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Peptide Aptamers That Bind to a Geminivirus

Replication Protein Interfere with Viral Replication


in Plant Cells

Luisa Lopez-Ochoa, Jorge Ramirez-Prado,


and Linda Hanley-Bowdoin

JOURNAL OF VIROLOGY
June 2006, p. 5841–5853
GEMINIVIRUS
• PLANT PATHOGEN
• LEGUMINOUS PLANTS
• AGRO-ECONOMIC LOSS

CR : Region common to A& B


component containing origin
of replication
AC1 : Replication initiator
AC2 :Transcriptional activator
AC3 :Replication enhancer
AV1 :Coat protein
BC1 :Intercellular movement
(MUNGBEAN YELLOW MOSAIC INDIA VIRUS) BR1 :Transport of viral DNA
across nuclear envelope
BEGOMOVIRUS
•Small genome
•Codes essential proteins only
•Exploits host cell machinery for its propagation
Baits for aptamer screens.
Diagrams of the TGMV AL1 coding regions (TAL11-352 and
TAL11-130) cloned downstream of the LexA DBD
Baits were tested for oligomerization activity
1. TAL11-352 plus AD:TAL11-352
2. TAL11-352 plus AD:Jun
3. TAL11-130 plus AD:TAL11-352
4. TAL11-130 plus AD:Jun
5. GUS plus AD:TAL11-352
PGal ATG NLC AD HAg Ter
6. GUS plusAD:Jun
7. CaAL11-349 plus AD:TAL11-352
8. CaAL11-349 plusAD:Jun
Stringent Yeast Two-hybrid Screening

• Gal HWUL selection


• Repression by transfer onto Glu HWU
• Assay of lacz and growth on Gal-HWUL
• Retransformation
• 11/350 clones (TAL11-352 )
• 88/597 clones (TAL11-130 )

Effect of FL-TrxA peptides on TGMV A


DNA accumulation
The 88 plasmids recovered from the screen of
the JM-1 library using TAL11-130 as bait
were retransformed into different bait strains
to confirm the specificity of the interaction.

Aptamers that bind to TAL11-130 also interact with TAL11-352


Mechanism of Geminiviral replication

• Geminivirus replicates via rolling circle replication and


recombination
•Rep initiates RCR by binding at the direct repeats and nicks the
origin
• Rep religates the newly synthesized ssDNA genome

A A T
A
T T
A T Replication initiation /
A Rep nicking site
T
C C
G C
A T
C G
T A
C G
A T
C G
G C
G C
G C CGCGACCGGTGTATTG
ATCGGTGTACAC//TCGGTGTATCGGTGTCTTA//GCCCATAG
Replication Interference Assay

Input replicon Released TGMV A replicon


cassette
Replication Interference Assay
Statistical significance of pairwise alignments. Pairwise alignments were performed for 100 sets of three random databases of
computer-generated 20-mer aptamers containing 88, 31, or 57 members. The frequencies of hits were compared to equivalent alignments of
the databases corresponding to all, interfering, and noninterfering N-TrxA peptide databases, respectively. (A) The left panel shows the
frequency distribution (expected mean, 54) of a random 20-mer peptide having at least one hit against a database comprised of 88 random
20-mer peptides. The right panel shows the frequency distribution (expected mean, 101) of the total number of hits per peptide for all the 88
random 20-mer peptides. The dashed lines represent the observed values for the all N-TrxA peptide database for each analysis. Similar
analyses were performed for the interfering and noninterfering TrxA peptide databases and their random 20-mer control databases (not
shown). (B) The observed and expected means and standard errors of the pairwise alignments of the three TrxA peptide databases are given.
The observed values for the three databases are significantly higher than the expected values derived from the random 20-mer databases (P
values of 0.0001).
Motifs in Trx A peptides

I
Criterion for motif classification:
I
I
1. Motifs include atleast 5 members
N,I 2. Members interact with CaAL1
I 3. Aas typically involved in Pro-Pro
I interactions
4. Motifs are related to plant protein
N
Significance of Aptamer Approach for Developing
Virus Resistant Plants
1. Stringent in vivo screening most likely selects peptides that fold
correctly, stably expressed and bind with high affinity

2. Peptides selected were bound to N terminus of the protein that


doesn’t resemble the host proteins (Non-toxic to host)

