L13 - RNAi and viruses Flashcards

Intended Learning Outcomes • Describe the concept of RNA-interference. • Describe the roles of the endonucleases DICER and Argonaute-2. • Explain how the RISC (RNA-induced Silencing Complex) complex inhibits virus replication in plants and insects. • Explain the role of viral encoded suppressors of RNAi (VSRs) in viral infection. • Discuss the role of RNAi in the regulation and inhibition of virus replication in mammalian cells.

1
Q

What are the key RNAi molecules involved in gene silencing?

A

dsRNA, shRNA, siRNA, and miRNA.

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2
Q

What is the main function of siRNA?

A

It guides the RNA-induced silencing complex (RISC) to degrade complementary mRNA.

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3
Q

What enzyme processes dsRNA into siRNA?

A

Dicer.

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4
Q

What is the function of the RISC complex?

A

It silences gene expression by degrading target mRNA.

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5
Q

How does R2D2 assist in RISC loading?

A

It binds the more stable end of siRNA, recruiting Dicer-2.

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6
Q

What happens to the passenger strand of siRNA?

A

It is destroyed during RISC activation.

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7
Q

What is the function of the PIWI domain in Ago-2?

A

It cleaves RNA in a sequence-specific manner.

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8
Q

How does Ago-2 contribute to RNAi?

A

It binds siRNA and guides mRNA degradation.

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9
Q

What type of RNA does the PIWI domain act on?

A

Single-stranded RNA (ssRNA) guided by siRNA.

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10
Q

What determines whether RISC degrades mRNA or suppresses translation?

A

The complementarity between siRNA and mRNA.

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11
Q

What happens if siRNA has a perfect match to its target?

A

The mRNA is cleaved and degraded.

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12
Q

What happens if siRNA has a bulged mismatch?

A

It results in translational suppression rather than cleavage.

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13
Q

How is RNAi amplified in plants and nematodes?

A

Through RNA-dependent RNA polymerase (RdRP).

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14
Q

What does RdRP do?

A

It synthesizes secondary siRNAs from target mRNAs, enhancing RNAi effects.

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15
Q

Why is this amplification significant?

A

It strengthens and prolongs the RNAi response against viruses.

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16
Q

What type of genome does Tombusvirus have?

A

A single-stranded RNA genome (~4800 bases).

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17
Q

How is Tombusvirus transmitted?

A

Through direct sap contact and contaminated soil.

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18
Q

What is a key feature of its replication strategy?

A

Its genome can be directly translated into proteins.

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19
Q

What is p19, and why is it important for Tombusvirus?

A

It is a viral suppressor of RNAi (VSR) that binds siRNA to prevent degradation.

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20
Q

How does p19 recognize siRNA?

A

It specifically binds 21-nucleotide dsRNA fragments.

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21
Q

What is the structural feature of p19?

A

It acts like a caliper, measuring and holding dsRNA.

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22
Q

What happens when p19 is deleted?

A

The virus is controlled by RNAi, leading to reduced spread and degradation.

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23
Q

What are the effects of a p19-deficient virus in tobacco plants?

A

No systemic symptoms and limited viral replication.

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24
Q

What does this indicate about p19’s role?

A

It is crucial for viral evasion of RNAi defenses.

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25
Q

How does RNAi function as an antiviral defense?

A

It degrades viral RNA via siRNA-guided RISC activity.

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26
Q

What type of viruses commonly encode VSRs?

A

Plant and insect viruses, and possibly some mammalian viruses.

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27
Q

How do plant virus VSRs interact with mammalian cells?

A

Some can still exhibit RNAi suppression activity.

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28
Q

What is the debate regarding VSRs in mammalian cells?

A

Whether they suppress RNAi or are actually involved in interferon (IFN) suppression.

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29
Q

Why is the IFN response significant in mammals?

A

It is the primary antiviral defense system instead of RNAi.

30
Q

What is a key question about VSRs in mammalian viruses?

A

Researchers are investigating whether VSRs actively suppress RNAi in mammalian cells or if their effects are related to interferon suppression.

31
Q

What is HEV71, and why is its 3A protein important?

A

HEV71 is a human enterovirus, and 3A is a VSR.

32
Q

What happens when HEV71 lacks 3A?

A

It induces viral siRNA (viRNA) production, leading to viral RNA degradation.

33
Q

How can a VSR-deficient HEV71 be rescued?

A

By knocking out Dicer, preventing siRNA processing.

34
Q

How does Zika virus (ZIKV) interact with the RNAi system?

A

ZIKV-derived viRNAs are detected in human neural progenitor cells (hNPCs) but not in differentiated neurons.

35
Q

What does this suggest about RNAi in mammals?

A

It may function in embryonic cells but not in mature somatic cells.

36
Q

What happens when key RNAi proteins (Dicer, Ago2) are knocked down?

A

ZIKV infection increases in hNPCs, confirming an antiviral role for RNAi.

37
Q

What did Shabihah & Ding (2021) discover?

A

Mammalian embryonic stem cells have a specialized Dicer isoform for viRNA production.

