Adaptation of a Potyvirus Chimera Increases Its Virulence in a Compatible Host through Changes in HCPro

Abstract

A viral chimera in which the P1-HCPro bi-cistron of a plum pox virus construct (PPV-GFP) was replaced by that of potato virus Y (PVY) spread slowly systemically in Nicotiana benthamiana plants and accumulated to levels that were 5−10% those of parental PPV-GFP. We tested whether consecutive mechanical passages could increase its virulence, and found that after several passages, chimera titers rose and symptoms increased. We sequenced over half the genome of passaged chimera lineages infecting two plants. The regions sequenced were 5′NCR-P1-HCPro-P3; Vpg/NIa; GFP-CP, because of being potential sites for mutations/deletions leading to adaptation. We found few substitutions, all non-synonymous: two in one chimera (nt 2053 HCPro, and 5733 Vpg/NIa), and three in the other (2359 HCPro, 5729 Vpg/NIa, 9466 CP). HCPro substitutions 2053 AUU(Ile)→ACU(Thr), and 2359 CUG(Leu)→CGG(Arg) occurred at positions where single nucleotide polymorphisms were observed in NGS libraries of sRNA reads from agroinfiltrated plants (generation 1). Remarkably, position 2053 was the only one in the sequenced protein-encoding genome in which polymorphisms were common to the four libraries, suggesting that selective pressure existed to alter that specific nucleotide, previous to any passage. Mutations 5729 and 5733 in the Vpg by contrast did not correlate with polymorphisms in generation 1 libraries. Reverse genetics showed that substitution 2053 alone increased several-fold viral local accumulation, speed of systemic spread, and systemic titers.

Adaptive substitutions at two amino acids of HCPro modify its functional properties to separately increase the virulence of a potyviral chimera

“…Compared to the parental PPV-GFP, the chimera accumulated and moved poorly (Sun et al, 2022), but after several mechanical passages it increased in virulence (titres, symptoms, speed of infection) in some plants. This adaptation correlated with the detection in the viral consensus sequences of those plants of non-synonymous mutations in some cistrons, two of them inside HCPro, changing amino acid 352 (Ile→Thr) or 454 (Leu→Arg) (Sun et al, 2022). Multifunctional HCPro suppresses sRNA-based silencing defences against potyviruses and is therefore important in allowing compatible infections and in setting viral titres (Li & Wang, 2019;Yang et al, 2021).…”

“…From the viral consensus sequences in those plants, adaptation correlated with the presence of either of two naturally acquired amino acid substitutions in the HCPro, the presence of substitutions in other viral proteins, such as the VPg and the coat protein (CP), or of mutations in non‐coding regions. We also found that the two amino acid substitutions inside HCPro, amino acids 352 Ile→Thr or 454 Leu→Arg, coincided with sites that displayed recurrent nucleotide polymorphisms in libraries of small RNA (sRNA) reads of viral sequences generated in first infections (Sun et al., 2022 ). This suggested that from the start of infection selective pressures were favouring within the viral population genomic copies that carried those specific mutations, in addition to limited variability elsewhere (Sun et al., 2022 ).…”

“…We previously found that a cloned chimera based on a PPV variant in which its P1‐HCPro bi‐cistron had been replaced by a modified one from potato virus Y (PVY) was infectious, though its titres were very low (5%–10% those of the parental PPV–GFP) and systemic dispersal within Nicotiana benthamiana was very slow, the chimera taking over 20 days to go systemic compared to 7 days for the parental strain (Sun et al., 2022 ). This chimeral virus was thus unadapted to the compatible host and, because of its clonal nature, lacked initial genetic variability.…”

In Silico Identification of Sugarcane Genome-Encoded MicroRNAs Targeting Sugarcane Mosaic Virus

“…It has been reported as a viral suppressor. HC-Pro is required to enhance expression via the fusion of P1, symptom development, and viral replication [103][104][105][106][107][108]. Until now, the potential for exploiting the regulation of sugarcane genome-encoded miRNA to abate infection by SCMV has not been investigated as a strategy for developing tolerant or resistant sugarcane cultivars.…”

The In Silico Sugarcane Genome-Encoded MicroRNA and Target Network Prediction for Targeting the SCMV

“…It has been reported as a viral suppressor. Enhanced expression by fusion of P1, symptoms development and viral replication are the key functions [61][62][63][64][65][66]. The miRanda and RNA22 algorithms predicted target site of sof-miR168 (a, b) at nucleotide position 1827.…”