inhibition of rabies virus replication by microrna n.israsena, n. ratanasetyuth, p.supavonwong, p....
TRANSCRIPT
![Page 1: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/1.jpg)
Inhibition of rabies virus Inhibition of rabies virus replication by microRNAreplication by microRNA
N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA
Department of Pharmacology, Chulalongkorn University Hospital.Queen Saovabha Memorial Institute, Thai Red Cross Society, Bangkok, Thailand.Department of Medicine, Chulalongkorn University Hospital.
![Page 2: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/2.jpg)
SiRNA
miRNA
![Page 3: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/3.jpg)
BMJ 2004;328:1245–48
![Page 4: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/4.jpg)
![Page 5: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/5.jpg)
Targets for gene silencing
Host proteins Leader RNA (Yang et al 1998,1999) Viral proteins
N ( Wunner et al., 1991, Wu et al 2002) P (Morimoto et al. 2005, Blondel et al.,2006) L (Tordo et al., 1998) G M (Finke,Conzelmann 2003)
Viral genome (Barik 2002)
![Page 6: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/6.jpg)
wash
Plating 70% conf
12 hours
Plasmid siRNA virus
transfection
12 hours
infection
8 hours
24 hours 24 hours 24 hours
2 viral titers
Cellular RNA Cellular RNA
Supernatant
IF
Western blot
Cellular RNA
Supernatant
Western blotReal-time PCR
![Page 7: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/7.jpg)
Pcmv 10-4 24 hr in cell
Viral mRNA &GAPDH in neuro-2a cells 24 hours after challenged with rabies virus
0
20
40
60
80
100
120
ps17 ps49 ns45
rela
tive
am
ou
nt
of
mR
NA
N mRNA
GmRNA
GAPDH
Viral genome in neuro-2a cells 24 hours after challenged with virus
100.0
11.417.7
70.7
0.0
20.0
40.0
60.0
80.0
100.0
120.0
control PS17 PS49 NS45
Rel
ativ
e am
ou
nt
of
RN
A
We tested
5 anti-N
2 anti-leader
and
3 anti negative strands
![Page 8: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/8.jpg)
Viral genome in supernatant 48 hours after challenged with rabies virus
100.0
7.7
63.1
38.0
0.0
20.0
40.0
60.0
80.0
100.0
120.0
control ps17 ps49 ns45
Rel
ativ
e am
ou
nt
of
RN
A
SiRNA PS17CONTROL
Control and transfected neuro-2a cells that express the PS17 siRNA were infected with CVS rabies virus for 48 h before they were fixed in acetone and stained with a FITC-labeled anti-N monoclonal antibodies
![Page 9: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/9.jpg)
Conclusions from siRNA studies
• Specific viral mRNA and viral replication can be reduced by siRNA
• Inhibition of leader RNA and negative strand RNA is less effective than inhibiting N mRNA
• All methods that we tested (22 nt siRNA, PCR-based, vector with pol II or pol III promoter ) normally yield only 70% reduction of viral protein mRNA and viral genome
• Stable NA cell line expressing anti-N siRNA slow the growth rate of the virus
![Page 10: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/10.jpg)
• the siRNA is extremely stringent in its specificity such that a single nucleotide mismatch may abrogate its function
To develop therapeutic tool- Need method that is more effective and less stringent
![Page 11: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/11.jpg)
miRNA
![Page 12: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/12.jpg)
miRNA
Approximately 60%of miRNAs are expressed independently, 15%of miRNAs are expressed in clusters, and 25% are in introns.
