Heterotrophic protists as a genomic hub for and other mobile genetic elements Protist host

supports Giant CroV parasitizes Mavirus

Matthias Fischer MPI for Medical Research Heidelberg, Germany Host: Cafeteria roenbergensis

www.eol.org

• marine heterotrophic nanoflagellate • ecologically important bacterivore • Ø 5-10 µm • 30 Mpb diploid genome, asexual (?) Cafeteria is a deep-branching stramenopile

Cafeteria

known hosts of giant DNA Parasite: Cafeteria roenbergensis virus (CroV)

Nuc

Viral factory

• capsid Ø = 300 nm • T number = 499 Electron microscopy by Ulli Mersdorf, MPImF • 691,790 bp dsDNA • > 600 proteins • 57 tRNA genes • member of Mimiviridae Fischer et al (2010) PNAS 107:19508 Xiao, Fischer et al (2017) Sci Rep 7:5484 Hyperparasite: the virophage mavirus

VF

50 nm EM by U. Mersdorf, MPImF 50 nm

capsid Ø = 75 nm 19 kb dsDNA à Mavirus is highly virulent for CroV 20 ORFs

Fischer & Suttle (2011) Science 332:231 Virulence towards giant viruses varies among virophages strong inhibition Mavirus CroV

Sputnik Mimivirus

Zamilon Mimivirus

no inhibition

Fischer & Suttle (2011) Science 332:231 La Scola, Desnues et al (2008) Nature 455:100 Gaia et al (2014) PLoS One 9:e94923 Virophages genomes are modular

Born et al (2018) PNAS Jun 25 Mavirus Cys protease rve integrase SF3 helicase DNA pol B

FtsK/HerA minor major ATPase capsid capsid DNA replication/integration virion morphogenesis Sputnik

ATPase PRO mCP MCP Tyr recombinase TVpol/S3H

0 5,000 10,000 15,000 20,000 bp

Conserved morphogenesis genes:

DNA-pumping ATPase Cys protease minor capsid protein major capsid protein Mavirus is related to endogenous eukaryotic elements

Mavirus Cys protease rve integrase FtsK/HerA minor major SF3 helicase DNA pol B ATPase capsid capsid

DNA replication/integration virion morphogenesis

Maverick/ DNA transposons

POL INT

• 15-20 kb long • flanked by inverted repeats • found in protists, nematodes, insects, vertebrates, fungi

Trichomonas vaginalis: 30% of genome are Feschotte & Pritham (2005) Trends Genet 21 Kapitonov & Jurka (2006) PNAS 103 Virophage genome integration

Sputnik integrated in mimivirus: (2012) PNAS 109

Endogenous virophages in an algal genome:

(2015) PNAS 112

Endogenous mavirus in Cafeteria genome?

Mavirus + ? Mavirus integrates into the host genome

endogenous mavirus

+ reactivation

genome integration exogenous endogenous state state virion integrated

Thomas Hackl

Host DNA Host DNA

Fischer & Hackl (2016) Nature 540:288 The mavirus infection cycle Mavirus

entry by mavirus cell entry clathrin-mediated endocytosis & integration occur independently of CroV

endogenous virophage

mavirus genome integration rve-integrase CroV virophage genome replication & transcription giant virus core giant virus transcriptase

giant virus factory

Born et al (2018) PNAS Jun 25 The mavirus infection cycle

mavirus cell entry & integration occur independently of CroV

endogenous virophage

CroV

virophage genome giant virus replication & core assembly giant virus transcriptase reactivation of endogenous mavirus giant virus factory is triggered by CroV Cost imposed by endogenous virophages?

- cost of replicating an endogenous virophage genome (20 kb in 30 Mb à 0.07%) - positional effects? • insertion into heterochromatin à not reactivatable • insertion into host genes à less fit / lethal phenotype - multiple insertions à genomic rearrangements à instability or novelty? - recombination with other endogenous viruses/transposons à new properties Virophages may provide adaptive population immunity

+ + Interference 4 3

• Specific + • Indirect memory • Acts on population level Activation • Altruistic + Adaptation 1 2 see also: Koonin & Krupovic (2017) Curr Opin Virol 25:7 How widespread are endogenous virophages?

Four Cafeteria roenbergensis strains Genome sequencing (MiSeq & PacBio) Assembly (Proovread)

A C

B

D Endogenous virophages in natural Cafeteria strains

Six distinct versions of Endogenous Mavirus-Like Elements (EMALEs)

host genome GC content: ~70% endogenous virus genome GC content: 30-50%

EMALE # Present in host strains

1 A,B,C,D

VV A32-like ATPase 2 A

3 A,C

4 B,C,D

5 A

6 B

Mavirus S3H INT POL ATPase PRO mCP MCP

0 5,000 10,000 15,000 20,000 bp

Hackl, Pfitzer & Fischer, in preparation Major capsid protein phylogeny of virophages

MCP maximum likelihood

≥80% branch support FtsK-HerA-type ATPase phylogeny of virophages

ATPase maximum likelihood

≥80% branch support Endogenous virophages in natural Cafeteria strains

Six distinct versions of Endogenous Mavirus-Like Elements (EMALEs)

host genome GC content: ~70% endogenous virus genome GC content: 30-50%

EMALE # Present in host strains

1 A,B,C,D gag pol YR

2 A

3 A,C

4 B,C,D

5 A

6 B

Mavirus S3H INT POL ATPase PRO mCP MCP

0 MV14 is conserved and its protein is packaged!5,000 10,000 15,000 20,000 bp Hackl, Pfitzer & Fischer, in preparation Some EMALEs carry Tyr recombinase retrotransposons

Ngaro retrotransposons (DIRS-like) Non-LTR retrotransposons with split direct repeats

0 6.4 kb

ORF 1 ORF 2 ORF 3 A1 B1 A2 B2 gag??? Tyr rec. RT domain RNase H-like domain Heterotrophic protists and their mobile genetic elements

Polinton-like viruses mCP MCP ATPase ? YR retrotransposons gag pol YR PgVV rare integration?

POL INT mCP MCP ATPase PRO ? Mavericks/Polintons

Giant viruses Virophages transpovirons

PLVs: Yutin et al (2015) BMC Biol 13:95 PgVV: Santini et al (2013) PNAS 110:10800 YR-RT: e.g. Piednoël et al (2011) BMC Genom 12:621 Transpovirons: Desnues et al (2012) PNAS 109:18078 Endog. giant viruses: e.g. Gallot-Lavallée et al (2017) Viruses 9:17 Take home points

Integrated virophage genome mavirus

Reactivation Integration CroV

Virophage

1. Virophages are parasites of giant viruses and MGE in heterotrophic flagellates

2. Mavirus leads a dual lifestyle and displays high mobility

3. Virophages may act as an adaptive immune system in Cafeteria-like flagellates

Open questions: • ecological significance? • diversity/specificity/stability/interactions? • virophages as HGT agents? • … Acknowledgements MPI for Medical Research Karina Barenhoff Anna Koslová Sarah Duponchel Thomas Hackl Ilme Schlichting Alexa Weinmann Kastė Jurgaitytė Mónica Berjón-Otero Kai Fenzl Ulrike Mersdorf Lena Pfitzer

Collaborators

MPI Evol. Biol.: Lutz Becks Ana Del Arco

MPImF: Jochen Reinstein Diana Born

UTEP, USA: Chuan Xiao

Georgia Tech, USA: Joshua Weitz

U. Amsterdam: Corina Brussaard