Natural self-attenuation of pathogenic viruses by deleting the silencing suppressor coding sequence for long-term plant-virus coexistence

Abstract

Potyviridae is the largest family of plant-infecting RNA viruses. All members of the family (potyvirids) have single-stranded positive-sense RNA genomes, with polyprotein processing as the expression strategy. The 5'-proximal regions of all potyvirids, except bymoviruses, encode two types of leader proteases: the serine protease P1 and the cysteine protease HCPro. However, their arrangement and sequence composition vary greatly among genera or even species. The leader proteases play multiple important roles in different potyvirid-host combinations, including RNA silencing suppression and virus transmission. Here, we report that viruses in the genus Arepavirus, which encode two HCPro leader proteases in tandem (HCPro1-HCPro2), can naturally lose the coding sequences for these two proteins during infection. Notably, this loss is associated with a shift in foliage symptoms from severe necrosis to mild chlorosis or even asymptomatic infections. Further analysis revealed that the deleted region is flanked by two short repeated sequences in the parental isolates, suggesting that recombination during virus replication likely drives this genomic deletion. Reverse genetic approaches confirmed that the loss of leader proteases weakens RNA silencing suppression and other critical functions. A field survey of areca palm trees displaying varied symptom severity identified a transitional stage in which full-length viruses and deletion mutants coexist in the same tree. Based on these findings, we propose a scenario in which full-length isolates drive robust infections and facilitate plant-to-plant transmission, eventually giving rise to leader protease-less variants that mitigate excessive damage to host trees, allowing long-term coexistence with the perennial host. To our knowledge, this is the first report of potyvirid self-attenuation via coding sequence loss.

Fig 1. ANSSV lost nearly the complete coding region of HCPro1-HCPro2 during infection in an areca palm tree.

(A) Leaf symptoms of an ANSSV-infected tree observed in 2017 and 2023. Virus isolates from the tree in 2017 and 2023 were designated as ANSSV-17 and ANSSV-23, respectively. Mild chlorosis is indicated by black arrows. (B) Summary of clean reads derived from RNA-seq data corresponding to the infected tree in 2017 and in 2023. (C) Alignment of clean reads with ANSSV-BT17 genome (MH330686). RNA-seq data from the infected tree in 2017 (upper panel) and in 2023 (lower panel) were used for the alignment. The horizontal axes represent nucleotide positions in the ANSSV-BT17 genome, while vertical axes indicate coverage. Nucleotide differences from the reference genome are highlighted with color bars: cytosine in blue, guanine in brown, adenine in green, and uridine in red. (D, E) RT-PCR analysis of RNA samples from the infected tree in 2023 using the indicated primer pairs. All leaves in the infected tree, L1-L6 (D), were tested. Equivalent amplicons from pSS-I-G, the ANSSV-BT17 infectious cDNA clone [34], were used as control. The RT-PCR product corresponding to Areca catechu Actin (AcActin) served as an internal control (lower panel).

Fig 2. ANRSV lost the complete coding region of HCPro1-HCPro2 during infection in an areca palm tree.

(A) Leaf symptoms of an ANRSV-infected tree observed in 2021 and 2023. Virus isolates from the tree in 2021 and 2023 were designated as ANRSV-HK21 and ANRSV-HK23, respectively. (B) Alignment of clean reads with ANRSV-XC1 genome (MH395371). RNA-seq data from the infected tree in 2023 were used for the alignment. The horizontal axes represent nucleotide positions in the ANRSV-XC1 genome, while vertical axes indicate coverage. Nucleotide differences from the reference genome are highlighted with color bars: cytosine in blue, guanine in brown, adenine in green, and uridine in red. (C, D) RT-PCR analysis of RNA samples from the infected tree in 2023 using the indicated primer pairs. All leaves in the infected tree, L1-L4 (C), were tested. Equivalent amplicons from pRS-G, the ANRSV-ZYZ infectious cDNA clone [47], were used as control. The RT-PCR product corresponding to Areca catechu Actin (AcActin) served as an internal control (lower panel).

Fig 3. Cloning, sequencing and analysis of the complete genome of ANSSV-BT23.

(A) Overlapped amplicons covering the entire genome of ANSSV-BT23. Panel I, RT-PCR amplification of seven overlapping fragments (lanes 1-7) spanning nearly the full-length genome of ANSSV-BT23. Panels II and III, amplicons deriving from 5’-RACE and 3’-RACE, respectively. Lanes 1 and 2 in panels II and III correspond to RNA extracted from healthy and ANSSV-BT23-infected areca palm trees, respectively. (B) Sequence comparison between the genomes of ANSSV-BT17 and ANSSV-BT23. The mutated nucleotides are indicated by vertical red lines. The amino acid changes resulting from missense mutations are shown. The missing region in ANSSV-BT23 is represented by red slant lines. The sequence alignment of HCPro1-to-P3 region highlights the missing sequence in ANSSV-BT23, with the duplicated 5-nt sequences flanking the deletion shaded in yellow. (C) Summary of nucleotide and amino acid variations in each viral cistron when comparing ANSSV-BT17 and ANSSV-BT23.

Fig 4. Cloning, sequencing and analysis of the complete genome of ANRSV-HK23.

