Title: RNA interference of influenza virus production by directly targeting mRNA for degradation and indire
1RNA interference of influenza virus production by
directly targeting mRNA for degradation and
indirectly inhibiting all viral RNA transcription
- Qing Ge, Michael T. McManus, Tam Nguyen,
Ching-Hung Shen, Phillip A. Sharp, Herman N.
Eisen, and Jianzhu Chen - Contributed December 23, 2002
- Published March 4, 2003
- Presented by Kelly McCoy
2Why bother using RNAi to inhibit influenza
production?
- 10-20 of the U.S. population is infected each
year resulting in 40,000 deaths - Influenza is easily spread
- Antigenic drift (changes in HA and NA- viral
antigens) prevents immunity - Antigenic shift (mixing of viruses from 2
different species) creates new strains that also
prevent immunity
3Brief Review of Influenza A
- 8 segments of ssRNA- Segments 1-6 each encode 1
protein, 7 8 each encode 2 proteins - PB2, PB1, and PA are components of the RNA
transcriptase - HA, NA, and NP are the major glycoproteins
- Segment 7 encodes M1 and M2 and Segment 8 encodes
NS1 and NS2
4Brief Review of Influenza A
- Hemagglutinin (HA) is a viral protein anchored
in the lipid bilayer that recognizes and binds to
host cells sialic acid
Neuraminidase (NA) is a viral protein anchored in
the lipid bilayer that after infection, cleaves
off sialic acid from the host cell to prevent
recapturing of the newly created virions
5Designing siRNAs specific for Influenza A virus
- Remember RNAi uses dsRNA to direct sequence-
specific degradation of mRNA - Search for conserved sequences of 21 nucleotides
(any longer sequence would trigger an interferon
response) - There are 15 HA subtypes and 9 NA subtypes, none
of which have 21 ntide conserved sequences - Thus, Ge et al. designed 20 different siRNAs
specific for NP, PA, PB1, PB2, M, and NS genes
and tested them
6Testing for Influenza Virus Production
- 2 testing methods were used
- Inhibition of Flu production in cell lines
- Madin-Darby canine kidney (MDCK) cells were used
- siRNAs were introduced into the cell and then
either A/PR/8/34 (PR8) virus WSN/33 (WSN) virus - Inhibition of Flu production in Embryonated
Chicken Eggs - 10-day-old embryonated chicken eggs were used
- PR8 virus was introduced alone or in conjunction
with the siRNAs
7Inhibition of Influenza Production in Cell Lines
- Through electroporation, the 20 different siRNAs
were introduced into the MDCK cells. 8 hours
later, PR8 or WSN virus was added to the cell
with a multiplicity of infection (moi) of 0.001,
0.01, or 0.1. - Controls
- 1. GFP-949 (siRNA specific for GFP) was
introduced into MDCK cells expressing
GFP. Later the cells were infected by the
viruses. - 2. Mock transfection The virus was
introduced into cells with no siRNA - Using an HA assay, the virus titer was determined
at different times after the infection for the
controls and the tested cells
8Results of siRNAs in Cell Lines infected with
Influenza
- Figure A graphically represents the time vs. the
virus amount for both PR8 and WSN virus - Mock transfection virus titers increased over
time - GFP-949 did not affect virus production this
means that siRNA does NOT interfere
nonspecifically with flu virus production
9Results of siRNAs in Cell Lines infected with
Influenza
- Together, Figures A and D show 3 different types
of results - Approx. 45 of the siRNAs had no effect on the
virus titer - Approx. 40 of the siRNAs significantly inhibited
virus production - Approx. 15 of the siRNAs potently inhibited
production - NP-1496 and PA-2087 produced no detectable HA
activity
10Results of siRNAs in Cell Lines infected with
Influenza
- Figure B shows the potentcy of of siRNA- the
virus titer was determined when MDCK cells were
transfected with different concentrations of
NP-1496 siRNA - As the amount of siRNA decreased, virus titer
increased but still very potent
11Results of siRNAs in Cell Lines infected with
Influenza
- Figure C shows that the procedure also works when
reversed - First the MDCK cells were infected by the virus
and then by the siRNA - Virus titer increased steadily with mock
transfection but NP-1496 siRNA worked to keep the
virus titer levels low
12Summary of Cell Line Results
- Certain siRNAs potently inhibit flu production in
MDCK cells - Influenza production is inhibited by siRNAs
specific for different viral genes (notably NP,
PA, and PB1) - siRNA works in cells with ongoing infection
13Testing for Influenza Virus Production
- 2 testing methods were used
- Inhibition of Flu production in cell lines
- Madin-Darby canine kidney (MDCK) cells were used
- siRNAs were introduced into the cell and then
either A/PR/8/34 (PR8) virus WSN/33 (WSN) virus - Inhibition of Flu production in Embryonated
Chicken Eggs - 10-day-old embryonated chicken eggs were used
- PR8 virus was introduced alone or in conjunction
with the siRNAs
14Inhibition of Influenza Production in Embryonated
Chicken Eggs
- PR8 virus was injected into 10-day-old
embryonated chicken eggs either alone or in
conjunction with siRNA - 17 hours later, virus titers were measured
- The controls used were the same as the ones
testing the Cell lines- mock transfection (no
siRNA) and GFP-949
15Effects of siRNAs on Virus titer
- Mock transfection and GFP-949 had no effect on
virus production - The same siRNAs that potently inhibited virus
production in MDCK cells (PB1-2257, PA-2087,
NP-1496) considerably reduced virus titers in
chicken embryos
16What is most likely the direct target of RNAi?
