Phd Viva_ 5nov09

PhD Viva voce

Mechanism of Rolling Circle Replication in Geminivirus: Role of Replication Enhancer (REn/AC3)

Kalyan Kumar Pasumarthy Plant Molecular Biology Group ICGEB, New Delhi

Geminiviral Biology

Objectives of this study

Results

Summary

Geminiviral Biology

Diseases and Symptoms of Geminiviruses A

D

B

E

C

F

G

Geminiviruses infect a wide variety of crop plants Infection affects normal growth and crop yield

Whiteflies Transmit Geminiviruses

Whiteflies and leafhoppers trasmit geminiviruses from one plant to another Whiteflies are a major concern for ToLCV and ChLCV

Virion Structure of Geminiviruses A

B

• 38 × 22 nm • 1/100 of HIV size • Smaller than Hep B (42 × 27 nm) • 1/7th the length of TMV

Geminiviruses are incomplete icosahedral twinned virions Genome consists of covalently closed ssDNA One virion accommodates only one DNA molecule limited by size

Genomic Content of Geminiviruses

CR IR

CR

DNA β 1.4 kb

Monopartite

: Maize streak virus, Wheat dwarf virus

Monopartite + β DNA

:Papaya leaf curl virus, Tomato leaf curl virus

Bipartite

: Mungbean yellow mosaic India virus, Abutilon mosaic virus

Geminiviral Lifecycle

Vector transmission

Nucleus Cytoplasm

Geminiviral Lifecycle

Primer synthesis ssDNA

Complementary strand synthesis dsDNA

Transcription

Replication termination Replication initiation

Translation

ORFs of Geminivirus DNA A

AC1 → Replication Initiator (Rep)

CR

AC2 → Transcriptional Activator (TrAp) AV2 AC4

RNAi suppressor (Sup) AC3 → Replication Enhancer (Ren)

AC1

AC4 → Replication RNAi suppression AV1

AC2

AV1 → Coat Protein (CP) AC3

AV2 → RNAi suppression

Replication Initiator Protein (AC1)

Tyr

Oligomerisation (120-182 Aa)

Walker A

Walker B

Replication Initiator Protein/Rep/AC1 (362 Aa) DNA Nicking/Ligation (1-120 Aa)

ATPase/Helicase (183-362 Aa)

DNA binding (1-130 Aa)

• Rep initiates the replication of the viral DNA •Rep is a multifunctional protein • Induces replication in the cells by reprogramming the cell cycle • Rep interacts with various host proteins and viral proteins

Replication of Geminiviral DNA

Re p

Rep initiates replication by site specific nicking Rep releases newly formed DNA by religating

Geminiviruses replicate by rolling circle replication

Replication Enhancer Protein (AC3)

pRB Replication Enhancer Protein/REn/AC3 (134 Aa) pRB R R 7

PCNA

28

• Replication accessory protein • Interacts with Rep and various host factors • No enzymatic motif • No DNA binding domain

95

Rep

128

Geminivi ral AC3

Transgenics No significant change in viral DNA replication in the absence of AC3

Protoplasts AC3 mutation resulted in drastic reduction of viral DNA replication (~50 fold)

Objectives of this study

Objectives • To express the recombinant AC3 protein from geminiviruses • Biochemical effect of its interaction with Rep • Oligomerisation status • Exhaustive search for the AC3 interacting host proteins and identify other possible pathways where AC3 plays role • Role of AC3 in replication ex vivo and in planta

Results

Nucleotide Sequence Alignment of AC3 ORF

Amino Acid Sequence Alignment of AC3 Protein

ToLCKeV GST-AC3 Protein Expression and Purification Induced V

VI

ND1 MY

K2

M

K4

CL

kDa

• GST is 26 kDa

60 50 40

• AC3 is 134 Aa (~15 kDa)

