presenter:nguyen trong binh

Genetic analyses of the function of PB1 subunit of the influenza virus RNA-dependent RNA polymerase

Presenter: Nguyen Trong Binh

Structure of influenza A virus

M2

HA

NA

M1

NP

PB1

vRNP complex

PB2 PA

Envelope

PB1

PA NH2 COOH

1 716 a.a.

PB2

Cap binding

NH2 COOH

1 759 a.a.

NLS

PB1

RNA polymeraseconsensus motif

NH2 COOH

1 757 a.a.

NLS

NLS

448 496

318 483

PB1 binding

Endonuclease

The schematic diagram of influenza virus RNA polymerase

PAPB1

PB2

PB1

PB1


NH2 COOH

1 757 a.a.

NLS

The schematic diagram of influenza virus RNA polymerase

PAPB1

PB2

Putative cRNA promoter binding

Putative vRNA promoter binding

Putative nucleotide binding

Aim

Functional analysis of PB1 subunit using mutant viruses

By determining the functional site of PB1involved in polymerization, nucleotide recognition,and so on.

(1) Reverse-genetics: Using mutant viruses to determine the replicational and transcriptional activities

(2) Forward-genetics: Using ribavirin which inhibits RNA synthesis to determine the catalytic active site of PB1 by isolation of ribavirin-resistant PB1 mutants

The enzymatic activities are regulated by the interaction with promoter sequence

Flu A promoter

Viral polymerase activity is regulated by the viral promoter

PAPB1

PB2

Capping activity

RNA synthesis activity

FluB promoter

Flu A recognized only Flu A promoter

To find important residues

by comparing Flu A with Flu B

in promoter binding site

Regulatroy mechanism?

PB1 NH2 COOH

1 757 a.a.

NLS



A/Narita/98 (H9N2)

A/WSN/33(H1N1) A/England/67(H2N2) A/Hong Kong/68(H3N2)

B/Aichi/88 B/Alaska/92 B/Argentina/01 B/Hong Kong/93

MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYS 50MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYS 50 MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYS 50 MDVNPTLLFLKVPVQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYS 50 MNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTGTGYTIDTVIRTHEYS 50

MNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTGTGYTIDTVIRTHEYS 50

MNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTGTGYTIDTVIRTHEYS 50 MNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTGTGHTIDTVIRTHEYS 50

*::** :**:.** * ********** **********:*:*** ***:**

A/Narita/98 (H9N2)






*::** :**:.** * ********** **********:*:*** ***:**

16 27 44

The a.a. positions 1-50 was highly conserved between influenza A and B viruses, but the amino acid positions 16, 27, and 44 differ between two viruses.

PA binding site1-15

Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters among influenza A and B viruses

Homology 85% 50~60%

The viral RNA synthesis activity of mutant viruses N16A, D27V, and 44I

A. mRNA C. vRNA B. cRNA

0

0.5

1

1.5

2

2.5

3

mock wt N16A D27V N44I

Rel

ati

ve

am

ou

nt

of

mR

NA

0

0.5

1

1.5

2

2.5

3


Rel

ati

ve

am

ou

nt

of

cRN

A

0

0.5

1

1.5

2

2.5

3

3.5

4


Rel

ati

ve

am

ou

nt

of

vRN

A

These mutations affect the RNA synthesis activity of viral mRNA, cRNA, and vRNA equally, there could be two possibilities:

****

****

***

Student’s t test (*, P < 0.05; **, P < 0.01)

Binh et al, 2013. Frontiers in Virology

Hypothesis

(1) Amino acids at the positions 27 and 44 affect cRNA/mRNA synthesis from vRNA through recognition of the promoter on vRNA, but do not affect vRNA synthesis from cRNA through recognition of the promoter on cRNA

(2) These mutations affect independently the synthesis of mRNA, cRNA, and vRNA, but the sum of effects leads similar outputs in the synthesis of mRNA, cRNA, and vRNA.


16 27 44PA binding site

1-15

A/Narita/98 (H9N2)



MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYS 50MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYS 50 MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYS 50 MDVNPTLLFLKVPVQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYS 50




*::** :**:.** * ********** **********:*:*** ***:**

MDVNPTLLFLKVPAQNAISTTFPYTGNPPYSHGTGTGYTMDTVNRTHQYS 50 A/Lsp/SC Alaska (H4N8)A/Narita/98 (H9N2)







*::** :**:.** * ********** **********:*:*** ***:**

MDVNPTLLFLKVPAQNAISTTFPYTGNPPYSHGTGTGYTMDTVNRTHQYS 50 A/Lsp/SC Alaska (H4N8)

Q: structure of Q is to close to that of N

D: position 27

D

Q

The viral RNA synthesis of mutant viruses at the position 16

Negatively charged amino acid may decrease the viral RNA polymerase activity.