- Figure A illustrates Influenzas negative
polarity - The vRNA is copied into both cRNA (for
replication purposes) and mRNA (to synthesize
viral proteins) - Thus what is the target of siRNAmRNA, vRNA, or
cRNA?
17What is most likely the direct target of RNAi?
- Figure B show the effects on inhibition of virus
production by modifying either the sense or
antisense strands of the siRNA - -Antisense siRNA strand is
- complementary to mRNA
- and cRNA
- -Sense siRNA strand is
- complementary to vRNA
- Inhibition requires a wt antisense strand in
the siRNA- thus the target is either mRNA, cRNA
or both
18What is most likely the direct target of RNAi?
- Figure C distinguishes between the 3 RNAs
- MDCK cells were transfected with M-37 siRNA and
PR8 - RNA was isolated 1,2, and 3 hours later and
quantified by RT (using primer shown in fig. A)
and real-time PCR - Gamma actin was used to normalize the data
- The 50 reduction in mRNA in the presence of
siRNA indicates that mRNA is the target
19Some siRNAs Inhibit Accumulation of All Viral RNAs
- The same procedure was performed except that
NP-1496 siRNA was used rather than M-37 siRNA
20Some siRNAs Inhibit Accumulation of All Viral RNAs
- Figure A shows that 3 hours after infection, NP
mRNA was detected only in the absence of NP-1496 - But, NP-specific vRNA and cRNA were also
inhibited in the presence of NP-1496 - Figures B and C show that NP-specific siRNA
inhibits the accumulation of M- and NS- specific
mRNA, vRNA, and cRNA - Therefore, depending on their sequence and
specificity, some siRNAs have a global affect
21Some siRNAs Inhibit Accumulation of All Viral RNAs
- There are 2 possible causes for this global
inhibition of viral RNAs - An interferon response is triggered by the
presence of dsRNA - -The same experiments were carried out
in Vero cells (cells - with the entire interferon locus
deleted) and the results are - shown in figure D
- -These graphs show that the response is
the same with or - without interferon present PLUS
22Some siRNAs Inhibit Accumulation of All Viral RNAs
- The levels of transcripts from cellular genes was
also assayed in the presence of siRNA - The figure shows the level of NP (high after 3
hours with no NP-1496) compared to a ribosomal
L32 gene (no difference is detected in the
presence or absence of siRNA) - Therefore, the broad inhibition of viral RNA is
not due to a cellular interferon response
23Some siRNAs Inhibit Accumulation of All Viral RNAs
- 2. The inhibition of viral RNA is a result of
enhanced RNA degradation - -The presence of dsRNA triggers a
pathway that targets RNA for degradation. A major
component of this pathway, PKR, was assayed first
for cells with NP-1496 and no virus infection- no
effect . Then PKR was assayed in cells infected
with the virus. Virus levels increased both with
and without NP-1496 - -Therefore the increased level of RNA
degradation is not the reason for the RNA
inhibition
24Summary of Important Findings
- siRNAs potently inhibit influenza virus
production in both cell lines and embryonated
chicken eggs - siRNAs that target NP and PA are the most
effective - Some siRNAs exert their inhibitory effect by
interfering with the accumulation of mRNA AND
other viral RNAs (I.e. NP and PA are required for
virus replication and translation) - Viral mRNA is the direct target of siRNA-mediated
interference
25Implications
- The result of these analyses provide the
beginning of exploration of siRNAs possible role
in an eventual therapy to treat influenza - Epithelial cells of the respiratory tract are
affected by the virus and therefore siRNAs could
possibly be administered intranasally or
pulmonarally
26Sources
- Ge, Qing et al. RNA interference of influenza
virus production by directly targeting mRNA for
degradation and indirectly inhibiting all viral
RNA transcription PNAS March 4, 2003 vol. 100
no. 5 p. 2718-2723 - http//www.psc.edu/science/Herlocher/Herlocher.htm
l - http//microvet.arizona.edu/Courses/MIC419web/Case
4flu.html - Levine, Arnold J. Viruses 1992, New York p.
155-171 -