A zebrafish reporter line reveals immune and neuronal expression of endogenous retrovirus

TLDR: In this article, the authors identified 8 copies of the zebrafish endogenous retrovirus (zferv) and created and characterised the first in vivo ERV reporter line in any species.

Abstract: Endogenous retroviruses (ERVs) are fossils left in our genome from retrovirus infections of the past. Their sequences are part of every vertebrate genome and their random integrations are thought to have contributed to evolution. Although ERVs are mainly kept silenced by the host genome, they are found activated in multiple disease states such as auto-inflammatory disorders and neurological diseases. What makes defining their role in health and diseases challenging is the numerous copies in mammalian genomes and the lack of tools to study them. In this study, we identified 8 copies of the zebrafish endogenous retrovirus (zferv). We created and characterised the first in vivo ERV reporter line in any species. Using a combination of live imaging, flow cytometry and single cell RNA sequencing, we mapped zferv expression to early T cells and neurons. Thus, this new tool identified tissues expressing ERV in zebrafish, highlighting a potential role of ERV during brain development and strengthening the hypothesis that ERV play a role in immunity and neurological diseases. This transgenic line is therefore a suitable tool to study the function of ERV in health and diseases. Funding This work has been supported by a European Leukodystrophy Association fellowship (ELA 2016-012F4) to NH, an MRC Programme Grant (MR/M004864/1) to SAR and . JPL is supported by Agence Nationale de la Recherche (grant ANR-16-CE20-0002-03.. Imaging was carried out in the Wolfson Light Microscopy Facility, supported by an MRC grant (G0700091) and a Wellcome Trust grant (GR077544AIA). Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 3: Reporter line for zferv1a recapitulates endogenous expression. A. Diagram of gateway construct used to create the Tg(zferv1a:GFP) reporter line using the pDestCryCFP tol2 backbone. B. High resolution image of a 5dpf Tg(zferv1a:GFP:pA)sh533 embryos, displaying high expression of the transgene in the thymus (white box), brain and spinal cord (white arrowheads). Scale bar 500µm. C. Dorsal view of a maximum projection of the brain from 5 dpf Tg(zferv1a:GFP) embryo acquired with a the lightsheet microscope. D. High resolution single slice image from the optic tectum of Tg(zferv1a:GFP). Scale bar 15 µm.

Figure 4: zferv1a is expressed in cells from the lymphoid lineage in adult hematopoietic tissues. A. Single slice DIC and confocal fluorescence colocalisation image of the thymus (black star) situated underneath the ear in 5dpf embryos and the signal from the Tg(zferv1a:GFP)sh533 reporter. Scale bar 100µm. B. Cell tracking reveal dynamic behaviour of entry and exit of thymus by GFP positive cells in Tg(zferv1a:GFP)sh533. Whole stack analysis of cell movement by using TrackMate on FiJi. Scale bar 10µm. C. Expression of Tg(zferv1a:GFP)sh533 in a 25dpf zebrafish highlighting the triangular shaped thymus (white arrowhead). D. Side scatter (SSC) and GFP plot from single cell suspension of a whole kidney marrow from an adult Tg(zferv1a:GFP) with gate selecting GFP positive cells. Inset plot showing the distribution of cells from a non-transgenic adult whole kidney marrow. E. Forward and side scatter (FSC/SSC) plot of selected GFP positive cells from panel D. Inset plot showing the location of the GFP positive cells in the haematopoietic lineage FSC/SSC plot. F. FSC/SSC plot of a whole kidney marrow separating different haematopoietic lineages by size and granularity.