GST-AC3

• Fusion protein is ~ 41 kDa

30 25

• 18°C/0.1mM IPTG/12-16 hrs

Induction of GST-AC3 proteins ND1 S

K2 P

S

M P

K4 S

CL P

S

P

Expression profile of GST-AC3 proteins

I

FT

W

M

1

2

3

4

5

6

Purification profile of GST-AC3 protein

GST-AC3 is soluble and purified

ToLCKeV His-AC3 Protein Expression and Purification Induced VI

V

M

ND1

ND2

K2

K4

IC

kDa 40

• His tag is ~3 kDa

30 25 20

• AC3 is 134 Aa (~15 kDa) • Fusion protein is ~18 kDa

His-AC3

15

•18°C/0.1mM IPTG/12-16 hrs

Induction of His-AC3 proteins

ST

VI S

P

M

ST

IC S

P

ST

K2 S

S

P

FT

W

M

1

2

3

5

7

8

kDa 35 25 18 14

Expression profile of His-AC3 proteins

Purification profile of His-AC3 protein

His-AC3 is soluble

ToLCKeV MBP-AC3 Protein Expression and Purification M

VI

K2 CL

K2

S M

K4

P

S

CL

P

kDa

• MBP tag is 43 kDa

60

• AC3 is 134 Aa (~15 kDa)

MBP-AC3

50 40

• Fusion protein is ~ 58 kDa

20

•18°C/0.1mM IPTG/12-16 hrs

15

Induction of MBP-AC3 proteins

Expression profile of MBP-AC3 proteins UC

FT

W1

W2

W3

M

1

3

5

7

1

2

PC 3

4

UC

M

PC

1

CC

2

MBP-AC3 MBP

AC3

Purification profile of MBP-AC3 protein

MBP-AC3 is soluble and purified to 99% homogeneity

Partial cleavage by Factor Xa

Complete cleavage by Factor Xa

AC3 precipitates upon cleavage by Factor Xa

What is the Oligomeric status of AC3?

Oligomerisation of AC3

M

MBP-AC3 MBP GST-AC3

Input

Bound

AC3 forms homo-oligomer

Oligomerisation of AC3

M

MBP-AC3 MBP GST-AC3

Input

Bound

158kDa Fraction No.

2

5

8

11

14

17

20

23

449kDa

669kDa

26

30

28

32

34

36

38

40

42

[i] [ii]

AC3 forms a higher order homo-oligomer of 12-14 mer

Oligomerisation of AC3

M T2008Jul29no011:10_U V

M T2008Jul29no003:10_U V

M T2008Jul29no002:10_U V

M T2008Jul29no010:10_U V

m A u

mAu 350 250

300 200

250

• Superdex 200 5/150 • 3ml bed volume • 1.374 void volume • 200 μl/min flow rate

MBP-AC3 Dextran (2000 kDa )

150

200 150 100

100

Throglobulin (669 kDa ) Aprotinin (6.9 kDa )

50

50 0

0

0.0 0.0

1.0

1.0

2.0 2.0

3.0

3.0

4.0

4.0

ml

m l

AC3 forms a higher order homo-oligomer of 12-14 mer

How does AC3 affect the Biochemical properties of Rep?

ToLCKeV AC1 Cloning

ND1

Kan

XhoI NotI EagI HindIII SalI BamHI EcoRI SacI KpnI NcoI

pETM30 6346bp

N’-His Tag

GST

TEV-Site

C’-His Tag oI Xh

I

Lac Op

Nc o

T7 Pro

MCS

AC1

K2

M

ToLCKeV His-GST-AC1 Expression and Purification Induced ND1 K2

M

ND1 S P

M

kDa

K2 S

P

116

His-GST-AC1

66 45 35 18

Induction of His-GST-AC1 proteins I

FT

W1

W2

1

3

5

7

M

Purification profile of His-GST-AC1 protein by GST resin

Expression profile of His-GST-AC1 proteins I

S

P

FT

M

1

3

5

7

9

Purification profile of His-GST-AC1 protein by Ni-NTA resin

His-GST-AC3 is insoluble

ToLCKeV MBP-AC1 Expression and Purification UI

I

M

S

P

1

3

5

7

9

11 13

15

17

MBP-AC1

Induction and expression profile of MBP-AC1 protein

Purification profile of MBP-AC1

MBP-AC3 is insoluble

+M BP -AC

GS T-A C3

GS T-A C3

M

MB P-A C

1

+M BP

+M BP -AC

GS T-A C3

GS T-A C3

MB P

-AC 1

+M BP

1

1

AC1-AC3 Interaction Studies

kDa 116

MBP-AC1

66

45

MBP GST-AC3

Input

Bound

GST-AC3 interacts with MBP-AC1

Role of AC3 on ATPase Activity of AC1

Rep (pmole)

-

0.1

MBP (pmole)

-

-

MBP–AC3 (pmole)

-

-

0.1

0.1

-

-

0.02 0.06

0.1

0.1

0.1

0.1

-

0.1

-

-

-

-

0.1

0.1

0.1

0.2

0.3

0.5

-

-

-

-

-

-

0.02

-

0.2

Relative (%) ATPase activity

0.5 Released Pi

γ P32 Labelled ATP

1

2

3

4

5

6

7

8

9

10

11

12

13

Rep (pmole)

-

0.1

0.1

0.1

0.1

0.1

0.1

0.1

MBP–K2_AC3 (pmole)

-

-

0.02

0.06

0.1

0.2

0.3

0.5

AC3 enhances the ATPase activity of Rep by 50-80%

What are the AC3 interacting host proteins?

