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Isolation and infection cycle of a polinton-like virus virophage in an abundant marine alga

Nature Microbiology volume 8, pages 332–346 (2023)Cite this article

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Abstract

Virophages are small double stranded DNA (dsDNA) viruses that can only replicate in a host by co-infecting with another virus. Marine algae are commonly associated with virophage-like elements such as Polinton-like viruses (PLVs) that remain largely uncharacterized. Here we isolated a PLV that co-infects the alga Phaeocystis globosa with the Phaeocystis globosa virus-14T (PgV-14T), a close relative of the "Phaeocystis globosa virus-virophage" genomic sequence. We name this PLV ‘Gezel-14T. Gezel is phylogenetically distinct from the Lavidaviridae family where all known virophages belong. Gezel-14T co-infection decreases the fitness of its viral host by reducing burst sizes of PgV-14T, yet insufficiently to spare the cellular host population. Genomic screens show Gezel-14T-like PLVs integrated into Phaeocystis genomes, suggesting that these widespread viruses are capable of integration into cellular host genomes. This system presents an opportunity to better understand the evolution of eukaryotic dsDNA viruses as well as the complex dynamics and implications of viral parasitism.

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Fig. 1: Gezel-14T is a bona fide virus.
Fig. 2: Infection dynamics of P. globosa, PgV-14T and Gezel-14T.
Fig. 3: Gezel-14T proteomic features.
Fig. 4: PLVs associated with P. globosa.
Fig. 5: Phylogeny and gene content of Gezel-like PLVs.

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Data availability

The sequencing data are available from NCBI SRA SRR20333090 (Bioproject PRJNA835735). PgV-14T and Gezel-14T genome assemblies were deposited in NCBI Genbank under accession numbers OP080611 and OP080612. Annotated fragments of complete PLVs and NDDV from P. globosa and other algae are provided as Supplementary File 6 . Source data are provided with this paper. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD036892. Additional material is supplied in the Figshare repository at https://doi.org/10.6084/m9.figshare.21294852. Source data are provided with this paper.

Code availability

Code used for bioinformatic analyses is available at https://github.com/BejaLab/Gezelvirus and https://github.com/BejaLab/phaeocystis-viral-elements.

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Acknowledgements

We thank A. Noordeloos for providing advice on how to culture P. globosa and PgV-14T, L. Shaulov for expert technical assistance with TEM sample preparations and imaging, I. Pekarsky and N. Dahan for help with light microscopy, I. Navon and the Smoler Proteomics Center for help with the mass spectrometry analyses, the ICTV Virophage study group for nomenclature discussions, and S. Larom for technical assistance. This work was funded by a European Commission ERC Advanced Grant (321647, to O.B.), Israel Science Foundation grants 143/18 (O.B.), 1623/17 and 2167/17 (T.L. and O.K.), and the Ariane de Rothschild Women Doctoral Program (S.R.). O.B. holds a Louis and Lyra Richmond Chair in Life Sciences.

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Authors and Affiliations

  1. Faculty of Biology, Technion - Israel Institute of Technology, Haifa, Israel

    Sheila Roitman, Andrey Rozenberg, Tali Lavy, Oded Kleifeld & Oded Béjà

  2. Department of Marine Microbiology and Biogeochemistry, NIOZ Royal Netherlands Institute for Sea Research, Texel, The Netherlands

    Corina P. D. Brussaard

  3. Institute for Biodiversity and Ecosystem Dynamics (IBED), University of Amsterdam, Amsterdam, The Netherlands

    Corina P. D. Brussaard

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  1. Sheila Roitman
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Contributions

S.R. conceived the project, designed the experiments and performed the experimental work. A.R. performed bioinformatic analyses. S.R., T.L. and O.K. performed proteomics. C.P.D.B supplied the algal and viral strains. O.B. supervised the project. S.R. drafted the paper, which was critically revised and approved by all authors.

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Correspondence to Sheila Roitman or Oded Béjà.

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Extended data

Extended Data Fig. 1 Comparison of the sequences of the terminal inverted repeats (TIRs) in Gezel-16T (formerly PgVV) and Gezel-14T.

a. The sequences of the TIRs were subdivided into (near) identical units based on their appearance in the four flanking regions. Note that the ends of the Gezel-14T genome could not be fully assembled and are thus truncated. Positions of the dinucleotide variation in units of type A are indicated. A region with homology to the TIR sequences located in both genomes in the intergenic spacer between genes TVpol and pgvv05 is shown for comparison. b. Results of amplification of the Gezel-14T TIR regions using a forward primer in unit G and side-specific reverse primers (shown to the left). The two major PCR products correspond to two versions of the fragment differing by the number of DAE units.

Extended Data Fig. 2 Gezel-14T is a virophage.

Infection experiments on P. globosa cultures infected with a. a mixed PgV-14T/Gezel-14T lysate, b. a pure PgV-14T lysate, c. Gezel-14T only. Purple lines denote the uninfected control culture; full lines the cell survival measured by OD of chlorophyll A. Viral abundances were calculated by qPCR and are marked as dashed lines for PgV-14T and dotted lines for Gezel-14T. n = 3 biologically independent cultures and lysates. Data are presented as mean values +/− SD, exact values can be found in Supplementary File 1 ‘Gezel-14T only infection’.

Extended Data Fig. 3 Gezel-14T ORFs not detected by proteomics are transcribed during infection.

PCR on cDNA for the six ORFs with no significant hits in the proteomics analyses. M, Molecular Marker; -, non-template control; +, positive control (Gezel-14T DNA); samples were collected 2,4 and 6 hs post-infection. Two experiments (biological replicates) were analysed for each gene, only one is shown here.