0

0.5

1

1.5

2

2.5

3

mock N D Q A

Rel

ati

ve

am

ou

nt

of

mR

NA

(wt)

0

0.5

1

1.5

2

2.5

3

mock N D Q A

Rel

ati

ve

am

ou

nt

of

vRN

A

(wt)

0

0.5

1

1.5

2

2.5

mock N D Q A

Rel

ati

ve

am

ou

nt

of

cR

NA

(wt)


****

*





1-15

A/Narita/98 (H9N2)







*::** :**:.** * ********** **********:*:*** ***:**








*::** :**:.** * ********** **********:*:*** ***:**


The amino acid at the position 27 in the wild type virus is aspartate, and aspartate is conserved among influenza A viruses, except for an H4N8 strain.

E

(2 types of negative charge D and E)


Uncharged amino acids at the position 27 enhanced the viral RNA synthesis.

0

0.5

1

1.5

2

2.5

3

mock D E N V

Rel

ati

ve

am

ou

nt

of

mR

NA

(wt)

0

0.5

1

1.5

2

2.5

3

3.5

4

mock D E N V

Rel

ati

ve

am

ou

nt

of

vRN

A

(wt)

0

0.5

1

1.5

2

2.5

3

mock D E N V

Rel

ati

ve

am

ou

nt

of

cRN

A

(wt)


**** **

***

*





1-15

A/Narita/98 (H9N2)







*::** :**:.** * ********** **********:*:*** ***:**








*::** :**:.** * ********** **********:*:*** ***:**


Q: structure of Q is to close to that of N

D: position 27

D

Q


44Q stimulated only vRNA synthesis.

A. mRNA

0

0.5

1

1.5

2

2.5

3

mock N I D Q

Rel

ati

ve

am

ou

nt

of

mR

NA

(wt)

0

0.5

1

1.5

2

2.5

3

3.5

mock N I D Q

Rel

ati

ve

am

ou

nt

of

vRN

A

(wt)

Rel

ati

ve

am

ou

nt

of

cRN

A

0

0.5

1

1.5

2

2.5

3

mock N I D Q(wt)

C. vRNA B. cRNA

**

** ** **



The effect on the primary transcription activity by incoming vRNP

These mutations did not affect primary transcription activity.

D. Position 44

0

0.5

1

1.5

2

mock N I D Q

mR

NA

/vR

NA

(wt)

0

0.5

1

1.5

2

mock N A D Q

mR

NA

/vR

NA

(wt)

B. Position 16

0

0.5

1

1.5

2

mock D N E V

mR

NA

/vR

NA

(wt)

C. Position 27

A. influenza B amino acids

0

0.5

1

1.5

2


mR

NA

/vR

NA


Effect of mutations on assembly of viral RNA polymerase complexes

WB: anti-PB2

WB: anti-tubulin

mock wt D27N D27E D27V N16A N16D N16Q N44I N44D N44Q

Input (2%)

1 2 3 4 5 6 7 8 9 10 11

The assembly of PB1 with PA and PB2 was not affected by these mutations.

PA

WB: anti-PB1

WB: anti-PA

+ - + + + + + + + + + +

1 2 3 4 5 6 7 8 9 10 11 12

mock wt D27N D27E D27V N16A N16D N16Q N44I N44D N44Q

IP: anti-PB2 antibody

PB1PB2

1.0 1.0 1.1 0.9 0.9 1.0 1.0 0.9 1.1 1.2 PB1/PA ratio

anti-PB2

anti-PB2

Summary 1

• Amino acids at the position 27 and 44 are involved in RNA polymerase activity and may contribute to subtype specificity.

• As the primary transcription activity of mutant viruses were not affected, this result indicates that replication activity and/or vRNP stability might be affected.

• The assembly of PB1 with PA and PB2 was not affected by these mutations.

