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Reprogramming Viral Host Specificity To Control InsectPopulations

Guillaume Cambray

To cite this version:Guillaume Cambray. Reprogramming Viral Host Specificity To Control Insect Populations. SyntheticBiology 7.0, Jun 2017, Singapour, Singapore. pp.1. �hal-01869871�

Reprograming Viral Host Specificity To Control Insect PopulationsGuillaume CambrayUMR 1333 DGIMI, INRA-University of Montpellier, Franceguillaume.cambray@inra.fr

VP1 2 3 4

NS1 NS2 NS3

ITRITR

VP transcript with alternative start sites

NS transcript with splicing and embedded ORF

Viral Particle gene block (VP) Non-structural gene block (NS)

JcDV (6,032 nts)

P9

P93

insect pests

Variations Cloning Transfection Tagged library Functional screening

Sequencing

Enrichment = Performance

1x 1x Xx

1:1

Sequencing-based phenotyping of designer capsids

Parellel screening

123

1 2 3

4 5 6

7 8 9

Temperature

pH

ABIOTIC

WholeAnimal

Dissected midgutCell culture

Nucleus

BIOTIC

All

Construction of a mutable non-propagatable JcDV

Roadblock #1 : No PCR through ITRs Roadblock #2 : Unexpected loss of function

A winded detour through the golden gate

Non-enveloped capsid : • maintains genome integrity • essential for early infectious steps

JcDV’s capsid: • Icosahedron : T=1, Ø=22 nm • 60 monomeres (1 VP1 : 9 VP2 : 9 VP3 : 41 VP4) • Structural model based on 94% identical GmDV (Solved @ 3.7 Å) Simpson et al., Structure (1998)

Some surface polymorphism between JcDV and GmDV impacts host specificity Multeau et al., Journal of Virology (2012)

One of the most diverse and successful group of animals, Insects are an integral part of ecosystems. Yet, some represent great nuisances for Human’s health and development...

Such pests have been efficiently controlled using chemical insecticides, but the rise of resistances, the broadly untargeted environmental impacts and the increasing recognition of chronic toxicity call for the urgent development of safer and cleaner alternatives.

Biological control strategies that take advantage of natural antogonistic relationships between existing organisms and a target pest have been around for millenia. In spite of the inherent risks of unintended side effects, these approaches have recently gained renewed interest.

Perhaps because they evoke greater fears, surpisingly few microorganisms have been used in that perspective.

Densoviruses are small viruses capable—as a group—of infecting a broad range of insects with various degree of specificity. Their minute genomes comprise a handful of genes, which lend themselves to in-depth molecular dissection using synthetic biology approaches.

Our goal is to develop the tools and knowledge necessary to enable the use of densoviruses as safe, specific and efficient biocontrol agents.We focus on JcDV, which infects crop-devasting caterpillars and AalDV, which infects disease-vector mosquitoes.

Here, I present early efforts to systematically unravel the structural motifs responsible for capsid specificity.

The capsid of densoviruses are small (19-24 nm) non-enveloped icosahedrons (T=1) resulting from the self-assembly of 60 identical or highly similar capsid proteins. The DNA sequences coding these proteins represent roughly a third of the genome and are the prime determinant of specificity.I am using the genome of JcDV to setup a the high-throughput, cost-effective pipeline to deconstruct the phenotypic consequences of many precise capsid mutations. This will permit to better understand natural variations, to map evolutionary landscape, to discover uselful properties and to learn the rules to reprogram specificities.

Immunostaining 3 days after transfection in Ld652Y cell

VP NS1 NS2

Backbone with NS

ITR (x2)

VP1-2-3

Reporters

P93

Mutation acceptor}

BsaI

}Mutable VP4

BsmBI

**

pGCDV58

5 10 15 20 25

510

1520

25

VP (Cp)

NS

(Cp)

1e-011e-021e-031e-041e-05

0 1 2 3 4 5 6 7

500

1500

2500

Days Post-Transfection

Fluo

rese

cenc

e (m

Ven

us, a

.u.)

Control7373+4973+7773+7873+79

PopIE2P93 LP93 MP93 S

+

++

+

pGCDV58pGCDV77pGCDV78pGCDV79pGCDV73

pGCDV1pGCDV92pGCDV90pGCDV91

PopIE2+ pGCDV49

☺

☹☹☹☹☹☹☺☺☹

AbstractTargeting

with densoviruses

Stokstad, E., Science (2017)

• Crop pest • Eats >80 plants• From S. and N. America• >100 kms / day• Currently invading Africa at lightening speed

The fall armyworm (Spodoptera frugiperda)

Bonizzoni et al, Trends in Parasitology (2013)

• Disease vector : Yellow fever, Dengue, Chikungunya, Zika, ...• Originally from S.E. Asia• Spreading over the world for decades

The tiger mosquito (Aedes albopictus)

A typical infectious cycle

1

2

34

Midgut epithelium

Target cell

NucleusReplicationExpressionEncapsidation

CapsidGenome

Specificity

Sequence & StructurePolymorphism

1 2 30

PiDV

DsDV

GmDV

MlDVJcDV HaDV

0.08

Molecular Dissection Of A Complex Phenotype

Small, But Not So Simple : The Price Of Biosafety

ITRReporter and selection blockNeoR mVenus

rep JcDV (6,006 nts)

+P93PA3 PopIE2

-

VP1 2 3 4ITR Viral Particle gene block (VP)

P9 NS1 NS2 NS3Non-structural gene block (NS)

Abrupt polymerase fall-off

Of structure and repeats

Highly structuredterminal repeat

in ITRs

The reconstructed genome does not replicate

Complementation reveals NSs regulatory function and the role of VP transcript’s tail in replication

TEM imaging of supernatantCapsid

58 58+1

90 91

7 days post-Infection (virions from co-transfections)

ExpressionCapsid assemblyReplicationInfection

☺☺☹☹

• NSs increase P93 activity• Tail of VP transcript overlapping with NS transcript necessary for replication

NS block outside the ITR : not encapsidated, no further infection

Expecting more VP upon replication

AB C D E

FGHI

A

BN

C

Gly-rich

BC loop

CD loop

DE loopEF

loop

FG loop

GH loop

HI loop

Burried: not involved in specificity

5-fold axis motifs: known to be involved

in specificity

Multeau et al., Journal of Virology (2012)

3-fold axis : protruding β-Annulus

5-fold-axis tunnel:Genome delivery ?

VP4 secondary structure

Quantititive caracterization of several hundred of thousands precisely designed capsid variants based on high-throughput DNA synthesis and amplicon sequencing.Supports heavy multiplexing to screen many abiotic and biotic conditions.

• Impossible to use inverse PCR• Recombination-based assemblies dangerous• Tedious golden gate stup enable scarless, flexible and efficient cloning• Open the way for combinatorial assemblies of densoviral chimeras