Virus Host Jumping Can Be Boosted by Adaptation to a Bridge Plant Species

Abstract

Understanding biological mechanisms that regulate emergence of viral diseases, in particular those events engaging cross-species pathogens spillover, is becoming increasingly important in virology. Species barrier jumping has been extensively studied in animal viruses, and the critical role of a suitable intermediate host in animal viruses-generated human pandemics is highly topical. However, studies on host jumping involving plant viruses have been focused on shifting intra-species, leaving aside the putative role of "bridge hosts" in facilitating interspecies crossing. Here, we take advantage of several VPg mutants, derived from a chimeric construct of the potyvirus Plum pox virus (PPV), analyzing its differential behaviour in three herbaceous species. Our results showed that two VPg mutations in a Nicotiana clevelandii-adapted virus, emerged during adaptation to the bridge-host Arabidopsis thaliana, drastically prompted partial adaptation to Chenopodium foetidum. Although both changes are expected to facilitate productive interactions with eIF(iso)4E, polymorphims detected in PPV VPg and the three eIF(iso)4E studied, extrapolated to a recent VPg:eIF4E structural model, suggested that two adaptation ways can be operating. Remarkably, we found that VPg mutations driving host-range expansion in two non-related species, not only are not associated with cost trade-off constraints in the original host, but also improve fitness on it.

Keywords: Plum pox virus; RNA virus; VPg; eIF4E; host jumping; plant virus; potyvirus; trade-off; viral evolution.

Figure 1

Effect of VPg mutations on A. thaliana infection by Plum pox virus (PPV). A. thaliana plants were inoculated by biolistic with the chimeric clone pICPPV-VPgSwCM-R, its indicated mutant variants (two independent clones, 1 and 2), or the PPV-R clone pICPPV-NK-lGFP. Extracts from upper non-inoculated leaves collected at 15 days after inoculation were subjected to CP-specific immunoblot analysis. Three individual plants (P1, P2 and P3), inoculated with the specified viruses, were analyzed. An extract of healthy plants (H) was used as a negative control. Blots stained with Ponceau red showing the large subunit of the ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) are included as loading controls. The yellow star indicates that the progeny virus of this plant had incorporated a mutation in the VPg sequence (P114S).

Figure 2

Sequence analysis of viral progeny from Arabidopsis thaliana exposed to mixed infections with competing viruses. DNAs of pICPPV-VPgSwCM-R chimeric clones modified by the specified mutations were mixed at the indicated ratio and biolistically inoculated into six A. thaliana plants. Viral progenies were analyzed in pools of two plants by reverse transcription-polymerase chain reaction (RT-PCR), then sequencing a cDNA fragment covering the VPg coding sequence. Images show the chromatograms of VPg codons 114 (position 1968–1970 in the viral genome) or 163 (position 2017–2019 in the viral genome). Identified viruses are indicated beneath the chromatograms; smaller letters indicate lower accumulation. A similar result was obtained in a replicate assay.

Figure 3

Sequence analysis of viral progeny from Nicotiana clevelandii exposed to mixed infections with competing viruses. N. clevelandii plants, 8 or 10, were inoculated by hand rubbing with mixtures containing DNAs of pICPPV-VPgSwCM-R DNA and of one version of this chimera modified with the mutation F163L (A), P114S plus F163L (B) or P114S (C). Non-mutated/mutated chimera mixtures at 1.5:1 ratio, were employed for the three competitions. An additional 1:1 ratio mixture was used for the PPV-VPgSwCM-R vs P114S competition. Viral progenies were analyzed in pools of two plants by reverse transcription-polymerase chain reaction (RT-PCR) amplification and sequencing of a DNA fragment covering the VPg coding sequence. Images show the chromatograms of VPg codons 163 (position 2017–2019 in the viral genome) and/or 114 (position 1968–1970 in the viral genome). Viruses identified are indicated beneath the chromatograms; smaller letters indicate lower accumulation. A similar result was obtained in a replicate assay.

Figure 4

Effect of VPg mutations on Chenopodium foetidum infection by Plum pox virus (PPV). C. foetidum leaves were inoculated by hand rubbing with leaf extracts of Nicotiana clevelandii plants, previously infected with two independent clones of pICPPV-VPgSwCM-R, variants of this chimeric clone mutated as indicated, or pICPPV-NK-lGFP (PPV-R isolate). Eight plants (three leaves per plant) per construct (four per clone) were inoculated. Representative images taken at 12 dpi under visible light are shown. Bar, 10.0 mm.

Figure 5

(A) Alignment of the translation initiation factors 4E from Homo sapiens (h-eIF4E) (P06730.2) along with eIF(iso)4Es from Arabidopsis thaliana [At-eIF(iso)4E] (O04663.2), Chenopodium foetidum [Cf-eIF(iso)4E] (partial sequence specifically obtained for this study) and Nicotiana clevelandii [Nc-eIF(iso)4E] (KC625579.1). Residues of eIF(iso)4E plant factors aligning with those interacting residues of h-eIF4E appear in bold and, in case of no conservation among the three plant species, are orange highlighted. Amino acids encoded by primers used for PCR amplification of the Cf-eIF(iso)4E sequence are italicized. Asterisks in the numbering of the partial Cf-eIF(iso)4E sequence indicate that for the count, missing amino acids were replaced by the equivalent ones from the full-length sequence of the Chenopodium quinoa eIF(iso)4E protein (LOC110697254 and LOC110692931). (B) Alignment of VPg sequences from Potato virus Y (PVY) (QED90173.1) and Plum pox virus (PPV), R isolate (EF569215) and SwCMp isolate (SHARCO database, http://w3.pierroton.inra.fr:8060, accessed on 1 April 2021). Two mutations independently arisen at SwCMp VPg, as consequence of the adaptation in A. thaliana, are shown in red over a yellow-shaded box. In both panels, specific amino acids perturbed as a result of interaction between h-eIF4E and PVY VPg, according to Coutinho de Oliveira et al. [51], are highlighted in blue. Residues of PPV VPg aligning with the h-eIF4E -interacting PVY VPg residues appear in bold. Protein sequences were aligned using Clustal Omega program (European Bioinformatics Institute), then adjusted by minor manual corrections.