Repair Of Frameshift Mutation In The Viral Genome Of Areca Palm Necrotic Ringspot Virus Variant ANRSV-A

Areca palm(Areca catechu L.)nut has extremely high medicinal value and is known as one of the top four southern Chinese medicinal materials.However,Areca palm necrotic ringspot virus(ANRSV)is an emerging virus belonging to the genus Arepavirus of the family Potyviridae which infects areca palm trees and results in the development of necrotic ringspot on the leave.ANRSV poses a significant threat to the areca palm industry and highlights the importance of plant disease management in agriculture.Infectious c DNA clones are a powerful tool to explore and characterize new pathogens using reverse genetics.In previous study,we obtained an infectious c DNA clone of ANRSV-A that harbored an additional adenylate(+1A)in a stretch of A’s between nucleotides 2818 and 2821,resulting in a translational frameshift with a premature stop codon at nucleotide 2830 in the viral genome.In this study,we found that the infectivity of ANRSV-A was restored post agroinoculation with Nicotiana benthamiana plants due to the repair of the frameshift within A-stretches in ANRSV-A via nucleotide insertions/deletions that generated 5 infectious viral RNA variants including wild-type ANRSV.We hypothesize that ANRSV-A exploits unknown nucleotide insertion/deletion RNA editing of the host plant to repair the frameshift,maintain viral genome integrity and genetic stability,and evade nonsense-mediated m RNA decay.Further studies have revealed that the A nucleotide at position 2823 nt is the key nucleotide involved in frameshift repair of ANRSV-A.The resulting 4 repaired virus variants except wild-type ANRSV induced mild symptoms in N.benthamiana plants.One of 5 repaired ANRSV clones presented as symptomless was identified to be a candidate for eliciting crossprotection against severe ANRSV infection.The main findings are as follows:1.The symptoms and systemic infections were observed in N.benthamiana agroinoculated with ANRSV-A at 35 days post inoculation(dpi).RT-PCR and Sanger sequencing analysis revealed the frameshift mutation site in ANRSV-A genome underwent single and multiple nucleotide insertions/deletions which eliminates the shift mutation in cells,resulting in 5 different repaired infective virus RNA variants included:wild-type ANRSV-WT which is repaired by deleting one A;ANRSV-Del4,which deletes four bases(three A’s and one U,causing the loss of one amino acid,I);ANRSVDel13,which deletes 13 bases(eight A’s,four U’s,and one C,causing the loss of four amino acids,IKFI);and ANRSV-Ins2,which inserts two A’s(adding one amino acid);ANRSV-Del1-NTS2 aa which deleting one A and includes two synonymous mutations in P3 protein.2.The single and multiple nucleotide insertions/deletion in frameshift site of ANRSV-A genome also detected on inoculated leaves with ANRSV-A at 9 days dpi using RT-PCR and Sanger sequencing.The resulting 2 different repaired infective virus RNA variants were :ANRSV-Del10,which deletes ten bases(six A’s,three T’s and one C,causing the loss of three aminos acid,KIH);and ANRSV-Del13,which deletes 13 bases(eight A’s,four U’s,and one C,causing the loss of four amino acids,IKFI).The result suggested that frameshift repair in the ANRSV-A genome on inoculated leaves resulted in production of multiple infectious viral RNA variants which lead systemic movement and infection in different plants.3.The repair of the frameshift within A-stretches in ANRSV-A failed to occur when the 2823 nt A at frameshift position was substituted with T,G,or C.The result showed that the A nucleotide at position 2823 nt is the key nucleotide involved in frameshift repair of ANRSV-A.4.To investigate the infectivity of 5 repaired infective virus variants,we constructed their green fluorescent protein(GFP)-tagged infectious c DNA clones(ANRSV-WT-GFP,ANRSV-Del13-GFP,ANRSV-Ins2-GFP,ANRSV-Del4-GFP,and ANRSV-Del1-NTS2aaGFP).The symptom,fluorescent tracking of viruses,RT-q PCR and Western blot revealed that the single-or multiple-amino acid mutations in P3 of ANRSV-Del13-GFP,ANRSVIns2-GFP,ANRSV-Del-GFP,and ANRSV-Del1-NTS2aa-GFP decreased symptom expression and viral accumulation level in N.benthamiana plants except the repaired wild-type ANRSV-WT-GFP.5.ANRSV-Del13-GFP infected N.benthamiana plants presented symptomless leaves.Primary ANRSV-Del13-GFP infection provided effective protection against ANRSV-WT infection and had no effect on plant growth and development.Our result showed that ANRSV-Del13-GFP is a promising mild strain for the practical use of crossprotection for controlling viruses in Areca palm.