Figure 5: Immune expression of zferv1a is restricted to CD4 and T-cells. A. Single slice confocal image of the thymus from double transgenic Tg(zferv1a:GFP)sh533 x Tg(lck:mCherry) with merge image used for colocalisation analysis. B. Single slice confocal image of the thymus from double transgenic Tg(zferv1a:GFP)sh533 x Tg(mpeg1:mCherryCAAX)sh378 with merge image used for colocalisation analysis (white arrow pointing to a single macrophage). C. Histogram

Figure 1. Multiple copies of zferv are present in the zebrafish genome. A. Diagram of the original zferv genome found by Steiner et al. used as a reference for nucleotide identity (ID% nt) B. Diagram representing the two closest related zferv genome found in most recent zebrafish genome GRCz11, named zferv1a and zferv1b. C. Diagram of 6 pseudo zferv genes with degenerated genome (dotted line represents insertions). 5’-LTR: 5’ –Long terminal repeat, Rep.: Repetitive element, gag-pol: genes encoding the polyprotein and reverse transcriptase, env: envelope gene.

Figure 2: Reporter line for zferv1a recapitulates endogenous expression. A. Expression of the envelope gene (env) by in situ hybridisation from 2-cell stage until 5dpf. Black box highlighting strong expression around the thymus, black arrows highlighting brain and spinal cord expression. Scale bar 500µm. B. Zoomed image on the thymus area, single positive cells are visible in the vicinity of the thymus around the ear (white arrowheads) and alongside the branchial arches (white arrows). Scale bar 70µm. C. Dorsal view of env expression in the brain at 5dpf. Scale bar 100µm.

Full text

A"zebrafish"reporter"line"reveals"immune"and"neuronal"expression"of"endogenous"
retrovirus."
!
Noémie! Hamilton
1,2
*,! Amy! Clarke
1
,! Hannah!Isles
1
,! Euan! Carson
1
,!Jean-Pierre!Levraud
3
,!
Stephen!A!Renshaw
1!
!
!
1.
!
The!Bateson!Centre,!Department!of!Infection,!Immunity!and!Cardiovascular!Disease,!
University!of!Sheffield,!Sheffield,!UK!
2.!The!Institute!of!Neuroscience,!University!of!Sheffield,!Sheffield,!UK!
3.!Macrophages!et!Développement!de!l’Immunité,!Institut!Pasteur,!CNRS!UMR3738,!25!
rue!du!docteur!Roux,!75015!Paris!!
!
*Corresponding!author:!n.m.hamilton@sheffield.ac.uk
!
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Abstract"
"
Endogenous!retroviruses!(ERVs)!are!fossils!left!in!our!genome!from!retrovirus!infections!
of! the! past.! Their! sequences! are! part! of! every! vertebrate! genome! and! their! random!
integrations!are!thought!to!have!contributed!to!evolution.!Although!ERVs!are!mainly!kept!
silenced!by!the!host! genome,!they!are!found! a ctivated!in!mult iple!disease!stat es!such!as!
auto-in flammat ory! d isorders!and!neurological!diseases.!What!makes!defining!their!role!
in!health!and!diseases!challenging!is!the!numerous!copies!in!mammalian!genomes!and!
the! lack! of! tools! to! study! them.! In!this! study,! we! identified! 8! cop i es! of! the!zebrafish!
endogenous! retrovirus!(!"#$%).!We!created!and!characterised!the!first!&'(%&%)!ERV!reporter!
line!in!any!species.!Using!a!combination!of!live!imaging,!flow!cytometry!and!single!cell!
RNA!sequencing,!we!mapped!!"#$%!expression!to!early!T!cells! and!neurons.!Thus,!this!new!
tool!identified!tissues!expressing!ERV!in !zebrafish,!highlighting!a !p oten t ial!role!of!ERV!
during! brain! devel opment! and! strengthening! the! hypothesis! that! ERV! play! a! role! in!
immunity!and!neurological!diseases.!This!transgen ic!line!is!therefore!a!suitable!tool!to!
study!the!funct ion!of!ERV!in!health!and!diseases.!
"
Keywords"
Retroelement,!zebrafish,!endogenous!retrovirus,!reporter!line,!!"#$%,!LTR!
"
Funding"
This! work! has! been! supported! by! a! European! Leukodystrophy! Association! fellowship!