![Page 13: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/13.jpg)
miRNA - 24 hr in cell
Viral mRNA&GAPDH in neuro-2a cells 24 after challeged with virus
5.9 5.1 4.85.3 2.8 4.4
95.9
79.0
122.3
0.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0
mi169 mi379 mi859
Rel
ativ
e am
ou
nt
of
RN
A
NmRNA
GmRNA
GAPDH
Viral genome in neuro-2a cells 24 hours after challenged with rabies virus
100.0
1.49.9
2.50.0
20.0
40.0
60.0
80.0
100.0
120.0
control mi169 mi379 mi859
Rel
ativ
e am
ou
nt
of
RN
A
All anti-N miRNA tested can suppress 85- 95% of viral transcription
Not significantly affect house keeper gene
Can suppress 85- 98 % of viral genome production
![Page 14: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/14.jpg)
Viral genome in the supernatant 48 hours after challenged with rabies virus
100.00
0.45 0.98 0.340.00
20.00
40.00
60.00
80.00
100.00
120.00
control mi169 mi379 mi859
Rel
ativ
e am
ou
nt
of
RN
A
85 % to 99 % suppression of virus production as measured in supernate
![Page 15: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/15.jpg)
Stained with anti G Ab Stained with anti N Ab
miRNA-NP1
Control neuro-2a
IF 48 hours after challenged with same amount of CVS virus
![Page 16: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/16.jpg)
0
20
40
60
80
100
120
control G1 G2 G3
miRNA against G mRNA
Viral genome in cells 24 hours after infection
Rel
ativ
e am
ount
of
vira
l RN
A
![Page 17: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/17.jpg)
0 20 40 60 80 100 120
Control
NN1
NN2
NP1
NP1+NN1
miRNA against viral genome
Relative amount of viral RNA in neuro-2a 72 hours after infection
![Page 18: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/18.jpg)
• miRNA against G-mRNA and against negative strand viral genome are not effective
![Page 19: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/19.jpg)
Target 1
Target 2
Target 3
Are 3 copies of same miRNA more effective?
Is miRNA against 3 different targets more effective?
When N protein is inhibited, is the viral genome more vulnerable to miRNA?
Does incorporation of ineffective miRNA to the complex reduce its activities?
เ
3 targets -> more chances of counter genetic variability and mutation
![Page 20: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/20.jpg)
0
20
40
60
80
100
120
Control NP1 NP1+NP1+NP1
Effect of expressing more than one miRNA as a chain
Viral RNA in supernatant 48 hours after infection
Rel
ativ
e am
ount
of
vira
l RN
A
![Page 21: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/21.jpg)
0
20
40
60
80
100
120
Control NP1 NP1+NP1+rev NP1+NP2+NP3
NmRNA
GmRNA
Effect of expressing more than one miRNA as a chain
Relative amount of RNA in cell 24 hours after infection
![Page 22: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/22.jpg)
0 20 40 60 80 100 120
NP1+rev+rev
NP1+NP1+rev
1NP1
control
fect o
Effect of expressing effective miRNA together with ineffective miRNA
Relative amount of viral RNA in neuro-2a cells 72 hours after challenged with CVS
![Page 23: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/23.jpg)
100.0
30.835.4
2.4 1.1
Control NN1 NN2 NP1+NP2+NP3 NP1+NP2+NN1
Chaining miRNA against viral genome with miRNA against NmRNA
Viral RNA in supernatant 72 hours after infection
Rel
ativ
e am
ount
of
vira
l RN
A
![Page 24: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/24.jpg)
Can RNA interference be used for street rabies?
![Page 25: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/25.jpg)
0 20 40 60 80 100 120
ds-mock
dsRNA-N
miRNA-negativecontrol
miRNA N 3-plus
miRNA N 2-plus 1-minus
Effect of dsRNA and miRNA (designed against CVS virus)
on street virus NmRNA in neuro-2a cells 24 hours after infection
85 % reduction of viral transcription
![Page 26: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/26.jpg)
HEP-Flury –GFP
(from Dr. Morimoto)
control dsRNA
miRNA-N
Still reasonably effective against targets that have 2-3 nucleotides different from designed targets
![Page 27: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/27.jpg)
Conclusions
miRNA against N-mRNA is effective in reducing both transcription and replication
miRNA against viral genome alone is not effective
Expressing multiple miRNA as a single premiRNA transcript is possible and will not significantly reduce effectiveness of individual miRNA
![Page 28: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/28.jpg)
Conclusions
miRNA can inhibit (at least) some strains of virus that contain different nucleotide(s) at miRNA target site
![Page 29: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/29.jpg)
Acknowledgements
T. HEMACHUDHA
N. RATANASETYUTH
P.SUPAVONWONG
P. VIROJANAPIROM
P. KHAWPLOD
BIOTEC Thailand
![Page 30: Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,](https://reader035.vdocuments.net/reader035/viewer/2022070305/55153bf155034673228b5c40/html5/thumbnails/30.jpg)