• Amino acid residues at the position 27 and 44 are involved in the viral genome replication, possibly via the cRNA promoter recognition with little effects on the transcription activity and the assembly of the RNA polymerase complex

Aim

Functional analysis of PB1 subunit using mutant viruses

(1) Reverse-genetics: Using mutant viruses to determine the replicational and transcriptional activities

(2) Forward-genetics: Using ribavirin which inhibits RNA synthesis to determine the catalytic active site of PB1 by isolation of ribavirin-resistant PB1 mutants

Ribavirin

Screening of ribavirin-resistant PB1 mutant

transfection

PB1 PA

NP

PB2

pol.Ipro

vNS-EGFP

293T cell

CAGpro

PB2PA

PB1

NP

EGFP vRNP

transcription

m7Gppp EGFP

translation

EGFP

random mutation

ribavirin

transfection

PB1 PA

NP

PB2

pol.Ipro

vNS-EGFP

293T cell

CAGpro

PB2PA

PB1

NPPA

PB1

NP

EGFP vRNP

transcription

m7Gppp EGFP

translation

EGFP

random mutationrandom mutation

ribavirinribavirin

A. Mini-replicon assay system

for screening

G9

G10

G7

G8

G5

G6

-Ribavirin +Ribavirin Ratio

30%

26%

27%

16%

11%

6%

G9

G10

G7

G8

G5

G6

-Ribavirin +Ribavirin Ratio

30%

26%

27%

16%

11%

6%

mock

G1

G2

WT

-Ribavirin

G4

G3

+Ribavirin Ratio

27%

12%

20%

16%

34%

mock

G1

G2

WT

-Ribavirin

G4

G3

+Ribavirin Ratio

27%

12%

20%

16%

34%

B. EGFP fluorescence in the first screening

Binh et al, 2014. BBRC

B. EGFP fluorescence

C. Luciferase activity D. Effect of D27N mutation on assembly of PB1 subunit

A. Sequencing of ribavirin-resistant PB1 mutant

Characterization of D27N mutant

WT D27NWT D27N

69%

24%

ratio-Ribavirin +Ribavirin

WT

D27N 69%

24%

ratio-Ribavirin +Ribavirin

WT

D27N

WT

PB1

tubulin

D27NWT

PB1

tubulin

D27NWT

PB1

tubulin

D27NWT

PB1

tubulin

D27N

Lysate

Ribavirin - + - +WT

PB1

tubulin

D27NWT

PB1

tubulin

D27NWT

PB1

tubulin

D27NWT

PB1

tubulin

D27N

Lysate

Ribavirin - + - +

020406080

100120

0 10 20 30 40 50Ribavirin (µM)

Lu

cife

rase

act

ivit

y (%

of

con

tro

l) wt

D27N

020406080

100120

0 10 20 30 40 50Ribavirin (µM)

Lu

cife

rase

act

ivit

y (%

of

con

tro

l) wt

D27N

Binh et al, 2014. BBRC

Summary 2

D27N mutant was isolated by forward genetics in the presence of ribavirin.

The luciferase assay of influenza virus RNA polymerase activity of D27N mutant was higher than that of wild type in the presence of ribavirin.

The expression level of D27N in the presence of ribavirin was similar to that of wild type.

Models and Discussion

PB1


NH2 COOH

1 757 a.a.

NLS




Ribavirin

Promoter

Stimulation

Acknowledgments

Thank you for your attention!

Generation of recombinant viruses by reverse-genetics

293 T cellTransfection

CAGpro

PB1 PB2 PA NPseg1 seg3 seg4

seg5 seg6 seg7 seg8

Pol.I

seg2

Point mutation

Plasmids expressing influenza vRNA

Plasmids expressing influenza viral proteins

Budding

Recombinant virus

Nucleus

vRNPs

HA

NA

M2M1

Replication

vRNA (-)

cRNA (+)

mRNA (+)

Transcription

Encapsidation

vRNP

M1: NS2

Nuclear export

Translation

PB1 PB2 PA NP M1 NS1 NS2

HA NA M2

ER

Golgi

PackagingvRNP

Influenza Amino acid positions

16 27 40 44 48

A N(polar) D (polar) M (nonpolar) N(polar) Q (polar)

B A (nonpolar) V (nonpolar) I (nonpolar) I (nonpolar) E (polar)

Transcription and replication of influenza virus genome

Replication

vRNA

cRNA

Progeny vRNA

Transcription

Cap

UUUUU

Cap

Host cellular pre-mRNA

vRNA

5’

3’

UUUUU

UUUUU

Viral mRNA

3’

5’3’

Cap

5’