(ELA!2016-012F4)! to!NH,!an!MRC!Programme!Grant! (MR/M004864/1)!to!SAR! and!.!JPL!
is! supported! by! Agence! Nat ionale! de! la! R echerche! ( grant! ANR-16-CE20 -0002-03..!
Imaging!was!ca rried!out! i n!t he!Wolfson!Light!M i croscopy!Facility,!supported!by!an!MRC!
grant!( G0700091)!and!a!Wellcome!Trust!grant !(GR077544AIA).!!
"
Conflict"of"Interest"Statement"
The! authors!declare!that!the!research!was!conducted!in!the!absence!of!any!commercial!or!
financial!relationships !that!could!be!construed!as!a!potential!conflict!of!interest.!
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preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in
The copyright holder for thisthis version posted January 21, 2021. ; https://doi.org/10.1101/2021.01.21.427598doi: bioRxiv preprint

Introduction"
"
Over!40%!of!the!human!genome!comprises!endogenous!transposable!elements!capable!
of!recombination!and!disruption!of!genes!and!modification!of!their!expression!(Lander!et!
al.!2001).!Endogenous!retroviruses!(ERVs)!are!transposable!elements!originating!from!
old!integrations!of!retroviruses!so!successful!that!they!have!become!part!of!all!vertebrate!
genomes!st udied.!ERVs!replicate!autonomously!using!a!copy-and-pa ste!mechanism!and!
although!they! form!a!smaller!percentage!of! all!retroelements,!they!still!represent !8%!of!
the!human!genome!(Bourque!et!al.!2018;!Lander!et!al.!2001).! The!majority!of!known!ERVs!
have! lost! their! ability! to! replicate,! but! those! most! recently! acquired! still! h ave! intac t !
genomes! with!the!ability! to!produce!viral! RNA!and!particles.! However,!these! competent!
ERVs!are!under!strict!transcriptional!suppression!by!epigenetic! mechanisms!(Maksakova,!
Mager,! and! Reiss! 2008;! Rowe! et! al.! 2010;! Turelli! et! al.! 2014),! protecting! the! host!
organisms!against!potential!retroviral!insertions!and!viral!activities.!!!
!
Although!immobilised!by!mutations!or!transcriptiona lly!repressed,!ERVs!have!a!complex!
relationship!with!the!human!genome,!which!they!can!regulate!by!providing!cis-regulatory!
elements! to! surrounding! genes! and! by! lifting! their! transcriptionally! repressed! state.!
Through!these!mechanisms,!it!is!believed!that!transposable!elements!have!fuelled!some!
of! the! necessary!genetic!changes!for!evolution! (Feschotte!2008;!Kunarso!et!al.!2010).!!
*+',+-&'./,!an!ERV!envelope!gene! essential! for!the! vascularisation!of!the!placenta,! is! at!the!
origin! of! evolutionary! diversification! of! the! placenta! (Chuong! 2018;! Mi! et! al.! 2000).!
During!human!embryogenesis,!expression!of!specific!ERV!families!have!been!associated!
with!cell!identity!and!cell!pot ency!in!ea rly!stem!cells!(Göke!et!al.!2015).!Additionally,!ERV!
expression!has!been!reported!in!healthy!human!tissues!such!as!ovary!and!testis!for!ERV-
9! (Pi! et! al.! 2004),! pancreas! (Shiroma! et! al.! 2001),! breast! (Tavakolian,! Goudarzi,! and!
Faghihl oo!2019),! stomach! and! small! intestine! (Okahara!et!al.!2004).! Mainly! based! on!
transcriptional!studies,!the!expression!of!different!families!of!ERV!is!likely!to!be!extended!
to!more!tissues,!however!their!role!in!tissue!development!and!function!remains!largely!
unknown.!!
!
