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Frontiers in infectious diseases > 13. RNAi and viruses > Flashcards

13. RNAi and viruses Flashcards

1
Q

What is RNAi an essential form of viral defence in?

A
  1. Vertebrates
  2. Plants
  3. Insects
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2
Q

What types of RNAs can induce an RNAi response?

A
  1. dsRNA
  2. Short hairpin RNA
  3. Imported siRNA
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3
Q

What is siRNA?

A
  1. Short interfering RNA
  2. It is derived from an infecting virus
  3. It inhibits expression of 1 specific virus.
  4. Cleaves miRNA
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4
Q

What is miRNA?

A
  1. Micro RNA
  2. Produced from the host cell genome
  3. Regulates gene expression of multiple mRNAs
  4. Inhibits translation.
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5
Q

What are the key steps in the RNAi response?

A
  1. Dicer detects and cleaves up long dsDNA to produce siRNAs.
  2. siRNAs are then loaded into the RNA induced silencing complex (RISC).
  3. This leads to cleavage or degradation of viral RNA molecules and suppression of translation.
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6
Q

What is dicer?

A
  1. A key molecule involved in small RNA biogenesis.
  2. It is an RNA endonuclease (RNase).
  3. It can cleave siRNA and miRNA.
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7
Q

How is miRNA generated?

A
  1. miRNA is produced in all mammalian cells to regulate gene expression.
  2. It is produced as a hairpin and first cleaved by Drosha in the nucleus which produces 3’ overhangs.
  3. the miRNA is then exported to the cytoplasm and cleaved by dicer.
  4. This dicer cleavage generates the characteristic 22 nucleotide long miRNA.
  5. miRNA also have 2 nt 3’ overhangs which can bind to PAZ domains.
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8
Q

How is siRNA generated?

A
  1. Viral RNA like dsRNA or short hairpin RNA is introduced to the cell.
  2. Dicer cleaves the long RNAs to produce siRNA.
  3. These can then be loaded into the RISC complex.
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9
Q

What domains do dicer and drosha contain?

A
  1. Helicase binding domain to recruit helicases and unwind RNA.
  2. RNA binding domain to bind to dsRNA.
  3. PAZ domain which recognises and binds 3’ overhangs on siRNAs.
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10
Q

How does Dicer cleave dsRNA?

A
  1. It binds to to dsRNA through its binding domain.
  2. It recognises specific sequences for cleavage.
  3. This leads to specific RNA fragments generated due to the shape of the enzyme and interactions.
  4. These RNA fragments are always 22 nucleotides long.
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11
Q

How can tell if there is dicer activity in a cell?

A
  1. If you isolate and sequence all the RNA in the cell, you can tell the length of the fragments.
  2. If there is a massive abundance of 22nt long RNA fragments, you know there is dicer activity.
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12
Q

How does the RISC complex assemble using siRNAs?

A
  1. Dicer and argonaut 2 are the key molecules in this pathway.
  2. siRNAs are generated from viral RNA by dicer.
  3. R2D2 binds to the 5’ end of the siRNA and stabilise the molecule.
  4. Other enzymes also can modify the RNA at this stage
  5. Ago-2 recruits the siRNA to the RISC complex.
  6. Ago-2 also unwinds the RNA into the separate passenger and guide RNA strands.
  7. The passenger strand is destroyed.
  8. The RISC complex is then fully activated wih a guide RNA which can bind foreign RNA that is invading the cell.
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13
Q

What is the function of RNAi, siRNA and RISC complex?

A

To identify and destroy foreign RNA to prevent infection.

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14
Q

What is the structure of Ago-2?

A
  1. PIWI domain is an endonuclease that cleaves RNA.
  2. PAZ domain to bind to the 3’ end of the guide RNA
  3. These work together to recognise and bind viral RNA then cleave it.
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15
Q

How can siRNAs be used in experimental systems?

A
  1. They can be used to silence gene expression of specific genes.
  2. We can introduce siRNAs to destroy mRNAs to knock down gene expression.
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16
Q

How does the RISC complex cleave foreign RNA?

A
  1. RISC binds to ssRNA like mRNA through complementary base pairing between the guide RNA and target RNA.
  2. If the binding is totally complementary this results in cleavage and degradation.
  3. Non total complementary binding leads to translational suppression similar to miRNA system.
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17
Q

How can the RNAi response be amplified?

A

Plants and worms can generate RNA-dependent RNA polymerase that functions in the siRNA pathway.

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18
Q

How is the RNAi response amplified in plants?

