A Bipartite Geminivirus with a Highly Divergent Genomic Organization Identified in Olive Trees May Represent a Novel Evolutionary Direction in the Family Geminiviridae

Abstract

Olea europaea Geminivirus (OEGV) was recently identified in olive in Italy through HTS. In this work, we used HTS to show the presence of an OEGV isolate in Portuguese olive trees and suggest the evolution direction of OEGV. The bipartite genome (DNA-A and DNA-B) of the OEGV-PT is similar to Old World begomoviruses in length, but it lacks a pre-coat protein (AV2), which is a typical feature of New World begomoviruses (NW). DNA-A genome organization is closer to NW, containing four ORFs; three in complementary-sense AC1/Rep, AC2/TrAP, AC3/REn and one in virion-sense AV1/CP, but no AC4, typical of begomoviruses. DNA-B comprises two ORFs; MP in virion sense with higher similarity to the tyrosine phosphorylation site of NW, but in opposite sense to begomoviruses; BC1, with no known conserved domains in the complementary sense and no NSP typical of bipartite begomoviruses. Our results show that OEGV presents the longest common region among the begomoviruses, and the TATA box and four replication-associated iterons in a completely new arrangement. We propose two new putative conserved regions for the geminiviruses CP. Lastly, we highlight unique features that may represent a new evolutionary direction for geminiviruses and suggest that OEGV-PT evolution may have occurred from an ancient OW monopartite Begomovirus that lost V2 and C4, gaining functions on cell-to-cell movement by acquiring a DNA-B component.

Keywords: Geminiviridae; Olea europaea L.; evolution; high-throughput sequencing; recombination.

Figure 1

(A) Common region of OEGV-PT DNA-A and DNA-B alignment (5′–3′). Iterons and complementary iterons sequences are shown in gray, black arrows indicate their relative orientation; TATA box is shown in gray; the nonanucleotide sequence is shown in blue together with the sequences with the potential to form a stem-loop structure (in gray) to the right and to the left. The differences in the nucleotide sequences between both OEGV-PT DNAs (A and B) are shown in bold. The ↓ (black arrow) indicates position 1 in the viral genome corresponding to the predicted replication origin of the viral DNA. GC rich region in shown in yellow. (B) Iterons arrangement in OEGV-PT DNA-A and DNA-B alignment (5′–3′) positioned in relation to the TATA box and stem loop. Gray arrows indicate two iterons (virion-sense) and two complementary iterons (complement-sense).

Figure 2

Phylogenetic trees constructed by the neighbor-joining (NJ) analysis from the alignment of the (A) OEGV PT full DNA-A (nt), (B) AC1/Rep (aa) and (C) AV1/CP (aa) from this study plus 39 sequences, with most representative geminiviruses including some isolates that had not yet been classified into genera, retrieved from the GenBank database. Virus genera, species and abbreviations, and GenBank accession numbers used in the NJ analysis are NW Begomovirus (SLCV clade); EuMV, SMLCV0, SLCV, PepGMV, MCLCuV, NW Begomovirus; AbMV, COYSV0, TGMV, BGYMV, JacYMV, OW Begomovirus; CoGMV, HGYMV0, SACMV, ACMV, ICMV, Mastrevirus; MSV, CpCAV, TYDV-A, Topocuvirus; TPCTV0, Turncurtovirus; TCTV, TLRV00-1, TLRV00-2, Curtovirus; PeYDV, BCTV, HrCTV, SSCTV, Eragrovirus; ECSV00, Capulavirus; FBSLCV, EcmLV, ALCV, PLLV00, Becurtovirus; BCTIV, SCTV00, Grablovirus; PrGV-A, GRBV, Citlodavirus; CCDaV, Maldovirus; AGV, GGV-A, and unassigned MMDAV0. Multiple sequence alignments were generated using MEGA 7 and the neighbor joining (BioNJ algorithms), based on calculations from pairwise nucleotide (nt) and amino acid (aa) sequences distances for full genome (nt) or proteins (aa) analysis. Numbers above the lines indicate bootstrap scores out of 1000 replicates. Roman numbers indicate the groups clustered in each phylogenetic tree.

