Autoxidation Products of the Methanolic Extract of the Leaves of Combretum micranthum Exert Antiviral Activity against Tomato Brown Rugose Fruit Virus (ToBRFV)

Abstract

Tomato brown rugose fruit virus (ToBRFV) is a new damaging plant virus of great interest from both an economical and research point of view. ToBRFV is transmitted by contact, remains infective for months, and to-date, no resistant cultivars have been developed. Due to the relevance of this virus, new effective, sustainable, and operator-safe antiviral agents are needed. Thus, 4-hydroxybenzoic acid was identified as the main product of the alkaline autoxidation at high temperature of the methanolic extract of the leaves of C. micranthum, known for antiviral activity. The autoxidized extract and 4-hydroxybenzoic acid were assayed in in vitro experiments, in combination with a mechanical inoculation test of tomato plants. Catechinic acid, a common product of rearrangement of catechins in hot alkaline solution, was also tested. Degradation of the viral particles, evidenced by the absence of detectable ToBRFV RNA and the loss of virus infectivity, as a possible consequence of disassembly of the virus coat protein (CP), were shown. Homology modeling was then applied to prepare the protein model of ToBRFV CP, and its structure was optimized. Molecular docking simulation showed the interactions of the two compounds, with the amino acid residues responsible for CP-CP interactions. Catechinic acid showed the best binding energy value in comparison with ribavirin, an anti-tobamovirus agent.

Keywords: 4-hydroxybenzoic acid; Combretum micranthum; ToBRFV; catechinic acid; coat protein; molecular docking.

Figure 1

Crystal structure and active site of TMV CP. (a) The 20S disk is a four-layer cylindrical structure with 17 CP molecules in each ring [59,60]. (b) Architecture of the two CP subunits (chain A in green and chain B in orange) depicting the binding site residues in between them.

Figure 2

Interactions between chain A and chain B in TMV CP [74]. The protein residues are represented as follows: the negatively charged residues are indicated in red, positive residues are in cyan, neutral residues are shown in green; aliphatic residues are grey; Pro and Gly are orange. H-bonds are depicted as cyan lines; salt bonds are reported as red lines; non-bonded contacts are light orange dotted lines. The number of H-bond lines between any two residues indicates the number of potential hydrogen bonds between them. The width of the striped line of non-bonded contacts is proportional to the number of atomic contacts.

Figure 3

Plant inoculation test. (a): trial’s layout. (b–h): 35 days after the inoculation. (b,c): symptomatic apical leaves (plants inoculated with Positive Control); (d): not symptomatic apical leaves (plants inoculated with Negative Control); (e): not symptomatic apical leaves (plants inoculated with inoculum treated with 1); (f): not symptomatic apical leaves (plants inoculated with inoculum treated with AME); (g): not symptomatic apical leaves (plants inoculated with inoculum treated with 2); (h): not symptomatic apical leaves (plants inoculated with inoculum treated with NaClO).

Figure 4

Sequence alignment of CP sequences and homology model of ToBRFV CP; (a) Sequences alignment between ToBRFV CP and TMV CP; (b) 3D comparison between ToBRFV CP homology model (green) and TMV CP crystal structure (red).

Figure 5

The Ramachandran plot of ToBRFV CP homology model. There are 218 residues located in the most favored region (red), 51 residues in the additional allowable region (yellow), 14 residues in the generous allowable region (light yellow), and only 3 residues in the prohibited region (white). Gly is plotted as triangles, Pro is plotted as squares, and all other residues are plotted as circles.

Figure 6

Binding pose (a) and interactions (b) of catechinic acid at the ToBRFV CP active site. (a): the protein is reported as light-yellow ribbons; catechinic acid is reported as green capped sticks. H-bonds are presented as purple dotted lines. (b): catechinic acid is surrounded by the protein residues represented as follows: the negatively charged residues are indicated in red, polar residues are in cyan, hydrophobic residues are shown in green, and H-bonds are depicted as purple arrows.