A
  1. The primary cleavage by dicer produces siRNAs.
  2. RISC complex cleavage and formation then binding to target ssRNA.
  3. This target ssRNA is then made dsRNA by RNA-dependent RNA polymerase.
  4. this dsRNA is then used to generate a 2nd round of siRNAs and amplify the response.
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19
Q

What are the key points of the amplification of the RNAi response?

A
  1. Dicer cleaves the dsRNA to make siRNA.
  2. Formation of the RISC complex by Ago-2.
  3. A 2nd wave of siRNAs generated by RNA-dependent RNA polymerase.
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20
Q

How is the RNAi response amplified in worms?

A
  1. Worms don’t need the initial dicer cleavage to generates the RISC complex.
  2. RISC just binds to the target RNA
  3. The polymerase extends the RNA to make short dsRNA that then enters Ago-2.
21
Q

What organisms is the RNAi response essential for?

A
  1. It is important in viral response in plants, arthropods and nematodes.
  2. dsRNAs are produced in viral infection which triggers RISC complex formation.
  3. This primes the organism to prevent viral infection.
22
Q

Do mammals use the RNAi response?

A
  1. Mammals have all the components for siRNA recognition and defence.
  2. However they also have the more evolved type 1 interferon system that detects dsRNA and triggers a systemic interferon response through TLRs and RLR.
  3. When 1 cell is targeted it can protect the rest of the organism
23
Q

Is RNAi a systemic response?

A
  1. Yes
  2. The siRNA response has been shown to prevent viruses spreading systemically around plants.
24
Q

Can viruses suppress the RNAi response?

A
  1. Yes
  2. Even simple viruses can encode proteins that can suppress the RNAi response.
25
Q

What is Tombusvirus?

A
  1. A simple ssRNA virus with only 4 proteins.
  2. Infects orchids, pepper, basil, crops and tobacco.
  3. Causes systemic infection and its lethal in 2 weeks.
  4. Transmitted through direct contact with sap or contaminated soil.
  5. It contains a multifunctional p19 protein which can suppress the RNAi response.
26
Q

What is a VSR?

A

a viral suppressor of RNAi

27
Q

How does the tombusvirus p19 protein suppress the RNAi response?

A
  1. It is made in large quantities.
  2. It binds dsRNA very tightly.
  3. It is specific for 21nt RNA fragments that are produced by the RNAi response.
  4. This means they bind to siRNAs.
  5. This prevents siRNAs from being loaded into the RISC complex and no RISC complex is formed.
  6. This promotes viral infection.
28
Q

Does p19 always suppress the RNAi response?

A
  1. Yes but at different levels.
  2. There is a balance between levels of dicer, p19 and siRNA production.
  3. It also depends on the type of plant and virus?
29
Q

Is suppression of RNAi essential for successful viral infection?

A
  1. RNAi is a very important response in plants and other organisms.
  2. When p19 is deleted from tombusvirus the RNAi response can control the infection completely.
  3. This shows viruses need to control RNAi for successful infection.
  4. This is also important for infecting mammals but they also have the type 1 interferon response.
30
Q

How does flock house virus control the RNAi response from insects?

A
  1. It produces a protein called B2.
  2. B2 can bind to siRNAs and prevent them being loaded into the RISC complex.
  3. B2 can also bind its own RNA-dependent RNA polymerase to down regulate RNA production to avoid detection.
31
Q

How does flock house virus normally infect insect cells?

A
  1. Viral RNA is injected into the cell.
  2. Formation of antigenomic RNA. This is dsRNA which triggers the RNAi response. Most of the dsRNA is hidden by the replication structure.
  3. Some cleavage of this dsRNA by dicer and loaded into the RISC comple.
  4. This leads to clearance of ssRNA in the cells and protects the wider organism.
32
Q

What parts of the viral genome generate siRNA?

A
  1. Study done in Aedes aegyptii.
  2. showed siRNA was produced from the whole viral genome
  3. When B2 (VSR) was added no siRNAs were produced.
33
Q

What does suppression of the RNAi response lead to?

A
  1. Reduction of the immune response
  2. The virus killing the insect
34
Q

Why is too much suppression of RNAi bad for the virus?

A
  1. The virus doesn’t want to kill the insect as this is bad for transmission.
  2. The virus wants the insect to survive as long as they can so the virus can be transmitted onto humans and spread.
  3. Then both the virus and the insect survives.
  4. This is why balance is needed between suppressing the RNAi response and the virus.
  5. Only suppression enough to make sure the host doesn’t die from the repsonse.
35
Q

Are VSRs expressed by all viruses?