3. Small size enables the peptides to move passively into the nucleus

4. Reduces DNA accumulation more strongly than the well


characterzied trans dominant Rep mutant

5. Peptides are capable of binding to the divergent Rep protein from


TGMV and CaLCuV
Work Presentation

KALYAN KUMAR PASUMARTHY


Ph.D STUDENT(2004)
GEMINIVIRUS: REPLICATION
• Multiplies mostly in differentiated cells
• Replicates by rolling circle replication
• Only viral protein known to involve directly in replication is replication
initiator protein (Rep/AC1)
• Viral replication is enhanced in the presence of another viral protein -
Replication enhancer (REn/AC3)
•Depends almost on host cellular machinery for elongation of replication

?
1. How does virus get the replication machinary in a differentiated cell?
2. What factors induce the host cell to synthesize the proteins required for
virus?
3. What factors direct the host replication machinary to the work on viral
genome?
Thus, proper understanding of geminiviral replication
enables us to control its pathogenesis at the replication
step and also lets us understand the plant replication
mechanism
AC3 IS THE REPLICATION ENHANCER

• AC3 associates with Rep (TGMV, BGMV)Settlage et al., 1996)

• Viral protein AC3 increases the accumulation of viral genome


(Replication Enhancer, Accessory Protein) (Sunter et al., 1990)

• AC3 interacts with other host proteins also:


• Retinoblastoma Related Proteins (TGMV,Settlage et al., 2001)
• Proliferating Cell Nuclear Antigen (TYLCV,Castillo et al., 2003).
• NAC domain Transcription factor (TLCV,Selth et al., 2005)
OUR LAB DATA
• AC3 regulates the nicking/ligation activity of Rep in a
concentration dependant manner
• ATPase and Helicase activity are upregulated

Probable Role of AC3


Enhanced genome replication of Geminivirus in the presence
of AC3 is because of the accessory role of AC3
• AC3 might regulate the INITIATION by Rep by modulating its nicking
activity
•AC3 up regulates the ATPase and Helicasitye activ of Rep which are the
threshold factors for the progress of the replication (ELONGATION)
that has been initiated.
• AC3 might bring other necessary molecules at the site of replication
which also effect helicase and ATPase activity.
MECHANISM OF ROLLING CIRCLE REPLICATION
IN GEMINIVIRUSES: ROLE OF REPLICATION
ENHANCER (REn/AC3)

• Interaction with Rep


• Oligomerization status of AL3
• Fishing out the interacting host proteins
• Effect of AC3 interacting host proteins on nicking/ligation,
ATPase and Helicase activity of Rep
• Assaying the affect of interacting host proteins on the Replication
efficiency of viral replicon
• Yeast model
• Cell line based assay
• Domain characterization of AC3 and interacting host proteins
AL3 interaction with Rep: in vitro binding assay

Unbound Bound
(400mM Salt Wash) Unbound Bound
(600mM Salt Wash)

AL3 AL3 AL3 AL3


1 L A+ P B M

1 L A+ P B M

e nol a P B M

PB M

PB M
AL3 interaction with Rep: in vitro binding assay

MBP+AL1

MBP+AL1
MBP+AL1

AL3+AL1

AL3+AL1
AL3+AL1

AL1
AL1
AL1

300mM Salt Wash 600mM Salt Wash 1000mM Salt Wash

Bound Fractions
AL3 Oligomerization: Sucrose Gradient Ultracentrifugation

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

AL1+AL3 Oligomerization: Sucrose Gradient Ultracentrifugation


Gel Filtration
In progress……

• Cloning of AL3 with alternate tags


• His Tag (pET 28a)
• GST Tag (pGEX 4T-1)

• Cloning of Truncated AL3


• Full length is 128 Aa
• Truncated versions
» 1-30 Aa
» 1-59 Aa
» 1-90 Aa
» 1-120 Aa
PROPOSED WORK

To continue exhaustive search for the various AC3 interacting molecules


• Phage display
• Yeast two hybrid analysis with cDNA library
• In vitro pull-down assay
• Analysis of the interacting molecules from the view of viral replication

To figure out the AC3 domain(s) interacting with AC1


• N/C-terminal truncations
• Site directed mutagenesis
• Evaluate the efficiency of mutated AC3 by assaying the colony forming
ability in yeast and replication efficiency in protoplasts
Model for Mutagenesis of AC3 and Replication
Efficiency Assay

AC2
Rep/AC1 REn/AC3
Effect of AC3 interacting Host proteins on
Replication of Viral genome: Yeast Model

Mutant yeast (defective in the


Wild type yeast
homologous interacting protein)
Effect of AC3 interacting Host proteins on Replication of
Viral genome: Plant Cell Line base assay

• TBY-2 protoplast transfection with wild type and mutant AC3 constructs

CR AC1
TBY-2

• Analyzing the replication efficiency by Southern blotting


• Similarly the assay will be done by silencing the host interacting proteins
Thank U…...

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