C1 acting on PMMoV was PMMoV CP. Concentration-dependent, competitive inhibition assays and MST further confirmed that PMMoV CP was a target of C1.

Complementary molecular docking analyses confirmed that both C1 and C2 interacted with Tyr13 and Tyr140 residues of PMMoV CP (Fig. S134). Together, these computational and experimental data strongly suggested that C1 and C2 competed mechanistically for the overlapping binding sites on PMMoV CP and thus might interfere with viral processes through similar pathways. In order to investigate the effects of Tyr13 and Tyr140 on virus infection, infectious clones of GFP-PMMoV CP^Y13A and GFP-PMMoV CP^Y140A were constructed. RT-qPCR and Western blot revealed that the substitution of tyrosine with alanine at position 140 (Y140A) in the PMMoV CP delayed the systemic infection of PMMoV, while the mutation of Y13A inhibited the systemic infection. These findings highlighted that Tyr13 of PMMoV CP played an essential role than Tyr140 in viral infection, which was consistent with the result of molecular docking and MD simulations. Subsequently, Co-IP MS and RNA-seq were proposed to analyze the reasons for this diversity by looking for differences between PMMoV CP and PMMoV CP^Y13A.

A total of 5 proteins designated A0A248QEL2, B1PSM0, I3QHX5, LOC109221810, and A2IBL2 were identified through Co-IP MS, RNA-seq and RT-qPCR. From the results of LCA and BiFC, it can be seen that both PMMoV CP and PMMoV CP^Y13A interacted with A0A248QEL2, B1PSM0, and I3QHX5, but not with LOC109221810 and A2IBL2. It was entertaining that a large number of aggregates were formed after the interaction between PMMoV CP and I3QHX5, while no aggregates were produced with the interaction between PMMoV CP^Y13A and I3QHX5 both in vivo and in vitro (Fig. S142). The formation of aggregates was closely related to phase separation as reported in literature [59,60]. In cells, certain proteins and other biomolecules could concentrate together through phase separation to form aggregates. These aggregates might serve as “reaction centers” within cells, promoting specific biochemical reactions or signaling processes. Phase separation has become a fundamental principle for organizing and dividing cellular processes and played an important role in the lifecycle of viruses. It regarded as the foundation for the formation of certain virus factories, such as inclusion bodies (IB), which could achieve efficient virus replication and particle assembly, and served as targets for antiviral agents [59,60]. Based on the fusion, fission and FRAP, it was demonstrated that CP and I3QHX5 could form LLPS both in vivo and in vitro, which further indicated that the formation of LLPS might be the main reason for the difference in the systemic infection between GFP-PMMoV CP and GFP-PMMoV CP^Y13A. As shown in Fig. 6A, the addition of C1 at 30, 36, and 42 hpi also affected the interactions of PMMoV CP with I3QHX5. Further suggesting that the antiviral activity exhibited by C1 might be due to the inhibition of aggregate formation. Tyrosine has been proven to be crucial for phase separation. Mutations or deletions of tyrosine residues could reduce their phase separation tendency [[61], [62], [63]]. This result was consistent with that the mutation of amino acid at 13 from tyrosine to alanine in PMMoV CP inhibited the propensity to form phase separation with I3QHX5.

Since it was tentatively concluded that the diverse interactions in PMMoV CP and PMMoV CP^Y13A with I3QHX5 were responsible for their differences in viral infection, what role did I3QHX5 play in virus infestation? Subsequently, VIGS was utilized to investigate the role of I3QHX5 played in viral infection. As shown in Fig. 7C, TRV:00 controls exhibited pronounced leaf curling phenotypes and enhanced green fluorescent signals at 8 dpi inoculated with GFP-PMMoV, while TRV:I3QHX5 plants displayed diminished fluorescent intensity and absence of phenotypic abnormalities under identical experimental conditions. Western blot and RT-qPCR manifested that silencing of I3QHX5 in N. benthamiana significantly reduced the accumulation of PMMoV CP and CP mRNA, indicating a critical role of I3QHX5 in viral systemic spread. And the accumulation level of PMMoV RNAs in protoplasts of TRV:00 was significantly higher than that of TRV:I3QHX5 after transfection of pCB-GFP-PMMoV for 24 h, further suggesting that silencing of I3QHX5 impaired the efficiency of viral replication. This finding was consistent with previous reports in the literature that AHCY (or SAHH) played crucial roles in viral infection, replication, or immune evasion across diverse viruses by regulating methylation metabolism or directly participating in the viral life cycle, thereby offering potential molecular targets for antiviral strategies [[64], [65], [66]].

In summary, our research findings revealed that C1 had significant potential as an antiviral agent. Further molecular docking, Co-IP MS, RNA-seq, LCA, BiFC, FRAP, and VIGS indicated that PMMoV CP could form LLPS through interaction with I3QHX5, while PMMoV CP^Y13A could not, thereby explaining the observed differences in viral replication efficiency.