ERVs! have! been! linked! directly! and! indirectly! to! the! evolution! and! functioning! of! the!
immune!system.!Enhancer!regions!of!interferon!stimulated!genes!key!to!the!interferon!
pathway,!such!as!IRF1!and!STAT1,! were!found! i ntroduced! and!amplified!by!ERV! elements,!
with!the!human! inflammasome! failing!to!form! upon! the!deletion! of!a !subset! of!ERVs!
(Chuong,!Elde,!a nd! Feschotte!2 016) .!Ad aptive!immunity!also!benefits!from!the!presence!
of!ERVs.!Indeed,!ERV!peptide!recognition!is!used! in!T!cell !selection!to!optimise!antigen!
recognition! and! T! cells! have! a! higher! sensitivity! to! exogenous! virus! infection! when!
presented !with! ERV!peptides! during! their!initial!thymic!selection ! (Mandl! et! al.! 2013;!
Young!et!al.!2012).!The!human!ERV!(HERV)!envelope!gene!contains!immunosuppressive!
domains!that!reduce!the!Th1!response!during!pregnancy!and!therefore!promote!foetal!
development!(Knerr!et!al.! 2004;!Lokossou!et!al.! 2020).!The!role!of!ERVs!in!our!immune !
system,!particula rly!in!the!training!of!our!adap tive!immunity,!can!be!a!double-edge!sword!
as!ERVs!are!linked!to!a!range!of!different!disease!states,!including!autoimmunity.!!
!
Aberrant! expression! of! ERVs! contributes! to! multiple! pathologies.! ERVs! are! found! in!
abundance!in!multiple!forms!of!cancers!and!are!considered!tumour-promoting!fa ctors!
(extensively! reviewed! in! (Bermejo! et! al.! 2020).! The! pathology! of! auto-inflammatory!
diseases! has! also! been! strongly! associated! with! ERVs.! Syst emic! l upus! erythematosus!
(SLE)! is! an! autoimmune! disorder! with! increased! level! of! autoantigen! for! an! ERV!
(Jorgensen!et!al.!2014).! Recently,! one!of!the!murine! SLE! susceptibility!locus!was!i dentified!
as!a!key!suppressor!of!ERV!expression,!consolidating!the!role!of!ERVs!in!the!pathogenesis!
of!SLE!(Treger!et!al.!2019).!A!similar!disorder!is!Acardi-Goutieres!Syndrome!(AGS),!a !type!
1!interferonopathy!which!resembl es!congenital!cytome galovirus!infection!and!is!caused!
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by!mutation!in!several!genes!encoding!enzymes!responsible!for!nucleic!acid!metabolism!
(Crow!et!al.!2015).!Mutations!in!some!of!these! genes,!such! as!TREX1,!MDA5! and!ADAR1!
trigger!aberrant!presence!of!various!retroelements!and!the!upregulation!of!an!antiviral!
immune!response!(Ahmad!et!al.!2018 ;!Thomas!et!al.!2017).!Interestingly,!anti-reverse!
transcriptase!therapy!in!AGS!pa tients!can!decrease!the!IFN!response,!highl ighting!the!role!
of! aberrant! presence! of! ERVs! in!triggering!an!immune!response! (G.!Rice!et!al.!2018).!
Increased! expression! of! ERVs! has! been! found! in! brains! of! patient! suffering! from!
neurodegenerative!disea ses! such!as! Motor! Neuron!Disease!(Li! et!al.!2015)!and!multiple!
sclerosis!(Johnston!et!al.!2001;!Mameli!et!al.!2007).!Overexpression!of!a!human!ERV!in!
neurons!was!shown!to!be!neurotoxic,!suggesting!a!p otential!role!of!ERVs!in!t riggering!
neuronal!toxicity!(Li!et!al.!2015).!A!direct!link!to!the!pathology!of!these!disorders!has!yet!
to!be!made,!but!nonetheless!ERVs!appear!as!strong!causal!factors!for!autoimmune!and!
neurological!disorders.!