Putative Interacting Proteins Identified by Phage Display

AC3 Interacting Peptides from Phage Display Screening

Proteins involved in replication and recombination

Proteins involved in RNA silencing

AC3 Interacting Peptides from Phage Display Screening

Proteins involved in histone and DNA modification

Proteins involved DNA or RNA polymerisation

How does AC3 affect Replication of viral DNA?

Role of AC3 in Replication : Ex vivo Model system in Yeast Plasmid

Replication Efficiency in Yeast

p Am URA

YCp50 S AR

XhoI

CE N

Bgl II

Replication Efficient (+)

4

ARS

HindIII p m A

URA

YCpOXhoI Bgl II

CE N

Replication Inefficient (-)

4

CR-AC3 HindIII 3 -A C CR

Yeast Replicon CEN4

URA

Amp

HindIII

Replication Efficient (+)

Role of AC3 in Replication : AC3 Mutation

AC2 CR

AC1

AC3

1476 bp AC3 Start

Yeast tranformed with the ToLCKeV viral DNA grows slowly

Role of AC3 in Replication : In planta Model system

pCK2 Replicon ~15 kb MCS AC2 CR

AC1

AC3

35S Pro

CR

Role of AC3 in Replication : Plant Vector Construction

bp 250 200 150 100 50

Cloning of CR

Role of AC3 in Replication : Plant Vector Construction

Nde I - 11725 R P C

01

4 40

22

35S

2

Nde I - 9524

pCambia+35S+K2_CR pCAMBIA1391Z+35S+CR 12112 bp 12112bp 30

20

bp

24 8

250 200 150 100

9

Nde I - 4015

Nde I - 6504

50

Cloning of CR

Schematic diagram showing the restriction pattern

Role of AC3 in Replication : Plant Vector Construction

Nde I - 11725

bp

R P C

5000

01

4

3000 2500 2000

40

22

35S

2

Nde I - 9524

pCambia+35S+K2_CR pCAMBIA1391Z+35S+CR 12112 bp 12112bp

1000

20

100

3500

1500

30

bp

24 8

250 200 150

4000

9

Nde I - 4015

Nde I - 6504

500

250

50

Cloning of CR

Schematic diagram showing the restriction pattern

Confirmation of CR direction

Role of AC3 in Replication : Plant Vector Construction

bp 3000 2500 2000 1500 1000 750 500

250

Cloning of CR-AC3

Role of AC3 in Replication : Plant Vector Construction

Nde I - 13580

Nde I - 11047

1523

1500

1

92

0

44 02

3000 2500 2000

Nde I - 11660

P CR

613

bp

CR -A C3

35S

pCK2 ToLCV KReplicon II Replicon 13967 bp 13967 bp Nde I - 4015

1000 Nde I - 9524

8

9

750

30

20

24

500 Nde I - 6504

250

Cloning of CR-AC3

Schematic diagram showing the restriction pattern

Role of AC3 in Replication : Plant Vector Construction

Nde I - 13580

Nde I - 11047

1523

1500

1

92

0

5000

44

3500

pCK2 ToLCV KReplicon II Replicon 13967 bp 13967 bp

8

9

Nde I - 9524

30

20

24

3000 2500 2000 1500

Nde I - 4015

1000 750

4000

02

3000 2500 2000

Nde I - 11660

bp

P CR

613

bp

CR -A C3

35S

500

1000 750 500

Nde I - 6504

250

Cloning of CR-AC3

250

Schematic diagram showing the restriction pattern

Confirmation of CR-AC3 direction

n Re pli co

pC K2 M

Re pli co n

n lico

pC K2

Re pli co n

pC K2 M Re p

M

pC K2

Re pli co n

pC K2 M

Re pli co n

pC K2

Re pli co n

pC K2 M

pC K2

Re pli co n

Role of AC3: Replication Assay in Tobacco

Episome

Actin 4 dpi

10 dpi

AC3 mutation does not affect viral replication in Tobacco leaves

Role of AC3 in Replication : AC3 Mutation (Second Strategy)