AAAAA

Cap

Cap AAAAA…

PB2: recognize and bind the cap

PA: cleave the cap

PB1: nucleotide addition

Method 2. Analysis of primary transcription activity by qRT-PCR

mRNA, and vRNA

were analyzed by qRT- PCR

MDCK cells Infection with WSN, MOI=2.5

RNA extraction by AGPC method 9 h post infection

Adding cycloheximide (CHX) into medium

Cytoplasm

Nucleus

mRNA

Incoming vRNP

Progeny vRNP

X

CHX

XPolymerase + NP

M2

HA

NA

M1

M2

HA

NA

M1

MDCK cell

X

CytoplasmNucleus

YMTX

MTX

MTX

MTX(Glu)n

FH2

FH4

CH2FH4

dUMP

TMP

TTP

DNA

Purine biosynthesis

RNA

Mechanism of action methotrexate

Reduced folic acid

receptor

YYY

Folic acid

receptor

MTXPreferred pathway Alternate pathway

MTX polyglutamate [MTX (Glu)n]MTX

Folylpolyglutamate synthase

FH2 FH4

Dihydrofolate reductase

A. Mini-replicon assay system

transfection

PB1 PA

NP

PB2

pol.I pro

vNS-Luc

293T cell

CAGpro

PB2PA

PB1

NP

Luc

vRNP

transcription

m7Gppp Luciferase

translation

Luciferase

random mutation

Methotrexate (MTX)

B. Luciferase activity

Effect of methotrexate on D27N mutant

0

20

40

60

80

100

120

0 0.3 1 3 10 30

Methotrexate (µM)

Lu

cife

rase

act

ivit

y

(% o

f co

ntr

ol)

WT

D27N

0

20

40

60

80

100

120

0 0.3 1 3 10 30

Methotrexate (µM)

Lu

cife

rase

act

ivit

y

(% o

f co

ntr

ol)

WT

D27N

Significant impact on our life by influenza virus infection

Wahlgren, 2011. Infection Ecology and Epidemiology.

A. Illustration of the host range of influenza A virus

H1-H3(H5, H7)

H7?

H1-H3

H4,-H7, H13

H10H3, H7

H5

H5,-H7, H9

H5, H7

H1-H3(H5, H7)

H7?

H1-H3

H4,-H7, H13

H10H3, H7

H5

H5,-H7, H9

H5, H7

Isolated by Shope and Lewis in

1930

B. Virus classification

Familly: Orthomyxoviridae

Genera: Influenza A

Influenza B

Influenza C

Nucleoprotein (NP) and matrix (M1) proteins

Other important characteristics

-FluA : a wide variety of hosts

-FluB : only humans

-FluC : humans but also from swine

-HA and NA of FluA viruses amino acid sequence variability than those of FluB viruses. FluC viruses: only a single hemagglutinin-esterase-fusion protein (HEF).

Models and Discussion

vRNA promoter

5’ AGU

A A

G A

AC

A G G C

3’ UC

G

U

U U

U

C

G

U C C G

A ………..

………..

.

......5’ A

GU

A A

G A

AC

A G G C

3’ UC

G

U

U U

U

C

G

U C C G

A ………..

………..

.

......

………..

………..

.

......

5’ AGC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......5’ A

GC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......

………..

………..

.

......

5’ AGU

A A

G A

AC

A G G C

3’ UC

G

U

U U

U

C

G

U C C G

A ………..

………..

.

......5’ A

GU

A A

G A

AC

A G G C

3’ UC

G

U

U U

U

C

G

U C C G

A ………..

………..

.

......

………..

………..

.

......

5’ AGU

A A

G A

AC

A G G C

3’ UC

G

U

U U

U

C

G

U C C G

A ………..

………..

.

......5’ A

GU

A A

G A

AC

A G G C

3’ UC

G

U

U U

U

C

G

U C C G

A ………..

………..

.

......

………..

………..

.

......

cRNA

cRNA promoter

5’ AGC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......5’ A

GC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......

………..

………..

.

......

Progeny vRNA

Strong activity

27N

27NG

27NG

5’ AGC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......5’ A

GC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......

………..

………..

.

......

Improved nucleotide

recognition

WT

R

WT

5’ AGC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......5’ A

GC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......

………..

………..

.

......

R

WT

Nucleotides

(A G U C R)

A. Panhandle model of vRNA and cRNA promoters of PB1 subunit between influenza A and B viruses

1. FluA vRNA 2. FluB vRNA 3. FluA cRNA 4. FluB cRNA

Why was the N-terminal region of PB1 chosen?