Although!ERV!enrichment!has!been!detected!in!neurological!pathol ogies,!little!is! known!
about!the!function!of!ERV!in!healthy!tissues.!The!exact!role!of!ERV!in!our!immune!system!
and!brain!pathologies!has!yet!to!be!understood!and!there!is!no!model!system!specifically!
looking! at!ERV!function!&'(%&%).!!Zebrafish! is!already!established!as!a!model!to!study!the!
immune! system,! with! significan t! homology! with ! mammals! in! innat e! and! ada ptive!
immunity! (Trede! e t!al.! 2004;! Renshaw! and!Trede! 2012).! The !genetic! tractability! and!
transparency!of!the!zebrafish! embryos!have!allowed!the!creation!of!transgenic!reporter!
lines,!some!of !which!have!elucidated!the!role!of!immune!cell!behaviour!&'(%&%)!(Renshaw!
et!al.!2006).!The!tractability!of!the!zebrafish!has!already!been!exploited!to!visualise!the!
expression!of!the!human!ERV-9!in!oocytes,!similarly!to!human!expression!(Pi!et!al.!2004).!
In! this! study,! we! used! the! zebrafish! as! a! tractable! in! vivo! model! to! characterise! the!
zebrafish!endogenous!retrovirus!( !"#$%).! We!identified! multiple! !"#$%! family! members,!
including!2!complete!genomes!!"#$%/0!and!!"#$%/1.!Using!the!tractability!of!the!zebrafish!
larvae! we! developed! a! reporter! line! for! !"#$%/0! and! imaged! for! the! first! time! ERV!
activation!in!healt hy!tissues! &'(%&%).!We!showed!that!!"#$%/0!is!expressed!in!the!thymus!
and! in! the! brain.! Colocalisation! analysis! and! single! cell! RNA! sequencing! revealed!
expression! of! !"#$%/0! specifica lly! in! T-cells,! suggesting! a! potential! role! for! ERV! in!
lymphocyte! development.! Brain! exp ression! of! ZFERV! appears! to! include! neuronal!
expression.!This!transgenic!line!can!be!used!as!a!tool!to!further!investigate!the!role!of!
ERVs!in!immunity!and!in!neurological!disorders.!
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Results"
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The"zebrafish"genome"contains"multiple"endogenous"retrovirus"integrations."
The!presence!of!an!ERV!in!zebrafish,!named!!"#$%,!has!been!reported!by!Shen!an d!Steiner!
while!screening!a!thymic!cDNA!library!(Shen!and!Steiner!2004)!(Figure"1A).!We!searched!
for!related!sequences!in!t he!most!recent!reference!zebrafish!genome!(GRCz11,!Tü!strain)!
using!BLASTN!searches,!with!the!original! !"#$%!sequence!as!a!query.!Limiting!ourselves!to!
sequences!flan ked!by!long!terminal!repeats! (LTRs)! on!both!sides,!we!retrieved!8 ! hits!
scattered!in! t he!zebra fish!genome,!but!n ot!t he! exact!!"#$%!sequence,!possibly! because!of!
strain! difference! ( Figure" 1+" Sup." Table" 1).! We! identified! 2 ! sequences! encoding!
apparently!fully!functional!ERVs,!with!91%!and!90%!identity!to!!"#$%,!that!we!respectively!
named !!"#$%/0!and!!"#$%/1!(Figure"1B).!These!t wo!ERVs!encode!almost!identical!proteins!
(95!to!97%!identity! at!the!amino-acid!level).! Their! LTRs! are!also!highly!simil a r!(95%!
identity! at! the! nucleot id e! level),! suggesting! that! t heir! promoters! probably! d rive!
expression!in!similar!cells.!An!additional!6!pseudo!!"#$%!genes!(here!call ed!!"#$%2!-!!"#$%3)!
were!identified!(Figure"1C),! !"#$%2!containing!a! frameshift,!!"#$%4!and! !"#$%5!with!large!
deletions! and! !"#$%6,! !"#$%7! and! !"#$%3! containing! a! large! insertion,! all! resulting! in! a!
degenerated!ERV!genome.!
