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. 2015 Apr 2;7(4):993-1001.
doi: 10.1093/gbe/evv034.

Multiple Layers of Chimerism in a Single-Stranded DNA Virus Discovered by Deep Sequencing

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Free PMC article

Multiple Layers of Chimerism in a Single-Stranded DNA Virus Discovered by Deep Sequencing

Mart Krupovic et al. Genome Biol Evol. .
Free PMC article

Abstract

Viruses with single-stranded (ss) DNA genomes infect hosts in all three domains of life and include many medically, ecologically, and economically important pathogens. Recently, a new group of ssDNA viruses with chimeric genomes has been discovered through viral metagenomics. These chimeric viruses combine capsid protein genes and replicative protein genes that, respectively, appear to have been inherited from viruses with positive-strand RNA genomes, such as tombusviruses, and ssDNA genomes, such as circoviruses, nanoviruses or geminiviruses. Here, we describe the genome sequence of a new representative of this virus group and reveal an additional layer of chimerism among ssDNA viruses. We show that not only do these viruses encompass genes for capsid proteins and replicative proteins that have distinct evolutionary histories, but also the replicative genes themselves are chimeras of functional domains inherited from viruses of different families. Our results underscore the importance of horizontal gene transfer in the evolution of ssDNA viruses and the role of genetic recombination in the emergence of novel virus groups.

Keywords: genetic recombination; metagenomics; origin of viruses; ssDNA viruses; virus evolution.

Figures

F<sc>ig</sc>. 1.—
Fig. 1.—
Confirmation of contaminated columns as origin of CHIV14 DNA by qPCR. (A) Verification of CHIV14 genome assembled from the Illumina deep-sequencing data by overlapping PCR and inverted PCR. Five sets of overlapping primer pairs and one set of inverted primer pair were designed and used to amplify overlapping DNA fragments. (Left) Schematic diagram of the positions of primer pairs for the overlapping PCR and inverted PCR. (Right) Amplification overlapping viral DNA fragments. The numbers above indicate the primer pair used for the PCR as illustrated on the left. The numbers on the left indicate the molecular weight in base pairs. (B) Scatterplot showing copy number of CHIV14 per microliter of DNA extraction. DNA from patients (n = 13), healthy controls (n = 13), and water (n = 31) was extracted using QIAamp mini spin columns (QIAamp Viral RNA Mini kit; Qiagen). In parallel, seven DNA extractions for each specimen type (patients, healthy individuals, and water) were performed using the UCP columns. Each dot represents one specimen. Bars show the average copy numbers of the viral genome.
F<sc>ig</sc>. 2.—
Fig. 2.—
Characterization of CHIV14. (A) Genome map of CHIV14. Predicted protein-coding genes are indicated with arrows, indicating the direction of transcription. A circle indicates the position of a potential origin of replication containing the nonanucleotide motif (AAGTATTAC) which is identical to the one found in BSL RDHV genome. (B) Maximum-likelihood phylogenetic analysis of the tombusvirus-like CPs. CHIVs are highlighted in red, tombusviruses in green and unclassified ssRNA viruses are either in blue when isolated, or in cyan when assembled from the Lake Needwood RNA virome. Tobacco necrosis virus A and Olive mild mosaic virus, both members of the genus Necrovirus within Tombusviridae, have CPs lacking the projection (P) domain and were used as an outgroup. Numbers at the branch points represent SH-like local support values. Nodes with support values less than 75% were collapsed. NCBI GI numbers are indicated for all reference sequences. The respective origins of the viromes from which the CHIV genomes were assembled are indicated next to the CHIV names. (C) Structural model of the CHIV14 CP. The P and shell (S) domains are indicated. The coloring represents sequence conservation among CHIV CPs. The color key is provided at the bottom of the panel.
F<sc>ig</sc>. 3.—
Fig. 3.—
Analysis of the chimeric RC-Rep protein of CHIV14. (A) Domain organization of the CHIV14 RC-Rep. Red ellipses indicate the positions of the three identified domains: The N-terminal endonuclease domain Gemini_AL1 (PF00799, residues 65–191), central domain Gemini_AL1_M (PF08283; residues 196–291), and the C-terminal SF3 helicase domain (PF00910; residues 278–390). The diagnostic motifs of the RC-Rep proteins (MI-III) as well as Walker A, B, and C (WA–C) motifs of the SF3 helicase domain are shown at the top. The position of the geminivirus RC-Rep-specific motif GRS (geminivirus Rep sequence) defined by Nash et al. (2011) is also indicated. (B) Distinct sets of best BLASTp hits for two different regions of the CHIV14 RC-Rep. The N-terminal and C-terminal domains were most similar to the corresponding regions of RC-Reps from geminiviruses and nanoviruses, respectively. Maximum-likelihood phylogenetic analysis of the full-length RC-Rep protein (C), endonuclease domain (D), and SF3 helicase domain (E) of CHIV14. CHIVs are highlighted in red, whereas members of the families Circoviridae, Geminiviridae, and Nanoviridae are shaded blue, cyan, and green, respectively. Numbers at the branch points represent SH-like local support values. Abbreviations and NCBI GI: RaCV, Raven circovirus (115334608); CaCV, Canary circovirus (18875310); SwCV, Swan circovirus (156079716); DuCV, Duck circovirus (71658852); RfCV, Rhinolophus ferrumequinum circovirus 1 (389568560); SGCV, Silurus glanis circovirus (365269059); PCV-1, Porcine circovirus-1 (94451274); PCV-2, Porcine circovirus-2 (404553515); CyCV-TB, Cyclovirus bat/USA/2009 (318069480); DfCyV-1, Dragonfly cyclovirus 1 (324309814); DfCyclV, Dragonfly cyclicusvirus (406870761); CircoTM-6c, Circoviridae TM-6c (297598949); LDMD-2013, Circo-2 LDMD-2013 (528320274); SaCV-13, Sewage-associated circular virus-13 (664651387); BBTV, Banana bunchy top virus (81993219); SCSV, Subterranean clover stunt virus (82005379); FBNYV, Faba bean necrotic yellows virus (20143454); MVDV, Milk vetch dwarf virus (82005916); NepaV, Nepavirus (403044759); NimiV, Niminivirus (404352299); MSV, Maize streak virus (14794722); BamiV, Baminivirus (403044751); PGMV, Pepper golden mosaic virus (22128601); HrCTV, Horseradish curly top virus (1255063); AbMV, Abutilon mosaic virus (39980674); JaMV, Jatropha mosaic virus (612184447); MYSV, Macroptilium yellow spot virus (417355462); TLCV-ND, Tomato leaf curl New Delhi virus (562890733); PedLCV, Pedilanthus leaf curl virus (224581833); BCTV, Beet curly top virus (46254388); TPCTV, Tomato pseudo-curly top virus (20564197).

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