B. Structure of PB1 subunit

PB2 binding

1 15 678286 483

PA binding RdRp catalytic domain

267 4931 139

757

757249 2561 83 494

179 297 458 519




Structure of PB1 subunit

Sugiyama et al, 2009. EMBO

1 35

757

678

Obayashi et al, 2008. Nature

239716

2

15

PB2 binding

1 15 678286 483

PA binding RdRp catalytic domain

267 4931 139

757

757249 2561 83 494

179 297 458 519




PAC-PB1NPB1C-PB2N

B cRNAA mRNA

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0.5 2.5 10 25

moi

Rel

ati

ve a

mo

un

t o

f cR

NA

C vRNA

0

2

4

6

8

10

12

0.5 2.5 10 25

moi

Rel

ati

ve a

mo

un

t o

f vR

NA

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0.5 2.5 10 25moi

Rel

ati

ve a

mo

un

t o

f m

RN

A

mock

wt

D27V

B cRNAA mRNA

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0.5 2.5 10 25

moi

Rel

ati

ve a

mo

un

t o

f cR

NA

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0.5 2.5 10 25

moi

Rel

ati

ve a

mo

un

t o

f cR

NA

C vRNA

0

2

4

6

8

10

12

0.5 2.5 10 25

moi

Rel

ati

ve a

mo

un

t o

f vR

NA

C vRNA

0

2

4

6

8

10

12

0.5 2.5 10 25

moi

Rel

ati

ve a

mo

un

t o

f vR

NA

0

2

4

6

8

10

12

0.5 2.5 10 25

moi

Rel

ati

ve a

mo

un

t o

f vR

NA

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0.5 2.5 10 25moi

Rel

ati

ve a

mo

un

t o

f m

RN

A

mock

wt

D27V

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0.5 2.5 10 25moi

Rel

ati

ve a

mo

un

t o

f m

RN

A

mock

wt

D27V

The viral RNA synthesis of mutant viruses at the position 27 (various moi )

when the amounts of viral RNAs of D27V were analyzed at various moi (0.5, 2.5, 10, and 25), those of D27V were increased

A. mRNA

0

0.5

1

1.5

2

3 6

hpi

Rel

ativ

e am

ou

nt

of

mR

NA

mockwtN16AD27VN44I

B. cRNA

0

0.5

1

1.5

2

3 6

hpi

Rel

ativ

e am

ou

nt

of

cRN

A

C. vRNA

0

0.5

1

1.5

2

3 6

hpi

Rel

ativ

e am

ou

nt

of

vRN

A

A. mRNA

0

0.5

1

1.5

2

3 6

hpi

Rel

ativ

e am

ou

nt

of

mR

NA

mockwtN16AD27VN44I

A. mRNA

0

0.5

1

1.5

2

3 6

hpi

Rel

ativ

e am

ou

nt

of

mR

NA

mockwtN16AD27VN44I

0

0.5

1

1.5

2

3 6

hpi

Rel

ativ

e am

ou

nt

of

mR

NA

mockwtN16AD27VN44I

mockwtN16AD27VN44I

B. cRNA

0

0.5

1

1.5

2

3 6

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Rel

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cRN

A

B. cRNA

0

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1.5

2

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hpi

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cRN

A

0

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2

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hpi

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cRN

A

C. vRNA

0

0.5

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1.5

2

3 6

hpi

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vRN

A

C. vRNA

0

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hpi

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vRN

A

0

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vRN

A

Comparison of RNA synthesis of mutant influenza A viruses containing amino acids specific for influenza B viruses (3 and 6 hpi)

A. mRNA

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2

3 6

hpi

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mR

NA

mock

wt

N16D

N16QN16A

B. cRNA

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cRN

A

C. vRNA

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vRN

A

A. mRNA

0

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2

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mR

NA

mock

wt

N16D

N16QN16A

A. mRNA

0

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2

3 6

hpi

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mR

NA

mock

wt

N16D

N16QN16A

mock

wt

N16D

N16QN16A

B. cRNA

0

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2

3 6

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cRN

A

B. cRNA

0

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cRN

A

0

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3 6

hpi

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cRN

A

C. vRNA

0

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2

3 6

hpi

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vRN

A

C. vRNA

0

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vRN

A

The viral RNA synthesis of mutant viruses at the position 16 (3 and 6 hpi)

C. vRNA

0

0.5

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vRN

A

B. cRNA

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cRN

A

A. mRNA

0

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3 6

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mR

NA

mockwtD27ED27ND27V

C. vRNA

0

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vRN

A

C. vRNA

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vRN

A

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A

B. cRNA

0

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cRN

A

B. cRNA

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3 6

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cRN

A

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cRN

A

A. mRNA

0

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3 6

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mR

NA

mockwtD27ED27ND27V

A. mRNA

0

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NA

mockwtD27ED27ND27V

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mR

NA

mockwtD27ED27ND27V

mockwtD27ED27ND27V


A. mRNA

0

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hpi

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mR

NA

mock

wt

N44I

N44D

N44Q

B. cRNA

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hpi

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A

C. vRNA

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vRN

A

A. mRNA

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mR

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mock

wt

N44I

N44D

N44Q

A. mRNA

0

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hpi

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mR

NA

mock

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N44I

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N44Q

0

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hpi

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mR

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mock

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N44I

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mock

wt

N44I

N44D

N44Q

B. cRNA

0

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2

3 6

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cRN

A

B. cRNA

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cRN

A

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cRN

A

C. vRNA

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vRN

A

C. vRNA

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vRN

A

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vRN

A


0

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4

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6

3h 6h 9h

hpi

Re

lati

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mR

NA

Mock

WT-R

WT+R

D27N-R

D27N+R

0

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NA

Mock

WT-R

WT+R

D27N-R

D27N+R

The viral RNA synthesis of mutant viruses at the position 27 in the presence of ribavirin (IC50= 20 μM)