!
!
Figure" 1.!Multiple" copies"of"zferv" are" present" in" the" zebra fish" genome.! A.! Diagram! of! the!
original!zferv!genome!found!by!Steiner!et!al.!used!as!a!r eference!for!nucleotide!identity!(ID%!nt)!
B.!Diagram! representing! the! two! closest! related! !"#$%! genome! found!in!most!recent!zebrafish!
genome!GRCz11,!name d!zferv1a!and!!"#$%/1.!C.!Diagram!of! 6!pseudo!zferv!genes!with!degenerated!
genome! (dotted! line! represents! insertions).! 5’-LTR:!5’!–Long! terminal! repeat,! Rep.:!Repetitive!
element,!gag-pol:!genes!encoding!the!polyprotein!and!reverse!transcriptase,!env:!envelope!gene.!
!
!
!
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zferv1a!is"actively"expressed"in"the"brain"and"in"the"thymus.!
!"#$%!expression!was!initially!found!in!the!thymus!at!5!days!post!fertilisation!(dpf)!using!
a!RNA!probe!against!the!envelope!(#'%)!gene!of!the!original!!"#$%!(Shen!and!Steiner!2004).!
To! verify! this! observation,! the! entire! !"#$%/0! genome! was! subcloned! from! a! fosmid!!
provided!by!the!Sanger!Center!and! we!used! this!sequence!to!create!an!&'( 8&-9!hybridisatio n!
(ISH)!RNA!probe!against!the!envelope!gene!(#'%)!of!!"#$%/0.!ISH!p erformed!on!a !time!
course! starting! from! 2! cell-stage! embryos! from! the! '0,$#! strain! con firmed! that! the!
strongest!signal! appeared!at!5dpf!(Figure"2).!At!5dpf,!the!thymus!was!strongly!labelled!
(Figure" 2A-B),! similarly! to! what! was! previously! reported! (Shen! and! Steiner! 2004).!
Individual!labelled!cells!were!visible!around!the!thymus!following!the!branchial!arches!
and!around!the!ear!(Figure"2B).!Additionally,!we!identified!a!clear!signal!of!the!#'%!probe!
in!the!brain!(Figure!2A,!2C)!and!the!spinal!cord!(Figure!2A).!!
!
!
Figure" 2:"Reporter"line"for"zferv1a"recapitulates"endogenous"expression.!A.!Expression!of!the!
envelope! gene!(#'%)!by!&'(8&-9(hybridisation!from!2-cell!stage!until!5dpf.!Black!box!highlighting!
strong!expression!around!the!thymus,!black!arrows!highlighting!brain!and!spinal!cord!expression.!
Scale!bar! 500µm.! B.!Zoomed!image!on! the ! thymus!area,!single!positive!cells! are!visible ! in! the!
vicinity!of! the! thymus! around! the! ear! (white! arrowheads)! and! alongside! the! branchial! arches!
(white!arrows).!Scale!bar!70µm.!C.!Dorsal!view!of!#'%!expression!in!the!brain!at!5dpf.!Scale!bar!
100µm.!!
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zferv1a"reporter"line"recapitulates"endogenous"expression.!
To!follow!the!expression!of!the!zebrafish!endogenous!retrovirus!we!identified!as!!"#$%/0,!
we!took!a!transgenic!approach!ta king!adva ntage!of!the!promoter!activity!of!retroviral!
LTR.!We!used!the!5’!viral!promoter!:-$6!from!!"#$%/0!to!drive!GFP!expres sion!by!Gateway!
recombination!(Figure"3A).!Injected!embryos!showing! expression!in!their!thymus!were!
raised!and!screened!in!adulthood!for!germline!transmission.!We!observed!a!strong!and!
consistent!GFP!expression!in!the!thymus!at!5dpf!in!F1!and!F2!larvae!(Figure"3B)!from!
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