(A) Overlapped amplicons covering the entire genome of ANSSV-BT23. Panel I, RT-PCR amplification of five overlapping fragments (lanes 1–5) spanning nearly the full-length genome of ANRSV-HK23. Panels II and III, amplicons deriving from 5’-RACE and 3’-RACE, respectively. Lanes 1 and 2 in panels II and III correspond to RNA extracted from healthy and ANRSV-HK23-infected areca palm trees, respectively. (B) Sequence comparison between the genomes of ANRSV-HK23 and ANRSV-XC1. The mutated nucleotides are indicated by vertical red lines. The amino acid changes resulting from missense mutations are shown. The missing region in ANRSV-HK23 is represented by red slant lines. The sequence alignment of 5′-UTR-to-P3 region highlights the missing sequence in ANRSV-HK23, with the duplicated 6-nt sequences flanking the deletion shaded in yellow. (C) Summary of nucleotide and amino acid variations in each viral cistron when comparing ANSSV-BT17 and ANSSV-BT23.

Fig 5. The absence of HCPro1-HCPro2 in ANSSV-BT23 and ANRSV-HK23 attenuates viral infection.

(A) Schematic diagrams of the indicated virus clones. The nearly complete (for pSS-BT23) and complete (for pRS-HK23) deletion of HCPro1-HCPro2 is indicated by red slant lines. For the hybrid clones, the regions indicated by red and blue rectangles are from ANSSV-BT17 and ANRSV-ZYZ, respectively. (B) Inoculation of pSS-BT23 and pSS-BT23-HCPro1-2¹⁷ in N. benthamiana. The indicated clones were agroinoculated into N. benthamiana plants (OD₆₀₀ = 0.5/ clone). Alternatively, the indicated plasmids were agoinoculated along a plasmid expressing TBSV P19 (final OD₆₀₀ = 0.5/ clone) in the infiltrated leaf. Representative plants were photographed at 18 days post-inoculation (dpi) under a UV lamp. Leaves indicated with rectangles in white dotted lines are enlarged. White arrows in (B) and (C) indicate scattered fluorescence spots. Bars, 5 cm. (C) Inoculation of pRS-HK23 and pRS-HK23-HCPro1-2^ZYZ. Idem to (B), except that representative plants were photographed at 20 dpi under a UV lamp. (D) Real-time RT-qPCR analysis of accumulation levels corresponding to the indicated viruses at 18 dpi (left) or 20 dpi (right). Two primer sets, SS23-qPCR-F/SS23-qPCR-R and RS23-qPCR-F/RS23-qPCR-R (S1 Table) targeting viral CPs, were designed to quantify the accumulation of ANSSV-BT23/ ANSSV-BT23-HCPro1-2¹⁷, and ANRSV-HK23/ ANRSV-HK23-HCPro1-2^ZYZ, respectively. A newly expanded leaf per plant was sampled for the assay. Error bars represent standard errors from three biological replicates. Statistically significant differences were determined by an unpaired two-tailed Student’s t test: ***, P < 0.001; *, 0.01 < P < 0.05; ns, P > 0.05. (E) Immunoblot detection of GFP at 18 dpi (left) and 20 dpi (right) in protein samples from plants infected with the indicated viruses. Coomassie blue staining of RbCL was used as a loading control.

Fig 6. The absence of HCPro1-HCPro2 compromises RNA silencing suppression activity and affects other critical functions.

(A) Inoculation of pSS-BT23 or pSS-BT23-HCPro1-2¹⁷ in wild-type (WT) and dcl2/4 N. benthamiana plants. The indicated clones were agroinoculated into plants (OD₆₀₀ = 0.5/ clone). Representative plants were photographed at 20 days post-inoculation (dpi) under a UV lamp. Leaves indicated with rectangles in white dotted lines are enlarged. Bars, 5 cm. (B) Inoculation of pRS-HK23 or pRS-HK23-HCPro1-2^ZYZ. Idem to (B). (C) Real-time RT-qPCR analysis of viral genomic RNA accumulation corresponding to the indicated viruses at 20 dpi. Two primer sets, SS23-qPCR-F/SS23-qPCR-R and RS23-qPCR-F/RS23-qPCR-R (S1 Table), were used to quantify the accumulation of ANSSV-BT23/ ANSSV-BT23-HCPro1-2¹⁷, and ANRSV-HK23/ ANRSV-HK23-HCPro1-2^ZYZ, respectively. A newly expanded leaf per plant was sampled for the assay. Error bars represent standard errors from three biological replicates. Statistically significant differences were determined by an unpaired two-tailed Student’s t test: ***, P < 0.001; **, 0.01 < P < 0.001; *, 0.01 < P < 0.05. (D) Immunoblot detection of GFP abundance at 20 dpi in protein samples from plants infected with the indicated viruses. Coomassie blue staining of RbCL was used as a loading control.

Fig 7. Pair-wise alignments of full-length and shorter ANRSV isolates found in the same tree.

For trees #10, #15 and #23, both the long and short amplicons (S3 Fig) were cloned. Plasmids from three independent colonies per plate were sequenced. Sequence from clones corresponding to the long amplicons were identical across samples. Nucleotide sequences of the long (FL) and various short (rounded numbers) amplicons were aligned. Short repeated sequences flanking the deleted fragments in the long amplicon are highlighted in yellow, while deleted regions in the short amplicons are marked with red slant lines.

Fig 8. A model illustrating the evolution of arepaviruses in infected trees.

The full-length parental viruses and isolates lacking HCPro1-HCPro2 are marked with red and blue dots, respectively. See the Discussion section for a detailed description of the evolutionary pathway.