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.
2
Q
What is the main function of siRNA?
A
It guides the RNA-induced silencing complex (RISC) to degrade complementary mRNA.
3
Q
What enzyme processes dsRNA into siRNA?
A
Dicer.
4
Q
What is the function of the RISC complex?
A
It silences gene expression by degrading target mRNA.
5
Q
How does R2D2 assist in RISC loading?
A
It binds the more stable end of siRNA, recruiting Dicer-2.
6
Q
What happens to the passenger strand of siRNA?
A
It is destroyed during RISC activation.
7
Q
What is the function of the PIWI domain in Ago-2?
A
It cleaves RNA in a sequence-specific manner.
8
Q
How does Ago-2 contribute to RNAi?
A
It binds siRNA and guides mRNA degradation.
9
Q
What type of RNA does the PIWI domain act on?
A
Single-stranded RNA (ssRNA) guided by siRNA.
10
Q
What determines whether RISC degrades mRNA or suppresses translation?
A
The complementarity between siRNA and mRNA.
11
Q
What happens if siRNA has a perfect match to its target?
A
The mRNA is cleaved and degraded.
12
Q
What happens if siRNA has a bulged mismatch?
A
It results in translational suppression rather than cleavage.
13
Q
How is RNAi amplified in plants and nematodes?
A
Through RNA-dependent RNA polymerase (RdRP).
14
Q
What does RdRP do?
A
It synthesizes secondary siRNAs from target mRNAs, enhancing RNAi effects.
15
Q
Why is this amplification significant?
A
It strengthens and prolongs the RNAi response against viruses.
16
Q
What type of genome does Tombusvirus have?
A
A single-stranded RNA genome (~4800 bases).
17
Q
How is Tombusvirus transmitted?
A
Through direct sap contact and contaminated soil.
18
Q
What is a key feature of its replication strategy?
A
Its genome can be directly translated into proteins.
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.
20
Q
How does p19 recognize siRNA?
A
It specifically binds 21-nucleotide dsRNA fragments.
21
Q
What is the structural feature of p19?
A
It acts like a caliper, measuring and holding dsRNA.
22
Q
What happens when p19 is deleted?
A
The virus is controlled by RNAi, leading to reduced spread and degradation.
23
Q
What are the effects of a p19-deficient virus in tobacco plants?
A
No systemic symptoms and limited viral replication.
24
Q
What does this indicate about p19’s role?
A
It is crucial for viral evasion of RNAi defenses.
25
How does RNAi function as an antiviral defense?
It degrades viral RNA via siRNA-guided RISC activity.
26
What type of viruses commonly encode VSRs?
Plant and insect viruses, and possibly some mammalian viruses.
27
How do plant virus VSRs interact with mammalian cells?
Some can still exhibit RNAi suppression activity.
28
What is the debate regarding VSRs in mammalian cells?
Whether they suppress RNAi or are actually involved in interferon (IFN) suppression.
29
Why is the IFN response significant in mammals?
It is the primary antiviral defense system instead of RNAi.
30
What is a key question about VSRs in mammalian viruses?
Researchers are investigating whether VSRs actively suppress RNAi in mammalian cells or if their effects are related to interferon suppression.
31
What is HEV71, and why is its 3A protein important?
HEV71 is a human enterovirus, and 3A is a VSR.
32
What happens when HEV71 lacks 3A?
It induces viral siRNA (viRNA) production, leading to viral RNA degradation.
33
How can a VSR-deficient HEV71 be rescued?
By knocking out Dicer, preventing siRNA processing.
34
How does Zika virus (ZIKV) interact with the RNAi system?
ZIKV-derived viRNAs are detected in human neural progenitor cells (hNPCs) but not in differentiated neurons.
35
What does this suggest about RNAi in mammals?
It may function in embryonic cells but not in mature somatic cells.
36
What happens when key RNAi proteins (Dicer, Ago2) are knocked down?
ZIKV infection increases in hNPCs, confirming an antiviral role for RNAi.
37
What did Shabihah & Ding (2021) discover?
Mammalian embryonic stem cells have a specialized Dicer isoform for viRNA production.
38
Why is Shabihah & Ding discovery important?
It suggests that RNAi plays a role in antiviral defense in embryonic cells.
39
What remains unclear?
Whether this function is retained in differentiated mammalian cells.
40
What is RNA interference (RNAi)?
RNAi is a biological process where double-stranded RNA (dsRNA) induces sequence-specific degradation of target RNA, silencing specific genes.
41
When was RNAi first discovered, and what recognition did it receive?
RNAi was first noted in plants and later identified in animals, earning the Nobel Prize in 2006.
42
What are the key components of the RNAi mechanism?
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
How does RNAi act as a defence against viral infections?
RNAi targets and degrades viral RNA, preventing viral replication.
44
In which organisms is RNAi a key antiviral defence?
RNAi is crucial in plants and insects but has a debated role in mammals.
45
How do viruses evade RNAi-mediated defences?
Viruses encode viral suppressors of RNAi (VSRs) to counteract RNAi and enhance their replication.
46
What host factors contribute to the RNAi response?
Host factors include lipid-modifying and membrane-shaping proteins that assist in viral replication structure formation.
47
How do plant viruses counteract RNAi?
Plant viruses produce VSR proteins that suppress RNAi, allowing the virus to evade plant defences.
48
What evidence exists for RNAi-based antiviral defences in insects?