A
  1. Many plant viruses and insect viruses express VSR.
  2. Some mammalian viruses can produce VSRs.
  3. Some plant virus VSRs can exhibit activity in mammalian cells.
36
Q

Are human virus VSR specific for inhibiting the RNAi response?

A
  1. Many human virus VSR bind to dsRNA so they could be inhibiting the RNAi response.
  2. But it is more likely they are inhibiting the type1 interferon response.3
  3. We cannot prove which it is specific for.
37
Q

What VSR activity does human enterovirus 71 do?

A
  1. HEV71 encodes protein 3A which is a viral suppressor of RNAi.
  2. VSR-deficient HEV71 induces a viral siRNA production in somatic cells.
  3. HEV71-derived siRNAs load into Ago and degrades HEV71 RNA.
  4. This shows HEV71’s VSR inhibits dicer and the RNAi response in order to infect human cells.
38
Q

How are mammalian miRNAs used by Hepatitis C?

A
  1. miRNAs are used by lots of viruses in different ways.
  2. Hepatitis C virus uses a liver specific miRNA that is essential for HCV replication.
  3. HCV binds the miRNA to prevent degradation and enhance replication.
  4. RNAi is important for protection but viruses can use our machinery to generate miRNAs for their benefit.
39
Q

How can miRNAs used by DNA viruses?

A
  1. Many DNA viruses like EBV can encode their own miRNAs.
  2. They are used to control their gene expression and host cell gene expression.
40
Q

What are siRNAs also known as?

41
Q

Do mammalian cells use the RNAi response and generate siRNAs?

A
  1. Mammalian viral VSRs are non-specific for the RNAi response and are more likely related to the type 1 interferon response.
  2. Mammalian cells lack RNA-dependent RNA polymerases to be able to amplify the siRNA so it is not as effective as it could be in plants.
  3. Mammals have the conserved cellular machinery to generate siRNA.
  4. But this mechanisms is not used in somatic cells due to the IFN response.
  5. Evidence shows the RNAi response is used in embryonic stem cells.
42
Q

How is the interferon response in somatic cells different to the interferon response in embryonic stem cells?

A
  1. Embryonic stem cells (ESCs) show reduced levels of induction of the interferon respnse.
  2. They have constitutive expression of parts of the interferon response.
  3. This is due to the fact ESC are more precious cells then somatic cells so require more protection.
  4. The RNAi response is also very important in these cells.
43
Q

How is the balance between the interferon response and the RNAi response struck in embryonic stem cells?

A
  1. Viruses suppress the RNAi response.
  2. The IFN response can also suppress the RNAi response.
  3. The IFN response recognises larger dsRNA if these were cleaved up they wouldn’t be recognised by the IFN response.
  4. So suppressing the RNAi response benefits the IFN response.
  5. In most cells the interferon response dominates for viral protection.
44
Q

Is the mammalian siRNA response only operative in embryonic stem cells?

A
  1. In neural progenitor cells, you get an effective RNAi response.
  2. This is shown by a massive increase in 22nt RNAs in these cells.
  3. Differentiated cells show no specific RNAs generated so a lack of RNAi response.
  4. Knock outs of the RNAi response showed zika virus replication in ESCs.
  5. This was shown in knock outs of Dicer and Ago2.
  6. This suggest that the RNAi response is very important in protecting against viral infection.
45
Q

What else do human embryonic stem cells have to protect themselves from viruses?

A

Anti-viral dicer

46
Q

What is anti-viral dicer and its role in protecting embryonic stem cells?

A
  1. Anti-viral dicer is generated through alternative splicing and is only found in embryonic stem cells.
  2. It is missing the Helicase 2i domain.
  3. It is much more efficient at cleavage then normal dicer and is produced much more abundantly.
  4. Some IFN genes are expressed but the RNAi response is the most important.
47
Q

How can the level and type RNAi response be measured?

A

High throughput sequencing

48
Q

What 3 observations indicate that RNAi is the major antiviral mechanism in plants and invertebrate?

A
  1. Viral infections in these organisms lead to accumulation of dicer dependent virus derived siRNA.
  2. Inactivation of key components of the RNAi pathway results in an increase in viral load in infected cells.
  3. Many plant and insect viruses encode viral suppressors of RNAi that interfere with distinct steps in the RNAi pathway.

Frontiers in infectious diseases (19 decks)

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