Virion of influenza A virus

NP

PB1

PB2 PA

HA

NA

M1

M2

NP

PB1

PB2 PA NP

PB1

vRNP complex

PB2 PA

HA

NA

M1

M2

HA

NA

M1

M2

Envelope

NP

PB1

PB2 PA

HA

NA

M1

M2

NP

PB1

PB2 PA

HA

NA

M1

M2

NP

PB1

PB2 PA NP

PB1

PB2 PA NP

PB1

vRNP complex

PB2 PA

HA

NA

M1

M2

HA

NA

M1

M2

HA

NA

M1

M2

Envelope

Segment 1

Segment 2

Segment 3

Segment 4

Segment 5

Segment 6

Segment 7

Segment 8

PB2

PB1

PA

HA0

NP

NA

M2

NS1

759 a.a.

2341 nt

757

716

2341

2233

HA1HA2

326222

1778

1565

498

454

1413

1027

M1 25297

NS2

890

237112

Segment 1

Segment 2

Segment 3

Segment 4

Segment 5

Segment 6

Segment 7

Segment 8

PB2

PB1

PA

HA0

NP

NA

M2

NS1

759 a.a.

2341 nt

757

716

2341

2233

HA1HA2

326222

1778

1565

498

454

1413

1027

M1 25297

NS2

890

237112

Structure of influenza A virus genome and viral proteins

Function of the influenza A virus componentsSegment Encoded

polypeptideRemarks

1 PB2 -Cap binding

-Component of RNA transcriptase complex

2 PB1 -Catalyzes nucleotide addition

-Component of RNA transcription, and replication complex

3 PA Endonuclease activity, component of RNA transcriptase and replicase complex

4 HA -Major surface glycoprotein

-Receptor (sialic acid) binding

-Proteolytic cleavage activation

5 NP -Monomer binds to RNA to form coiled ribonucleoprotein

6 NA Surface glycoprotein, neuramidase activity

7 M1 Major protein of virion

M2 Ion channel activity essential for virus uncoating

8 NS1 -Inhibits cellular pre-mRNA 3’ end cleavage and polyadenylation, inhibits pre-mRNA splicing

NS2 -Interacts with M1 and involved in nuclear export of RNPs

Hemmaglutinin (HA)

Matrix (M1)

Neuramidase (NA)

Polymerase complex (PB1, PB2, and PA)

Nucleoprotein (NP)

BM2 ion channel

Nuclear export protein (NEP)

NB ion channel

Virion of influenza B virus

Structure of influenza B virus genome and viral proteins

8 segments coding for 11 proteins. Segments size range from 890 to 2,341nt. Genome total size is 13.5Kb Viral RNA polymerase (PB1, PB2 and PA) transcribes one mRNA from each genome segment. Transcription is primed by cap cleaved from cellular mRNAS by Cap snatching. mRNA are polyadenylated by the viral polymerase stuttering on a poly U track. MP and NS mRNA can be spliced, giving rise to mRNA coding for BM2 and NEP proteins. PB1-F2 is translated from the +1 ORF of PB1 mRNA, NB is translated from the -1 ORF of NA mRNA.

Virion of influenza C virus

Structure of influenza C virus genome and viral proteins

No NA-encoding gene

Catalytic sites of influenza A virus PB1 subunit

http://www.uniprot.org/uniprot/P03431

Promoter of segment 2 (PB1) of influenza A and B

FluA: cDNA: 5’-AGCAAAAGCAGGCA-------GCCTTGTTTCTACT- 3’

FluA virus cRNA: 5’-AGCAAAAGCAGGCA-------GCCUUGUUUCUACU- 3’

FluA virus vRNA: 5’-AGUAGAAACAAGGC-------UGCCUGCUUUUGCU- 3’

FluA PB1: GenBank: M25932.1

FluB PB1: GenBank: M20479.1

FluB: cDNA: 5’- AGCAGAAGCGGAGC-------CTCGTGTTTCTACT -3’

FluB virus cRNA: 5’- AGCAGAAGCGGAGC-------CUCGUGUUUCUACU -3’