Studies on viruses like flock house virus in Drosophila show that VSRs prevent RNAi activation, balancing viral replication and host defence.
49
Why is the role of RNAi in mammalian antiviral defence debated?
Mammals primarily rely on the type I interferon response, though RNAi may still play a role, especially in embryonic stem cells.
50
How can the RNAi response be amplified?
Some organisms have mechanisms that enhance RNAi, increasing its effectiveness against viral infections.
51
What is the relationship between RNAi and interferon responses in mammals?
There is a complex interplay between RNAi and type I interferon responses, requiring further research to understand their full impact on viral infections.
52
Why is studying RNAi important for antiviral strategies?
Understanding the balance between host defences and viral suppressors could lead to new antiviral therapies.
53
What is a key area of future research in RNAi?
Investigating RNAi's role in mammals and its potential therapeutic applications.
54
What are virus replication factories?
Specialized intracellular compartments that create a favourable environment for viral replication while evading host defences.
55
Why do positive-sense RNA viruses alter host cellular architecture?
They replicate in association with cellular membranes, modifying host structures to support their replication.
56
What are the two main mechanisms of bacterial entry into host cells?
The trigger mechanism (dramatic membrane reorganisation) and the zipper mechanism (subtle surface modifications).
57
How do viruses manipulate the actin cytoskeleton?
By using host GTPases (e.g., Rho, Rac, Cdc42) to generate membrane ruffles, aiding viral entry.
58
How do bacterial proteins contribute to viral entry?
They mimic host factors to modulate cytoskeletal pathways and create replication niches.
59
What are the limitations of traditional transmission electron microscopy in viral imaging?
Sample preparation and resolution constraints.
60
What advanced imaging techniques allow for high-resolution study of viral replication structures?
Cryo-electron tomography and focused ion beam milling.
61
What are double-membrane vesicles, and why are they important for RNA virus replication?
Virus-induced organelle-like structures that protect viral RNA replication from host defences.
62
What have recent imaging studies revealed about replication factories?
Specific viral and host protein interactions crucial for viral replication.
63
How do researchers identify host factors essential for viral replication?
Using siRNA libraries to knock down host proteins and observe changes in the viral lifecycle.
64
What are some common host factors involved in viral replication?
RNA-binding proteins, chaperones, membrane trafficking proteins, and phosphoinositide-associated proteins.
65
How does poliovirus hijack host cellular pathways for replication?
It disrupts the ER-Golgi network via the 3A protein, altering host recycling pathways.
66
What replication strategies does SARS-CoV-2 use?
It forms double-membrane vesicles and viral factories, using unique viral proteins to evade host defences.
67
How does hepatitis C virus create replication structures?
It forms membranous webs via the NS5A protein, modifying host membranes for enhanced replication and evasion of degradation.
68
How do RNA viruses exploit host cellular processes?
They use sophisticated strategies to hijack host pathways, ensuring efficient replication while avoiding immune defences.
69
How have recent imaging advancements contributed to virology?
They provide detailed insights into viral replication complexes, improving our understanding of virus-host interactions.
70
Why is understanding viral replication structures important?
It helps identify potential therapeutic targets for antiviral treatments.
71
What are the two key enzymes involved in the RNAi pathway?
Dicer and Argonaute (particularly Ago2) are the key enzymes. Dicer processes dsRNA into siRNAs, while Argonaute incorporates siRNA into RISC and mediates mRNA degradation or translation suppression.
72
What is the function of Drosha in RNAi?
Drosha is a nuclear RNase III enzyme that cleaves precursor microRNAs (pri-miRNAs) into hairpin-shaped pre-miRNAs before export to the cytoplasm for further processing by Dicer.
73
What is the difference between the antiviral response in embryonic stem cells and differentiated mammalian cells?
Embryonic stem cells have a limited interferon response and rely more on RNAi, whereas differentiated cells predominantly use the interferon system for antiviral defense.
74
How do viruses use host microRNA machinery for their own benefit?
Viruses such as HCV and Epstein-Barr virus exploit host or encode viral microRNAs to stabilize their genomes, enhance replication, or regulate host gene expression.
75
What role does RNAi play in embryonic stem cells during viral infection?
RNAi provides a crucial antiviral defense in embryonic stem cells, especially due to the limited function of the interferon system in these cells.
76
How do viruses like Flock House Virus regulate their replication through VSRs?
FHV uses B2 protein to suppress RNAi by binding siRNA and interacting with viral RdRP to reduce replication, balancing replication and immune evasion.
77
What is the functional significance of alternate splicing of Dicer in embryonic stem cells?
Alternate splicing produces a shortened, more antiviral Dicer isoform in embryonic stem cells, enhancing siRNA production and antiviral defense.
78
What evidence suggests RNAi suppressors may exist in mammalian viruses?
Proteins like HEV 3A have shown suppression of Dicer activity, and introducing mutations in such VSRs leads to increased siRNA production and viral RNA degradation.
79
How does the RNAi response differ between progenitor and differentiated neural cells in response to Zika virus?
Human neural progenitor cells produce 21–22 nt viRNAs in response to ZIKV, indicating an active RNAi response, which is absent in differentiated neurons.
80
How can siRNA profiles be used as evidence for antiviral RNAi activity?
Detection of 21–22 nt siRNAs mapping to both strands of viral genomes (e.g., ZIKV or Sindbis virus) indicates active Dicer processing and RNAi engagement.
