RNA interference of influenza virus production by directly targeting mRNA for degradation and indire

1
RNA 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

2
Why 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

3
Brief 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

4
Brief 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
5
Designing 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

6
Testing 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

• Fifth level

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Inhibition 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

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Results 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

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Results 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

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Results 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

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Results 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

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Summary 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

13
Testing 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

• Fifth level

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Inhibition 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

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Effects 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

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What 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?

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What 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

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What 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

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Some siRNAs Inhibit Accumulation of All Viral RNAs

• The same procedure was performed except that
  NP-1496 siRNA was used rather than M-37 siRNA

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Some 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

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Some 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

22
Some 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

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Some 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

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Summary 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

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Implications

• 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

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Sources

• 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