FluB virus vRNA: 5’- AGUAGAAACACGAG--------GCUCCGCUUCUGCU -3’

RTP (ribavirin triphosphate) could inhibit the enzyme by competing with the natural substrate without being a substrate for the enzyme

G R

Termination

5’

3’

vRNA

C C

Termination

5’

3’

vRNA

RTP could be an alternative substrate for the RNA polymerase and cause chain termination

R

CC

5’

3’

vRNA

RTP could be an alternative substrate for the RNA polymerase without causing chain termination

Cycle 1

3’

5’

cRNA

R R

C C

3’

5’

R

RTP could be an alternative substrate for the RNA polymerase without causing chain termination

R

5’

3’

cRNA Cycle 2

5’

3’

Wild- type Mutant-type

vRNAvRNA

C C U U

Investigation of effective concentrations of ribavirin in inhibition of A influenza virus (WSN) growth using MDCK cell

Plaque assay: 0, 10, 33, 100, 300 µM ribavirin

inhibitory effect of ribavirin

y = -30.032Ln(x) + 140.13

R2 = 0.9974

0

20

40

60

80

100

1 10 100 1000

µM ribavirin

% o

f c

on

tro

l (p

laq

ue

n

um

be

r) IC50=20 µMIC90 =75 µM

Result

Screening the PB1 mutant library

PB1 mutant library

divide into 10 groups after transformation

1 2 3 4 5 6 7 8 9 10

mini-replicon assay ± 75 µM choose 1 group

isolate single plasmid

repeat

LB agar kan+

isolate plasmids of each group

3x104 clones

Today’s report

Screening 1st 2nd 3rd 4th 5th

WT 27% 16% 19% 7% 29%

G1 12% 25% 26% 35% 19%

G2 20% 28% 20% 21% 17%

G3 16% 43% 33% 30% 66%

G4 34% 43% 33% 41% 59%

G5 30% 26% 32% 32% 33%

G6 26% 31% 47% 24% 54%

G7 27% 44% 35% 17% 86%

G8 16% 38% 29% 25% 40%

G9 11% 38% 40% 28% 40%

G10 6% 29% 31% 24% 53%

Screening of ribavirin-resistant PB1 by mini-replicon assay (EGFP)

Sequencing of mutant viruses at the position 16

N A D Q

WT N16A N16D N16Q


D N E V

WT D27N D27E D27V


N I D Q

WT N44I N44D N44Q

pfu/ml

viral typeaverage

wt 3x107 (± 2.8 x107)

N16A 8x107 (± 3.46 x107)

N16D 5x107 (± 4.16 x107)

N16Q 3x107 (± 2.52 x107)

D27N 3x107 (± 2.61 x107)

D27E 5x107 (± 4.16 x107)

D27V 5x107 (± 4.58 x107)

N44I 3x107 (± 2.31 x107)

N44D 5x107 (± 2.31 x107)

N44Q 7x107 (± 3.06 x107)

Titer check

The effect on the primary transcription activity of mutant viruses substituted to FluB (MOI =2.5, 9 hpi)

A. mRNA B. vRNA

0

0.5

1

1.5

2

2.5

3

Mock wt N16A D27V N44I

Rel

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mR

NA

0

0.5

1

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2

2.5

3

Mock wt N16A D27V N44I

Rel

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vRN

A

A. mRNA B. vRNA

The effect on the primary transcription activity of mutant viruses at the position 16 (MOI =2.5, 9 hpi)

0

0.5

1

1.5

2

2.5

3

Mock N A D QRel

ati

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am

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of

mR

NA

(wt)

0

0.5

1

1.5

2

2.5

3

Mock N A D QRel

ati

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of

vRN

A

(wt)


A. mRNA B. vRNA

0

0.5

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2

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3

Mock D N E VRel

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mR

NA

(wt)

0

0.5

1

1.5

2

2.5

3

Mock D N E V

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vRN

A

(wt)


A. mRNA B. vRNA

0

0.5

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2

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3

Mock N I D Q

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vRN

A

(wt)

0

0.5

1

1.5

2

2.5

3

Mock N I D QRel

ati

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of

mR

NA

(wt)

Reviewed by Nagata K, et al

(2008)

Influenza A cRNA Influenza B virus cRNA

Influenza A vRNA

Influenza B vRNA

5’ AGU

A A

G A

AC

C G A G

3’ UC

G

U

C U

U

C

G

C C U C

A ………..

………..

.

......

5’ AGC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......

5’ AGU

A A

G A

AC

A G G C

3’ UC

G

U

U U

U

C

G

U C C G

A ………..

………..

.

......