Figure 2

Phylogenetic trees constructed by the neighbor-joining (NJ) analysis from the alignment of the (A) OEGV PT full DNA-A (nt), (B) AC1/Rep (aa) and (C) AV1/CP (aa) from this study plus 39 sequences, with most representative geminiviruses including some isolates that had not yet been classified into genera, retrieved from the GenBank database. Virus genera, species and abbreviations, and GenBank accession numbers used in the NJ analysis are NW Begomovirus (SLCV clade); EuMV, SMLCV0, SLCV, PepGMV, MCLCuV, NW Begomovirus; AbMV, COYSV0, TGMV, BGYMV, JacYMV, OW Begomovirus; CoGMV, HGYMV0, SACMV, ACMV, ICMV, Mastrevirus; MSV, CpCAV, TYDV-A, Topocuvirus; TPCTV0, Turncurtovirus; TCTV, TLRV00-1, TLRV00-2, Curtovirus; PeYDV, BCTV, HrCTV, SSCTV, Eragrovirus; ECSV00, Capulavirus; FBSLCV, EcmLV, ALCV, PLLV00, Becurtovirus; BCTIV, SCTV00, Grablovirus; PrGV-A, GRBV, Citlodavirus; CCDaV, Maldovirus; AGV, GGV-A, and unassigned MMDAV0. Multiple sequence alignments were generated using MEGA 7 and the neighbor joining (BioNJ algorithms), based on calculations from pairwise nucleotide (nt) and amino acid (aa) sequences distances for full genome (nt) or proteins (aa) analysis. Numbers above the lines indicate bootstrap scores out of 1000 replicates. Roman numbers indicate the groups clustered in each phylogenetic tree.

Figure 3

Multiple alignment of OEGV-PT CP protein sequences with most representative geminiviruses including some isolates that had not yet been classified into genera, retrieved from the GenBank database. Gray boxes are highlighting several conserved regions (CR) or motifs, and blue indicate 100% homology between the geminivirus. Conserved regions (CR) or motifs; CR I, motif PWRsMaGT (conserved on NW Begomovirus), CR II, CR III or putative motif candidates R, CR IV, CR V (amino acids enriched region in OEGV-PT) and CR VI or putative motif candidates ALY. Virus species and genera are NW Begomovirus (SLCV clade); EuMV, SMLCV0, SLCV, PepGMV, MCLCuV, NW Begomovirus; AbMV, COYSV0, TGMV, BGYMV, JacYMV, OW Begomovirus; CoGMV, HGYMV0, SACMV, ACMV, ICMV, Mastrevirus; MSV, CpCAV, TYDV-A, Topocuvirus; TPCTV0, Turncurtovirus; TCTV, TLRV00-1, TLRV00-2, Curtovirus; PeYDV, BCTV, HrCTV, SSCTV, Eragrovirus; ECSV00, Capulavirus; FBSLCV, EcmLV, ALCV, PLLV00, Becurtovirus; BCTIV, SCTV00, Grablovirus; PrGV-A, GRBV, Citlodavirus; CCDaV, Maldovirus; AGV, GGV-A, and unassigned genera; MMDaV.

Figure 3

Multiple alignment of OEGV-PT CP protein sequences with most representative geminiviruses including some isolates that had not yet been classified into genera, retrieved from the GenBank database. Gray boxes are highlighting several conserved regions (CR) or motifs, and blue indicate 100% homology between the geminivirus. Conserved regions (CR) or motifs; CR I, motif PWRsMaGT (conserved on NW Begomovirus), CR II, CR III or putative motif candidates R, CR IV, CR V (amino acids enriched region in OEGV-PT) and CR VI or putative motif candidates ALY. Virus species and genera are NW Begomovirus (SLCV clade); EuMV, SMLCV0, SLCV, PepGMV, MCLCuV, NW Begomovirus; AbMV, COYSV0, TGMV, BGYMV, JacYMV, OW Begomovirus; CoGMV, HGYMV0, SACMV, ACMV, ICMV, Mastrevirus; MSV, CpCAV, TYDV-A, Topocuvirus; TPCTV0, Turncurtovirus; TCTV, TLRV00-1, TLRV00-2, Curtovirus; PeYDV, BCTV, HrCTV, SSCTV, Eragrovirus; ECSV00, Capulavirus; FBSLCV, EcmLV, ALCV, PLLV00, Becurtovirus; BCTIV, SCTV00, Grablovirus; PrGV-A, GRBV, Citlodavirus; CCDaV, Maldovirus; AGV, GGV-A, and unassigned genera; MMDaV.

Figure 4

Alignment of OEGV-PT DNA-B MP aa sequences with begomoviruses showing (in gray) the putative tyrosine phosphorylation site [RK]-x(2,3)-[DE]-x(2,3)-Y in the NW Begomovirus and the homologous OW and OEGV-PT region. On top, codons of OEGV-PT sequence are shown. In green is shown how a single nt substitution (C by A) in the first codon of the site would be enough to change the functionality of tyrosine phosphorylation (T by K) in OEGV-PT. Blue shaded aa represent conserved aa among begomoviruses.

Figure 5

Schematic representation of the recombination events detected by RDP4.101 in OEGV-PT genome and corresponding p-values. A simplified linearized map was used to represent OEGV-PT full DNA-A. The floating boxes above the genome show the position of the two recombination events in OEGV-PT DNA-A. The floating arrows below the genome correspond to the position of each of the putative OEGV genes.