Genetic analyses of the function of PB1 subunit of the influenza virus RNA-dependent RNA polymerase Presenter: Nguyen Trong Binh
Dec 30, 2015
Genetic analyses of the function of PB1 subunit of the influenza virus RNA-dependent RNA polymerase
Presenter: Nguyen Trong Binh
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
RNA polymeraseconsensus motif
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
Putative cRNA promoter binding
Putative vRNA promoter binding
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)
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
*::** :**:.** * ********** **********:*:*** ***:**
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
mock wt N16A D27V N44I
Rel
ati
ve
am
ou
nt
of
cRN
A
0
0.5
1
1.5
2
2.5
3
3.5
4
mock wt N16A D27V N44I
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.
Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters among influenza A and B viruses
16 27 44PA binding site
1-15
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
*::** :**:.** * ********** **********:*:*** ***:**
MDVNPTLLFLKVPAQNAISTTFPYTGNPPYSHGTGTGYTMDTVNRTHQYS 50 A/Lsp/SC Alaska (H4N8)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
*::** :**:.** * ********** **********:*:*** ***:**
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)
A. mRNA C. vRNA B. cRNA
****
*
Student’s t test (*, P < 0.05; **, P < 0.01)
Binh et al, 2013. Frontiers in Virology
Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters among influenza A and B viruses
16 27 44PA binding site
1-15
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
*::** :**:.** * ********** **********:*:*** ***:**
MDVNPTLLFLKVPAQNAISTTFPYTGNPPYSHGTGTGYTMDTVNRTHQYS 50 A/Lsp/SC Alaska (H4N8)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
*::** :**:.** * ********** **********:*:*** ***:**
MDVNPTLLFLKVPAQNAISTTFPYTGNPPYSHGTGTGYTMDTVNRTHQYS 50 A/Lsp/SC Alaska (H4N8)
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)
The viral RNA synthesis of mutant viruses at the position 27
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)
A. mRNA C. vRNA B. cRNA
**** **
***
*
Student’s t test (*, P < 0.05; **, P < 0.01)
Binh et al, 2013. Frontiers in Virology
Alignment of amino acid sequences of putative RNA binding region common to vRNA and cRNA promoters among influenza A and B viruses
16 27 44PA binding site
1-15
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
*::** :**:.** * ********** **********:*:*** ***:**
MDVNPTLLFLKVPAQNAISTTFPYTGNPPYSHGTGTGYTMDTVNRTHQYS 50 A/Lsp/SC Alaska (H4N8)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
*::** :**:.** * ********** **********:*:*** ***:**
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 44
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
**
** ** **
Student’s t test (*, P < 0.05; **, P < 0.01)
Binh et al, 2013. Frontiers in Virology
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
mock wt N16A D27V N44I
mR
NA
/vR
NA
Binh et al, 2013. Frontiers in Virology
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
RNA polymeraseconsensus motif
NH2 COOH
1 757 a.a.
NLS
Putative cRNA promoter binding
Putative vRNA promoter binding
Putative nucleotide binding
Ribavirin
Promoter
Stimulation
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
Putative cRNA promoter binding
Putative vRNA promoter binding
Putative nucleotide binding
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
Putative cRNA promoter binding
Putative vRNA promoter binding
Putative nucleotide binding
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
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
B. cRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
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
C. vRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
vRN
A
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
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
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mock
wt
N16D
N16QN16A
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
mock
wt
N16D
N16QN16A
A. mRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mock
wt
N16D
N16QN16A
mock
wt
N16D
N16QN16A
B. cRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
B. cRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
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
C. vRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
vRN
A
The viral RNA synthesis of mutant viruses at the position 16 (3 and 6 hpi)
C. vRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
vRN
A
B. cRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
A. mRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mockwtD27ED27ND27V
C. vRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
vRN
A
C. vRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
vRN
A
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
vRN
A
B. cRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
B. cRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
A. mRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mockwtD27ED27ND27V
A. mRNA
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mockwtD27ED27ND27V
0
0.5
1
1.5
2
2.5
3
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mockwtD27ED27ND27V
mockwtD27ED27ND27V
The viral RNA synthesis of mutant viruses at the position 27 (3 and 6 hpi)
A. mRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mock
wt
N44I
N44D
N44Q
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
mock
wt
N44I
N44D
N44Q
A. mRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mock
wt
N44I
N44D
N44Q
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
mR
NA
mock
wt
N44I
N44D
N44Q
mock
wt
N44I
N44D
N44Q
B. cRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
B. cRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
cRN
A
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
C. vRNA
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
vRN
A
0
0.5
1
1.5
2
3 6
hpi
Rel
ativ
e am
ou
nt
of
vRN
A
The viral RNA synthesis of mutant viruses at the position 44 (3 and 6 hpi)
0
1
2
3
4
5
6
3h 6h 9h
hpi
Re
lati
ve
am
ou
nt
of
mR
NA
Mock
WT-R
WT+R
D27N-R
D27N+R
0
0.2
0.4
0.6
0.8
1
1.2
3h 6h 9h
hpi
Re
lati
ve
am
ou
nt
of
mR
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.
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)
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
ati
ve
am
ou
nt
of
mR
NA
0
0.5
1
1.5
2
2.5
3
Mock wt N16A D27V N44I
Rel
ati
ve
am
ou
nt
of
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
ve
am
ou
nt
of
mR
NA
(wt)
0
0.5
1
1.5
2
2.5
3
Mock N A D QRel
ati
ve
am
ou
nt
of
vRN
A
(wt)
The effect on the primary transcription activity of mutant viruses at the position 27 (MOI =2.5, 9 hpi)
A. mRNA B. vRNA
0
0.5
1
1.5
2
2.5
3
Mock D N E VRel
ati
ve
am
ou
nt
of
mR
NA
(wt)
0
0.5
1
1.5
2
2.5
3
Mock D N E V
Rel
ati
ve
am
ou
nt
of
vRN
A
(wt)
The effect on the primary transcription activity of mutant viruses at the position 44 (MOI =2.5, 9 hpi)
A. mRNA B. vRNA
0
0.5
1
1.5
2
2.5
3
Mock N I D Q
Rel
ati
ve
am
ou
nt
of
vRN
A
(wt)
0
0.5
1
1.5
2
2.5
3
Mock N I D QRel
ati
ve
am
ou
nt
of
mR
NA
(wt)
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
least sandpiper
This species has greenish legs and a short thin dark bill
Influenza A virus H4N8 (A/least sandpiper/South Central)
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/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)
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
*::** :**:.** * ********** **********:*:*** ***:**
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
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
Putative cRNA promoter binding
Putative vRNA promoter binding
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)
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
*::** :**:.** * ********** **********:*:*** ***:**
16 27 44PA binding site
1-15
Homology 85% 50~60%