Provirophages and transpovirons as the diverse mobilome of giant viruses

Abstract

A distinct class of infectious agents, the virophages that infect giant viruses of the Mimiviridae family, has been recently described. Here we report the simultaneous discovery of a giant virus of Acanthamoeba polyphaga (Lentille virus) that contains an integrated genome of a virophage (Sputnik 2), and a member of a previously unknown class of mobile genetic elements, the transpovirons. The transpovirons are linear DNA elements of ~7 kb that encompass six to eight protein-coding genes, two of which are homologous to virophage genes. Fluorescence in situ hybridization showed that the free form of the transpoviron replicates within the giant virus factory and accumulates in high copy numbers inside giant virus particles, Sputnik 2 particles, and amoeba cytoplasm. Analysis of deep-sequencing data showed that the virophage and the transpoviron can integrate in nearly any place in the chromosome of the giant virus host and that, although less frequently, the transpoviron can also be linked to the virophage chromosome. In addition, integrated fragments of transpoviron DNA were detected in several giant virus and Sputnik genomes. Analysis of 19 Mimivirus strains revealed three distinct transpovirons associated with three subgroups of Mimiviruses. The virophage, the transpoviron, and the previously identified self-splicing introns and inteins constitute the complex, interconnected mobilome of the giant viruses and are likely to substantially contribute to interviral gene transfer.

Fig. 1.

Lentille virus and Sputnik 2. (A) (Inset) In situ hybridization using Sputnik 2 probe (green), Lentille virus probe (red), and DAPI nucleic acid staining (blue) after 7 h p.i.; the merged image shows intense replication of both Lentille virus and the virophage inside the same viral factory of A. polyphaga cells; the arrow indicates colocalization of the virophage and Lentille virus signals. (B and C) PFGE (B) and Southern blot (C) using DIG-labeled Sputnik 2 probes showing the Sputnik genome integrated into that of Lentille virus. Lane 1: λ marker; lane 2: low-range marker; lane 3: native Mimivirus genome; lane 4: native Lentille virus genome; lane 5: native Sputnik 2 genome; lane 6: ApaI-digested Mimivirus genome; lane 7: ApaI-digested Lentille virus genome; lane 8: EagI-digested Mimivirus genome; lane 9: EagI-digested Lentille virus genome; lane 10: λ marker; lane 11: low-range marker; lane 12: ApaI-digested Sputnik 2 genome; lane 13: EagI-digested Sputnik 2 genome. (D) Map of the Sputnik 1 (outer circle) and Sputnik 2 genomes (inner circle). The ORF number is indicated above the genome along with orientations, clockwise in blue and counterclockwise in red. A line indentifies the EagI restriction site, which cuts once into the Sputnik 1 and 2 genomes. Inside: Sputnik 2 genome GC skew (orange/yellow) and GC content.

Fig. 2.

Identification of the transpoviron. (A) (Inset) In situ hybridization using a Lentille virus probe (green), transpoviron probe (red), and DAPI nucleic acid staining (blue) at 7 h p.i. in A. polyphaga cells. (B) PFGE showing the Lentille virus genome (asterisk) and the transpoviron as a second band between the 6.55- and 9.42-kb bands of the LR marker (lane 3) identified with a white arrow. (C) Southern blot using DIG-labeled transpoviron probes. Lane 1: λ marker; lane 2: low-range marker; lane 3: Lentille virus genome; lane 4: Sputnik 2 genome; lane 5: A. polyphaga; lane 6: A. castellanii; lane 7: A. polyphaga infected with Lentille virus and Sputnik 2; lane 8: A. polyphaga infected with Sputnik 2; lane 9: A. polyphaga infected with Mimivirus.

Fig. 3.

The spread of transpovirons and virophages in giant viruses. The figure shows the pattern of presence–absence of transpovirons and virophages among the three groups of giant viruses isolated in our laboratory (black) and by others (blue). The identified transpovirons and virophages are mapped onto the phylogenetic tree obtained from concatenated alignments of DNA polymerase B, D5-like ATPase/helicase, and a transcription factor of giant viruses. Accession numbers are indicated in SI Appendix, Table S8.

Fig. 4.

Organization of genes in transpoviron genomes. The approximate position and strand orientation of each ORF (>150 nt) is indicated by an arrow. The ORFs with homologs outside the transpovirons are color-coded and their predicted biochemical activities are indicated (TM, protein with a predicted transmembrane helix; C2H2, Zn-finger protein; Super Family I (SFI) Helicase). An ORF that is homologous between different transpovirons is rendered in blue, and completely uncharacterized ORFs (ORFans) are rendered in white.