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Host genome integration and giant virus-induced reactivation of the virophage mavirus

Nature volume 540, pages 288–291 (2016)Cite this article

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Abstract

Endogenous viral elements are increasingly found in eukaryotic genomes1, yet little is known about their origins, dynamics, or function. Here we provide a compelling example of a DNA virus that readily integrates into a eukaryotic genome where it acts as an inducible antiviral defence system. We found that the virophage mavirus2, a parasite of the giant Cafeteria roenbergensis virus (CroV)3, integrates at multiple sites within the nuclear genome of the marine protozoan Cafeteria roenbergensis4. The endogenous mavirus is structurally and genetically similar to eukaryotic DNA transposons and endogenous viruses of the Maverick/Polinton family5,6,7. Provirophage genes are not constitutively expressed, but are specifically activated by superinfection with CroV, which induces the production of infectious mavirus particles. Virophages can inhibit the replication of mimivirus-like giant viruses and an anti-viral protective effect of provirophages on their hosts has been hypothesized2,8. We find that provirophage-carrying cells are not directly protected from CroV; however, lysis of these cells releases infectious mavirus particles that are then able to suppress CroV replication and enhance host survival during subsequent rounds of infection. The microbial host–parasite interaction described here involves an altruistic aspect and suggests that giant-virus-induced activation of provirophages might be ecologically relevant in natural protist populations.

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Figure 1: Generation and characterization of endogenous mavirus virophages in Cafeteria roenbergensis.
Figure 2: Gene expression analysis of the endogenous mavirus genome.
Figure 3: CroV infection induces replication and virion production of the endogenous mavirus.
Figure 4: Reactivated mavirus inhibits CroV and promotes host survival in subsequent infections.

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Acknowledgements

This research was supported by the Max Planck Society. We are grateful to C. Suttle for access to host and virus strains, and to the Roscoff team for maintaining and distributing protist strains. We thank K.-A. Seifert, K. Fenzl and K. Barenhoff for technical assistance, U. Mersdorf for electron microscopy, C. Roome for IT support, L. Czaja and the Max Planck Genome Centre in Cologne for bioinformatic assistance, S. Higgins for suggestions, K. Haslinger and J. Reinstein for comments on the manuscript, and I. Schlichting for mentoring and support.

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  1. Thomas Hackl

    Present address: †Present address: Massachusetts Institute of Technology, 15 Vassar Street, Cambridge, Massachusetts 02139, USA.,

Authors and Affiliations

  1. Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Heidelberg, 69120, Germany

    Matthias G. Fischer & Thomas Hackl

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  1. Matthias G. Fischer
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Contributions

M.G.F. conceived the study, designed and performed experiments, collected, analysed, and interpreted data, and wrote the manuscript. T.H. corrected and assembled sequence data, and analysed, interpreted, and visualized data. Both authors revised and approved the manuscript.

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Correspondence to Matthias G. Fischer.

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The authors declare no competing financial interests.

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Nature thanks J.-M. Claverie, E. Koonin and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 CroV-mavirus co-infection experiment of C. roenbergensis strain E4-10P to generate the mavirus-carrying strain E4-10M1.

C. roenbergensis strain E4-10P was either mock-infected, infected with CroV, or co-infected with CroV and mavirus. Cell densities are based on microscopy counts and were monitored for 8 days, viral numbers were monitored for 6 days and are derived from qPCR data assaying short amplicons of the mavirus MV18 gene (MCP) and the crov283 gene (D11-like transcription factor), respectively. The detection limit for both methods was about 103 per millilitre. These experiments were performed in single copies. The mavirus-positive host strain E4-10M1 was isolated from the pool of surviving cells in the +mavirus, +CroV infection. See also Supplementary Spreadsheet 4.

Source data

Extended Data Figure 2 The k-mer frequency distribution of the diploid genome of Cafeteria roenbergensis strain E4-10P.

Frequency distribution of in silico generated random 19-mers in the genomic read set of E4-10P. The distribution exhibits a major peak at 120× coverage (solid red line) corresponding to the majority of homozygous k-mers of the underlying diploid genome, a smaller peak at half the diploid coverage (60×, dotted green line) comprising haplotype-specific k-mers, and a weak third peak at three times the haploid coverage (180×, dashed blue line) indicating a primarly diploid, partly triploid genome structure. Low-coverage k-mers derive from sequencing errors and bacterial contamination.

Extended Data Figure 3 Gene expression of the endogenous mavirus genome is inhibited by cycloheximide (CHX) and aphidicolin (APH).