AC2 CR

AC1

AC3

Mutation sites 1414, 1412 bp

Role of AC3 in Replication : AC3 Mutation (Second Strategy)

AC2 CR

AC1

AC3

Mutation sites 1414, 1412 bp

Nucleotide alignment showing the AC3 mutations

Role of AC3 in Replication : AC3 Mutation (Second Strategy)

AC2 CR

AC1

AC3

Mutation sites 1414, 1412 bp

Nucleotide alignment showing the AC3 mutations

Protein alignment showing the AC3 mutations

Role of AC3: Replication Assay in Tomato

Replicon

Episome Actin 5dpi

10dpi

15dpi

AC3 enhances replication of viral DNA in tomato plants

Does AC3 affect RNAi in plants?

Role of AC3 in RNAi : In vivo Model system in Plant pCK2 Replicon ~15 kb

PCN MC A S

AC2 CR

AC1

AC3

Episome 2.8 kb

35S Pro

PCN CR CR A

Role of AC3 in RNAi : PCNA Silencing in Tomato

Wild type VIGS vector AC3 mutated VIGS vector

Control vector

AC3 enhances PCNA gene silencing in Tomato plants

Role of AC3 in RNAi : PCNA Silencing in Tomato

Reduced flowering observed in wildtype VIGS infiltrated tomato plant

Normal flowering and fruit formation observed in AC3 mutated VIGS infiltrated tomato plant

Deformities observed in leaves of wild type VIGS infiltrated tomato plant

PCNA silenced plant has reduced flowering and altered leaf morphology

Summary

Summary • Recombinant ToLCKeV AC3 protein was purified as an MBP fusion protein to a homogeneity of ~ 99% • AC3 forms higher order homo-oligomer consisting of 12-14 mer (major) and dimer (minor) • AC3 oligomerises with AC1 and enhances the ATPase activity of AC1 to an extent of 50-80% • Phage display identified various putative AC3 interacting host proteins from various metabolic pathways including DNA and histone modification, DNA replication and RNAi • Ex vivo and In planta vector systems were developed to study the role of viral replication. AC3 enhances replication in planta by 2-3 fold in tomato leaves • In planta replicon vector was utilised for virus induced gene silencing of PCNA in tomato plants. Silencing of PCNA was phenotypically strong in wildtype VIGS vector in comparision to AC3 mutated VIGS vector

Proposed Model

Interacts with host proteins (PCNA, pRBR, SlNAC1 etc.,) and Rep protein to enhances DNA replication by 2-3 fold

ToLCKeV AC3

AC3 enhances silencing probably by interacting with host proteins (Ago1, Ago7, Dcl1, Dcl2, HEN1 etc.,)

Acknowledgements

Dr Nirupam R Choudhury Dr Sunil Mukherjee Dr Neeti Padmashree Sudhir K Sopory Prof VS Chauhan Dr Shahid Jameel Dr Krishnamuthy Natarajan

Dr MK Reddy Dr Tuteja Dr Sneh lata Dr Suresh Nair Dr Pawan Malhotra Dr Raj Bhatnagar Dr Shiva Reddy Dr Leelavathi Dr Nirupama Bhatnagar Suman Ranjan Das Kannan Dharmendra Kosalai Ajay Rabi

Acknowledgements Vikash Pranjal Jamil Geetika Prerna Afsar Vipin Bhuban Deepti Pankaj Rashmi Meetu Sameer Susheel Urvashi Dharmendra Pratap Jyoti Nishant

Tanh Tien Archana Preeti Sameena Deepak Ravikanth Pooja Nujhat Vandita Sudhakar Mallikarjun Chandrasekhar Satish Yukti Hung Members of PMB Members of II floor My friends in ICGEB

Acknowledgements

School of Biotechnology Madurai Kamaraj University

ICGEB

Siddhartha College of Arts & Science Vijayawada

Kishore Vaigyanik Protsahan Yojana DST & IISc

Navodaya Vidyalaya Chalackurthy Camp

CSIR

Acknowledgements

ICGEB 2004-09

Acknowledgements

Tarun K Bhatt Diwakar Kumar

My Family