Source data

Extended Data Fig. 4 Secondary structure and domain composition of the protein coded by Gezel-16T ORF PGVV_00014.

First track: per-position PSIPRED secondary structure prediction with blue lines corresponding to beta sheets and red lines to alpha-helices (coils not shown), Y-axis reflects confidence values (0–9). Second track: position of the large deletion in Gezel-14T. Third track: amino acid repeats discovered with RADAR with each colour corresponding to a repeat type. Fourth track: locations of the hhsearch matches to Pfam profile PF03903 (Enterobacteria phage T4 tail-fiber protein gp36) when searched against the Pfam database distributed with HH-Suite. Fifth track: hhsearch matches to Uniprot records: VP1_MPRVN – protein VP1 of Micromonas pusilla reovirus (Q1I0V1); FIBL1_BPT5 – L-shaped tail-fiber protein pb1 of Escherichia phage T5 (P13390).

Extended Data Fig. 5 Proteomic analysis of PgV-14T particles and during infection.

Proteins found by mass spectrometry in purified PgV-14T viral particles (P - rhomboids) and 4, 6 and 8 hs  post-infection (circles). Relative quantification as described in the methods section. Dots mark samples where relevant peptides were found, but below the significance threshold. Right panel includes all detected uncharacterized proteins. Raw data can be found in Supplementary File 3.

Extended Data Fig. 6 Early mimiviral promoter motif among mesomimiviruses.

Results of the MEME search for common motifs in sequences upstream of ORFs in mesomimiviruses. Only motifs fitting the pattern WWWWWTGW are shown, supplemented by the unusually high-frequency palindromic motif TCCGGA of Tetraselmis virus 1. For each motif, a consensus sequence, number of sites and E-value are provided (to the left). Per-position weblogos and frequency distribution of distances from the start codon are shown in the middle and to the right.

Extended Data Fig. 7 Phylogenetic analysis of MCPs from NCLDVs and NCLDV-like dwarf viruses.

The clade including the MCPs of NCLDV-like dwarf viruses (NDDVs) is highlighted in green and MCPs appearing in mesomimiviral genes are highlighted in cyan. The tree is midpoint-rooted. Host groups are indicated when known. Numbers of sequences for collapsed clades are shown in parentheses.

Extended Data Fig. 8 Viruses associated with Phaeocystis globosa and other haptophtes.

a. Schematic representation of the P. globosa-PgV-Gezel system. b. Distribution of NCLDVs, PLVs and NDDVs among haptophytes. The cladogram is after references85,86,87. Strains available with genomic data suitable for analysis of integrated viruses are indicated (asterisks mark strains for which only transcriptomes are available). CeV – Chrysochromulina ericina virus, CpVs – Chrysochromulina parva viruses; EhVs – Emiliania huxleyi viruses, IgV – Isochrysis galbana virus88, ‘PaV’ – ‘Phycodnaviridae Antarctica virus’ (mesomimivirus hypothesized to infect P. antarctica89), PgVs – Phaeocystis globosa viruses, PpV – Phaeocystis pouchetii virus.

Extended Data Fig. 9 Clustering of Gezel-group PLVs.

a. Bipartite network of gene cluster sharing between Gezel-group PLVs. Triangles represent individual PLV genomes. Genes were clustered based on profile-profile matches (see Materials and Methods) and each cluster is represented as a dot. Red labels are provided for clusters that could be associated with widespread and/or functional families (see Supplementary Table 1 for definitions of the widespread families). b. Clustering structure according to vcontact2. VC subclusters are indicated, partial genomes of integrated P. globosa PLVs are indicated with asterisks.

Extended Data Fig. 10 Incidence and mobility of genes coding for putative non-intronic homing endonucleases located between genes for capsid proteins.

From top to bottom: GIY-YIG endonuclease gene seg2 between mCP and MCP genes (ORFs pgvv10 and pgvv12) in Gezel and its absence in closely related viruses as evidenced by the three metagenomic contigs; HNH endonuclease gene in the PLV Montjoie2259 and its lack in members of the same subgroup as exemplified by PLV-YSL1; a similar case of segD, a gene for a GIY-YIG-family endonuclease located between genes coding for the hexon and penton proteins present in Enterobacteria phage T4 but absent from Enterobacteria phage T2. ORF numbers are provided, percentages show similarity at the DNA level.

Supplementary information

Supplementary Information

Supplementary Text, Table 1, Figs. 1 and 2, and References.

Reporting Summary

Supplementary File 1

Primers list, P. globosa-PgV-Gezel infection dynamics.

Supplementary File 2

Genome comparison of PgV and Gezel -16T and -14T viral strains, virions measurements.

Supplementary File 3

Proteomics data, PCRs for poly-cystronic transcripts.

Supplementary File 4

Metagenomic and Metatranscriptomics assemblies analysed in this project, integrated and standalone viruses index and sequences, PCR for P. globosa PLVs.

Supplementary File 5

Cluster affiliation and best hhsearch Pfam hits for protein sequences from mesomimiviruses, Lavidaviruses, PLVs and NCLDV-like dwarf viruses.

Supplementary File 6

Annotated viruses and viral fragments found in algal genomes.

Source data

Source Data Fig. 1

Unprocessed DNA gel, unprocessed images (TEM-SYBR).

Source Data Extended Data Fig. 3

Unprocessed gels.

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Roitman, S., Rozenberg, A., Lavy, T. et al. Isolation and infection cycle of a polinton-like virus virophage in an abundant marine alga. Nat Microbiol 8, 332–346 (2023). https://doi.org/10.1038/s41564-022-01305-7

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