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The Nanovirus-Encoded Clink Protein Affects Plant Cell Cycle Regulation

through Interaction with the Retinoblastoma-Related Protein

Sebastien Lageix,1Olivier Catrice,2Jean-Marc Deragon,1,3Bruno Gronenborn,2Thierry

Pelissier,1*and Bertha Cecilia Ramrez2*

CNRS UMR 6547 BIOMOVE, UniversiteBlaise Pascal, 63177 Aubie`re Cedex,1 Institut des Sciences du Vegetal,

CNRS, 91198 Gif-sur-Yvette,2 and LGDP, UMR 5096, Universitede Perpignan, 66860 Perpignan Cedex,3 France

JOURNAL OF VIROLOGY,

Apr. 2007, p. 41774185 Vol. 81, No. 8

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General DescriptionThe genus Nanovirus is one of two genera in the family

Nanoviridae, which consists of viruses with small (18-19 nm)

virions and are multipartite (6 to 8 or more segments of

closed ssDNA), each segment of which is circular and about

1kb in size.

DNA SegmentSize(nt) Putative product

DNA-M (1) 1001 movement protein

DNA-C2(2) 1003 Replication associated protein

DNA-C (3) 991 cell-cycle link (Clink) protein

DNA-N (4) 1002 nuclear shuttle protein

DNA-S (5) 998 coat proteinDNA-V (6) 1017 Replication associated protein

DNA-U1 (7) 988 non-structuralprotein U1

DNA-R (8) 1005 replication initiatorprotein M-Rep

Nanovirus

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>aj005967|Nanovirus|FBNYV|Cell cycle link protein Clink|

MGLKYFSHLPEELRQKIVHDHLQQERKKEFLEKAIEDSCRRHVSLLKSDPSPSELYALSKFLDSLADYVGKQFNTRCLIKWKKDVPANIKFEVMEEQHLRLYGFVDMDDLLCREVLPPEEDDDITYEDG

MIVNCSELDKLFEALGIKVVYITVSKNCICTPLNKDIVIS

Cell cycle link protein - Clink

F-Box : N of Clink is an F-Box domain characterised by LRR

LxCxE : Motif that binds to Retinoblastoma related proteins

Clink:

Clink is a 17 kDa protein

The protein binds to human RB and plant RBR invitro through LxCxE

It interacts with Skp1, a member ofprotein complex that targets proteins

for ubiquitin-dependent degradation by the 26S proteosome

Clink also acts as a replication enhancer

Does Clink interact with RBR in planta?

What is the effect of this interaction on the cell cycle proliferation?

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Gluco-corticoid inducible system

35S Gal 4 DNA BDVP 16

ADGR

No Gluco-corticoid

Inactive Transcription

Factor

35S Gal 4 DNA BDVP 16

ADGR

Gluco-corticoid

Active Transcription

Factor

Target Gene

Expression

Advantages :

Non-toxic hormone

Easilypermeable

Dose dependant gene expression

Target genes are induced without pleiotrophic effects

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T=24H

Clink specifically induces cell cycle regulated genes

Clink is stably expressed from the inducible Gluco-corticoid regulon

Samples are 6-7 week old fully expanded rosette leaves

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Impact of Clink on Cell Cycle

Samples are 6-7 week old fully expanded, detached rosette leaves treated with Dex/ethanol for 4 daysIn each case the sample size is 800-1,100 nuclei. Bar represents 15 microns

A : Ethanol treated Clink transgenic

B : Dex treated Clinkmut transgenic

33% 10%

19% 19%

7% 1%

No. represents the % of nuclei observed for each type in Clink expressing cells

95% 5%

Dex treated Clink transgenic

C

D

E

F

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A B

C D

Effect of Clink expression on cell size

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Effect of Clink expression on the Ploidy Level

Clink expressing cells have undergone more endoreduplication cycles

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Ploidy Levels in FBNYV Infected Tissues

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