Selected cellular and viral transcripts isolated at 0 and 24 h p.i. from mock-infected or CroV-infected E4-10P and E4-10M1 cultures in the presence of 5 μg/ml aphidicolin or 50 μg/ml CHX were quantified by qRT-PCR. Shown are the average quantification cycle (Cq) values of three independent experiments; error bars, s.d. The following genes were assayed: host AspRS, C. roenbergensis E4-10 aspartyl-tRNA synthetase; crov342, CroV major capsid protein; crov497, CroV DNA polymerase B; crov505, CroV isoleucyl-tRNA synthetase; MV03, mavirus DNA polymerase B; MV15, mavirus genome-packaging ATPase; MV16, mavirus maturation protease; MV17, mavirus minor capsid protein; MV18, mavirus major capsid protein. Cq values of the reverse transcriptase-negative (−RT) reactions are shown directly to the right of the respective +RT results. Accession numbers are listed in Extended Data Table 3. See also Supplementary Spreadsheet 1.

Source data

Extended Data Figure 4 Hypothesis for CroV-induced reactivation of endogenous mavirus.

Shown is a schematic C. roenbergensis cell displaying selected events of a CroV infection cycle in strains E4-10P (left) and E4-10M1 (right). Following CroV entry (1), the virion factory forms in the cytoplasm. At the onset of late phase, a CroV-encoded transcription factor recognizing the late CroV promoter motif is synthesized (2). We hypothesize that in E4-10M1 cells, the late transcription factor could enter the nucleus (3), bind the mavirus promoter sequences, and activate gene expression of the provirophage (4). Mavirus-specific transcripts would then be exported and translated (5) and some of the mavirus proteins would return to the nucleus to excise or replicate the provirophage genome (6). The mavirus genome could then translocate to the CroV factory (7), where genome replication, particle assembly, and genome packaging would occur (8). Cell lysis releases the newly synthesized CroV and mavirus particles (9) and the reactivated virophages inhibit CroV propagation during subsequent co-infections (10). By contrast, CroV infection of an E4-10P cell does not induce a virophage response and CroV continues to infect other host populations (11).

Extended Data Figure 5 Purification and characterization of reactivated mavirus particles.

a, Three-litre cultures of CroV- or mock-infected E4-10P and E4-10M1 cultures were concentrated 200-fold and stained with uranyl acetate for electron microscopy. Representative particles from each filtrate are boxed and shown at higher magnification. Mavirus-like particles are marked by arrows. Filtrate numbers refer to the infections shown in Fig. 3a. b, The concentrated samples were analysed on 1.1–1.5 g/ml linear CsCl density gradients. A concentrated sample of reference mavirus was run in parallel and yielded a band at approximately 1.29 g/ml CsCl (arrow). Only the CroV-infected E4-10M1 culture produced a band at a similar density. c, PCR analysis of band material extracted from the CsCl gradients shown in b at a density of about 1.29 g/ml CsCl. Primers MaV21F and MaV21R were used to generate a 956-bp-long product of the MV19 gene. d, Material from the 1.29 g/ml CsCl band or from equivalent positions was extracted from the gradients and visualized by negative-stain electron microscopy. Only the CroV-infected E4-10M1 culture contained mavirus-like particles.

Extended Data Figure 6 Ultraviolet light treatment abolishes infectivity of reactivated mavirus.

Reactivated mavirus contained in filtrate 4 from the infection experiments shown in Fig. 3a was irradiated with 500 J m−2 of ultraviolet-C light (λ = 254 nm). Ultraviolet light-treated and untreated mavirus suspensions were tested for infectivity by co-infection of host strain E4-10P with CroV. As shown in the lower right panel, reactivated mavirus treated with ultraviolet light was no longer able to replicate in the presence of CroV. Data are shown as the mean of biological triplicates ± s.d. See also Supplementary Spreadsheet 5.

Source data

Extended Data Table 1 Cell and virus concentrations in different size fractions of mock-infected and CroV-infected E4-10P and E4-10M1 populations
Full size table
Extended Data Table 2 Details on the 11 bioinformatically well-supported mavirus integration sites in C. roenbergensis strain E4-10M1
Full size table
Extended Data Table 3 PCR oligonucleotide primers used in this study
Full size table

Supplementary information

Supplementary Spreadsheet 1

This file contains source data for figure 2 and Extended Data Figure 3. (XLSX 53 kb)

Supplementary Spreadsheet 2

This file contains source data for figure 3. (XLSX 54 kb)

Supplementary Spreadsheet 3

This file contains source data for figure 4. (XLSX 58 kb)

Supplementary Spreadsheet 4

This file contains source data for Extended Data Figure 1. (XLSX 12 kb)

Supplementary Spreadsheet 5

This file contains source data for Extended Data Figure 6. (XLSX 23 kb)

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Fischer, M., Hackl, T. Host genome integration and giant virus-induced reactivation of the virophage mavirus. Nature 540, 288–291 (2016). https://doi.org/10.1038/nature20593

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