5’ AGC

A A

G A

GC

G G A G

3’ UC

A

U

C U

U

U

G

U G C U C

………..

………..

.

......

Function of the influenza virus components

Segment Encoded polypeptide

Remarks

1 PB2 -Cap binding

-Component of RNA transcriptase complex

2 PB1 -Catalyzes nucleotide addition

-Component of RNA transcription, and replication complex

3 PA Endonuclease activity, component of RNA transcriptase and replicase complex

4 HA -Major surface glycoprotein

-Receptor (sialic acid) binding

-Proteolytic cleavage activation

5 NP -Monomer binds to RNA to form coiled ribonucleoprotein

6 NA Surface glycoprotein; neuramidase activity

7 M1 Major protein of virion

M2 Ion channel activity essential for virus uncoating

8 NS1 -Inhibits cellular premRNA 3’ end cleavage and polyadenylation; inhibits pre-mRNA splicing

NS2 -Interacts with M1 and involved in nuclear export of RNPs

Side group of amino acidsQ: CH2: this structure or length of side group may

affect

least sandpiper

This species has greenish legs and a short thin dark bill

Influenza A virus H4N8 (A/least sandpiper/South Central)

http://en.wikipedia.org/wiki/File:Least_Sandpiper_Don_Edwards_WR_1.jpg

1918, Spanish influenza (H1N1): 40-50 million people (Taubenberger et al, 2006)

1957, Asian influenza (H2N2): ~2 million people ( WHO, 2005)

1968, Hong Kong influenza (H3N2): ~1 million people ( WHO, 2005)

2009, influenza pandemic (H1N1): ~ 18,500 people (Dawood et al, 2012)

2013, a new avian influenza A virus in China, 135 patients, including 44 deaths ( (H7N9) ) (WHO, August 12th, 2013)

Seasonal influenza virus infections in humans cause annual epidemics, leading to millions of human infections worldwide and having significant health and economic burdens; influenza pandemics can also have devastating effects globally, resulting in millions of deaths.

Influenza virus research plays an important role for protection of public health as well as economic developments globally. Also important to development of new vaccines and anti-influenza drugs.

Significance of viral research

A/Narita/98 (H9N2)






*::** :**:.** * ********** **********:*:*** ***:**

A/Narita/98 (H9N2)






*::** :**:.** * ********** **********:*:*** ***:**

Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters (1-83 a.a.) among influenza A and influenza B viruses

16 27 44

D

QQ

DN

EMutation

D and E (2 types of negative charge)

N and Q (Structure of N is close to that of Q, Q stimulate RNA synthesis)

Acidic amino acid D at the position 27 was substituted by uncharged polar amino acid N like H4N8 strain

N

N

N

R

NH2

O

N N

H2N

OR

N N

O

OR

N

N

N

R

H2N

O

Ribavirin

Ribavirin

Cytidine

Uridine

H

Ribavirin is an ambigously hydrogen-bonding purine mimic

Ribavirin is an inhibitor of inosine monophosphate dehydrogenase

(IMPDH)

Ribavirin RMP IMP

XMP Succinyl AMP

GMP

GDP

GTP

RNA synthesis

AMP

ADP

ATP

IMPDH

RDP

RTP

Adenosine kinase

Undefined kinase

Undefined kinase

Termination

An alternative substrate for the RNA polymerase and cause chain termination

5’ AGC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......5’ A

GC

A A

A A

GC

A G G C

3’ UC

A

U

C U

U

U

G

U U C C G

………..

………..

.

......

………..

………..

.

......

R

Interference of RTP (ribavirin triphosphate)

on viral RNA polymerase

The nucleotide recognition mechanism of influenza virus RNA polymerase is unknown. Therefore, isolation of the ribavirin-resistant PB1 mutants is useful to know the function of the viral RNA polymerase.

Structure of PB1 subunit

Whether these mutations affect the assembly of the polymerase or not

PB1 NH2 COOH

1 757 a.a.

NLS



A/Narita/98 (H9N2)






*::** :**:.** * ********** **********:*:*** ***:**

A/Narita/98 (H9N2)






*::** :**:.** * ********** **********:*:*** ***:**


1-15

Homology 85% 50~60%

RNA polymerase complexes in the supernatant fraction

Method 3. Immunoprecipitation

MDCK cells

Infection with WSN (wild type or mutant), MOI=1

Cell lysate by sonication

7 h post infection

Immunoprecipitation with rabbit anti-PB2 antibody

Western blotting analysis

presenter:nguyen trong binh

Documents

vrna synthesis

synthesis of mrna

viral rna polymerase

crna promoters

amino acid positions

affect crnamrna synthesis

b viruses homology

pa binding site1