The Nanovirus-Encoded Clink Protein Affects Plant Cell Cycle Regulation

through Interaction with the Retinoblastoma-Related Protein†

Se´bastien Lageix,1 Olivier Catrice,2 Jean-Marc Deragon,1,3 Bruno Gronenborn,2 Thierry Pe

´lissier,1‡* and Bertha Cecilia Ramı´rez2‡*

CNRS UMR 6547 BIOMOVE, Universite´ Blaise Pascal, 63177 Aubie`re Cedex, 1 Institut des Sciences du Ve´ge´tal,
CNRS, 91198 Gif-sur-Yvette,2 and LGDP, UMR 5096, Universite´ de Perpignan, 66860 Perpignan Cedex, 3 France

JOURNAL OF VIROLOGY,
Apr. 2007, p. 4177–4185 Vol. 81, No. 8
 Nanovirus

General Description
The 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 protein
DNA-V (6) 1017 Replication associated protein
DNA-U1 (7) 988 non-structural protein U1
DNA-R (8) 1005 replication initiator protein M-Rep
 Cell cycle link protein - Clink

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

MGLKYFSHLPEELRQKIVHDHLQQERKKEFLEKAIEDSCRRHVSLLKSDPSPSELYALSKFLDS
LADYVGKQFNTRCLIKWKKDVPANIKFEVMEEQHLRLYGFVDMDDLLCREVLPPEEDDDITYEDG
MIVNCSELDKLFEALGIKVVYITVSKNCICTPLNKDIVIS

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 of protein 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?
 Gluco-corticoid inducible system

VP – 16 VP – 16
35S Gal 4 – DNA BD GR 35S Gal 4 – DNA BD GR
AD AD

Advantages :
No Gluco-corticoid Gluco-corticoid

• Non-toxic hormone

• Easily permeable
Inactive Transcription Active Transcription
Factor Factor
• Dose dependant gene expression

• Target genes are induced without pleiotrophic effects

Target Gene
Expression
 Regulation of Transcription factor E2F by Rb

Chromocentre: Condensed heterochromatic region of a chromosome that stains

strongly with DAPI (stains centromeric and nucleolus organization).

Clink mut (C*) : Mutant Clink with the LxCxE mutated to LxRxA

PCNA , CDC6 : E2F regulated S phase genes

CDKB: B1,B2-type cyclin-dependent kinases thought to control entry and progression
through the M phase and expressed during G2 and M phases of dividing tissues.
These are markers for the G2/M phases.
 Clink is stably expressed from the inducible Gluco-corticoid regulon
T=24H

Clink specifically induces cell cycle regulated genes

Samples are 6-7 week old fully expanded rosette leaves

 Impact of Clink on Cell Cycle
33% 95% 10% 5%
A : Ethanol treated Clink transgenic
B : Dex treated Clinkmut transgenic
C
D Dex treated Clink transgenic
E
F
19% 19%

7% 1%

No. represents the % of nuclei observed for each type in Clink expressing cells
Samples are 6-7 week old fully expanded, detached rosette leaves treated with Dex/ethanol for 4 days
In each case the sample size is 800-1,100 nuclei. Bar represents 15 microns
 Effect of Clink expression on cell size

A B

C D
 Effect of Clink expression on the Ploidy Level

Clink expressing cells have undergone more endoreduplication cycles

Ploidy Levels in FBNYV Infected Tissues