38
Q

Why is Shabihah & Ding discovery important?

A

It suggests that RNAi plays a role in antiviral defense in embryonic cells.

39
Q

What remains unclear?

A

Whether this function is retained in differentiated mammalian cells.

40
Q

What is RNA interference (RNAi)?

A

RNAi is a biological process where double-stranded RNA (dsRNA) induces sequence-specific degradation of target RNA, silencing specific genes.

41
Q

When was RNAi first discovered, and what recognition did it receive?

A

RNAi was first noted in plants and later identified in animals, earning the Nobel Prize in 2006.

42
Q

What are the key components of the RNAi mechanism?

A

The key components are Dicer, which processes dsRNA into siRNAs, and Argonaute proteins, which form the RNA-induced silencing complex (RISC) to guide silencing.

43
Q

How does RNAi act as a defence against viral infections?

A

RNAi targets and degrades viral RNA, preventing viral replication.

44
Q

In which organisms is RNAi a key antiviral defence?

A

RNAi is crucial in plants and insects but has a debated role in mammals.

45
Q

How do viruses evade RNAi-mediated defences?

A

Viruses encode viral suppressors of RNAi (VSRs) to counteract RNAi and enhance their replication.

46
Q

What host factors contribute to the RNAi response?

A

Host factors include lipid-modifying and membrane-shaping proteins that assist in viral replication structure formation.

47
Q

How do plant viruses counteract RNAi?

A

Plant viruses produce VSR proteins that suppress RNAi, allowing the virus to evade plant defences.

48
Q

What evidence exists for RNAi-based antiviral defences in insects?

A

Studies on viruses like flock house virus in Drosophila show that VSRs prevent RNAi activation, balancing viral replication and host defence.

49
Q

Why is the role of RNAi in mammalian antiviral defence debated?

A

Mammals primarily rely on the type I interferon response, though RNAi may still play a role, especially in embryonic stem cells.

50
Q

How can the RNAi response be amplified?

A

Some organisms have mechanisms that enhance RNAi, increasing its effectiveness against viral infections.

51
Q

What is the relationship between RNAi and interferon responses in mammals?

A

There is a complex interplay between RNAi and type I interferon responses, requiring further research to understand their full impact on viral infections.

52
Q

Why is studying RNAi important for antiviral strategies?

A

Understanding the balance between host defences and viral suppressors could lead to new antiviral therapies.

53
Q

What is a key area of future research in RNAi?

A

Investigating RNAi’s role in mammals and its potential therapeutic applications.

54
Q

What are virus replication factories?

A

Specialized intracellular compartments that create a favourable environment for viral replication while evading host defences.

55
Q

Why do positive-sense RNA viruses alter host cellular architecture?

A

They replicate in association with cellular membranes, modifying host structures to support their replication.

56
Q

What are the two main mechanisms of bacterial entry into host cells?

A

The trigger mechanism (dramatic membrane reorganisation) and the zipper mechanism (subtle surface modifications).

57
Q

How do viruses manipulate the actin cytoskeleton?

A

By using host GTPases (e.g., Rho, Rac, Cdc42) to generate membrane ruffles, aiding viral entry.

58
Q

How do bacterial proteins contribute to viral entry?

A

They mimic host factors to modulate cytoskeletal pathways and create replication niches.

59
Q

What are the limitations of traditional transmission electron microscopy in viral imaging?

A

Sample preparation and resolution constraints.

60
Q

What advanced imaging techniques allow for high-resolution study of viral replication structures?

A

Cryo-electron tomography and focused ion beam milling.

61
Q

What are double-membrane vesicles, and why are they important for RNA virus replication?

A

Virus-induced organelle-like structures that protect viral RNA replication from host defences.

62
Q

What have recent imaging studies revealed about replication factories?

A

Specific viral and host protein interactions crucial for viral replication.

63
Q

How do researchers identify host factors essential for viral replication?

A

Using siRNA libraries to knock down host proteins and observe changes in the viral lifecycle.

64
Q

What are some common host factors involved in viral replication?

A

RNA-binding proteins, chaperones, membrane trafficking proteins, and phosphoinositide-associated proteins.

65
Q

How does poliovirus hijack host cellular pathways for replication?

A

It disrupts the ER-Golgi network via the 3A protein, altering host recycling pathways.

66
Q

What replication strategies does SARS-CoV-2 use?

A

It forms double-membrane vesicles and viral factories, using unique viral proteins to evade host defences.

67
Q

How does hepatitis C virus create replication structures?

A

It forms membranous webs via the NS5A protein, modifying host membranes for enhanced replication and evasion of degradation.

68
Q

How do RNA viruses exploit host cellular processes?

A

They use sophisticated strategies to hijack host pathways, ensuring efficient replication while avoiding immune defences.

69
Q

How have recent imaging advancements contributed to virology?

A

They provide detailed insights into viral replication complexes, improving our understanding of virus-host interactions.

70
Q

Why is understanding viral replication structures important?

A

It helps identify potential therapeutic targets for antiviral treatments.