Inhibition of rabies virus replication by microRNA N.ISRASENA, N. RATANASETYUTH, P.SUPAVONWONG, P. VIROJANAPIROM, T.HEMACHUDHA Department of Pharmacology,

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