Conclusion

In this study, thirty-eight purine morpholine nucleoside analogues incorporating a sulfonamide fragment with favorable activities against PMMoV were designed and synthesized. ABPP technology, Western blot and MST demonstrated that PMMoV CP was the target protein of C1. Molecular docking, MD simulations, confocal, Western blot and RT-qPCR revealed that Y13A in PMMoV CP could severely inhibit virus infection. Correlated mechanism studies revealed that PMMoV CP could aggregate after interacting with I3QHX5, while PMMoV CP^Y13A could not under confocal. Fusion, fission, and FRAP demonstrated that the condensed material formed by CP and I3QHX5 had the properties of LLPS both in vivo and in vitro. These findings indicated that C1 could inhibit the formation of LLPS by specifically targeting Tyr13 of PMMoV CP, thus achieving the purpose of antiviral effects. Therefore, this study not only pointed out the direction for the research and development of novel antiviral agents, but also gave a certain reference for the study of antiviral mechanisms.

Compliance with ethics requirement

This article does not contain any studies with human or animal subjects.

CRediT authorship contribution statement

Yuyuan Yang: Methodology, Investigation, Visualization, Data curation, Formal analysis, Writing – original draft. Runjiang Song: Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review & editing. Shaobo Wang: Formal analysis, Writing – original draft. Guangcheng Zu: Formal analysis, Writing – original draft. Baoan Song: Conceptualization, Supervision, Funding acquisition, Project administration, Writing – review & editing.

Funding

We acknowledge the financial support from the National Natural Science Foundation of China (No. 32330087 & 32302388), the Key Technologies R&D Program of Guizhou Province in China (No. 2017-5788-1), and the Scientific Research Innovation Team of Guizhou University (No. 202403).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

We thank Prof. Fei Yan (Ningbo University, Zhejiang, China) for generously providing antibody of PMMoV CP and plasmid of pCB-GFP PMMoV. We appreciate the assistance provided by Assoc. Prof. Fengxu Wu (Hubei University of Medicine, Hubei, China) in molecular docking and molecular dynamics simulations.

Footnotes

^{Appendix A}

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jare.2025.05.062.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary Data 1

mmc1.docx^{(267.6MB, docx)}

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PERMALINK

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

Yuyuan Yang

Runjiang Song

Shaobo Wang

Guangcheng Zu

Baoan Song

Graphical abstract

Highlights

Abstract

Introduction

Objectives

Methods

Results

Conclusion

Introduction

Materials and methods

Materials

General procedures for the preparation of target compoundsC1,C3-C38

General procedures for the preparation of target compoundC2

Antiviral activity assay

Microscale thermophoresis (MST)

Molecular modeling

Homology modeling

Molecular docking

Molecular dynamics (MD) simulations

Transient expression of agrobacterium tumefaciens

Co-immunoprecipitation mass spectrometry (Co-IP MS) and RNA-seq

Luciferase complementation assay (LCA) and bimolecular fluorescence complementation (BiFC)

Virus-induced gene silencing (VIGS)

Results

Design and synthesis of target compoundsC1,C3-C38

Fig. 1.

Inhibitory activities ofC1,C3-C38against PMMoV

Table 1.

Searching for the target protein ofC1acted on PMMoV

Concentration dependent experiment ofC2with PMMoV CP and competitive inhibition betweenC2andC1with PMMoV CP

Detection of binding affinities of compounds with PMMoV CP via MST

Searching for the pivotal amino acid residues ofC1acting on PMMoV CP via molecular modeling

Fig. 2.

Difference in symptoms among GFP-PMMoV CPY13A, GFP-PMMoV CPY140A, and GFP-PMMoV in N. benthamiana

Fig. 3.

Expression of CP and GFP by Western blot

Expression of CP gene via RT-qPCR

Looking for differential proteins that interacted with PMMoV CP and PMMoV CPY13Athrough Co-IP MS and RNA-Seq

Fig. 4.

Table 2.

Validation of differential proteins interacting with PMMoV CP and PMMoV CPY13Avia LCA

Fig. 5.

Confirmation of differential proteins interacting with PMMoV CP and PMMoV CPY13Avia BiFC

Fig. 6.

Interaction of PMMoV CP with I3QHX5 in vitro

Liquid-liquid phase separation (LLPS) of PMMoV CP with I3QHX5

Silencing of I3QHX5 attenuated the infection of GFP-PMMoV in N. benthamiana

Fig. 7.

Fig. 8.

Discussion

Conclusion

Compliance with ethics requirement

CRediT authorship contribution statement

Funding

Declaration of competing interest

Acknowledgement

Footnotes

Appendix A. Supplementary data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Inhibitory activities ofC1,C3-*C38**against PMMoV*

Difference in symptoms among GFP-PMMoV CP^Y13A, GFP-PMMoV CP^Y140A, and GFP-PMMoV in N. benthamiana

Looking for differential proteins that interacted with PMMoV CP and PMMoV CP^Y13Athrough Co-IP MS and RNA-Seq

Validation of differential proteins interacting with PMMoV CP and PMMoV CP^Y13Avia LCA

Confirmation of differential proteins interacting with PMMoV CP and PMMoV CP^Y13Avia BiFC