Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,
Post on 27-Mar-2015
214 Views
Preview:
Transcript
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.
SiRNA
miRNA
BMJ 2004;328:1245–48
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)
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
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
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
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
• 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
miRNA
miRNA
Approximately 60%of miRNAs are expressed independently, 15%of miRNAs are expressed in clusters, and 25% are in introns.
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
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
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
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
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
• miRNA against G-mRNA and against negative strand viral genome are not effective
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
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
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
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
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
Can RNA interference be used for street rabies?
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
HEP-Flury –GFP
(from Dr. Morimoto)
control dsRNA
miRNA-N
Still reasonably effective against targets that have 2-3 nucleotides different from designed targets
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
Conclusions
miRNA can inhibit (at least) some strains of virus that contain different nucleotide(s) at miRNA target site
Acknowledgements
T. HEMACHUDHA
N. RATANASETYUTH
P.SUPAVONWONG
P. VIROJANAPIROM
P. KHAWPLOD
BIOTEC Thailand
top related