Lecture 16 - Nucleic Acid-Activated Viral Defence Pathways Flashcards
How can ssRNA fold?
By having self-complementary sections (self-annealing)
How can ssRNA form structures even when it hasn't got self-complementary sections? 1) 2) 3) 4) 5)
1) Wobble G-U pairs
2) Sheared G-A pairs
3) U - H20 - C pairs
4) Reverse Hoogsteen AU
5) Base triples
Base triples
Where 3 G bind together
RNA structure that can cleave RNA
P4-P6 ribozyme
Chordate response to detection of viral nucleic acids
Protein-mediated response, leading to IFN expression
Invertebrate response to detection of viral nucleic acids
Use RNA PAMPs to directly generate virus-interfering RNAs (viRNAs) which can be loaded into an RNA-induced silencing complex to mediate silencing of viral RNA
How are viral dsRNAs degraded for loading into RISC?
By dicer family proteins
viRNA
Virus-derived interfering RNA
Which organisms use viRNA to protect against viral infection?
Arthropods, nematodes, plants
miRNA
22bp imperfectly-duplexed RNA that acts on eukaryotic host mRNA to affect host protein levels.
Expressed by host
Micro RNA
Generally inhibits mRNA transcription, but sometimes can activate. Degrades target mRNA when there is a high level of duplex.
Is siRNA naturally-occurring in humans?
No
shRNA
Short-hairpin RNA.
Artificial, designed to be a dicer substrate that produces a functional siRNA
How was a plant immune system first observed?
Plant infected with tobacco ringspot virus showed a decline in symptoms on the upper leaves of the plant (further from the site of infection)
Plant immune system
Post-transcriptional gene silencing (RNA-based)
Invertebrate immune system
RNA interference
How does RNA-induced silencing complex (RISC) interfere with viral RNA? 1) 2) 3) 4) 5)
1) Viral RDRP makes dsRNA in cytoplasm
2) Dicer and RNase III generate small RNAs from dsRNA genome
3) Strand of dsRNA with the less tightly-bound 5’ end is loaded into RISC as the guide strand of RNA. TRBP associates with dicer, loads guide strand.
4) Guide strand binds to mRNA, RISC cleaves RNA. Ago2 is the endonuclease in RISC
5) Guide strand is released, can complex again with RISC
Common pathway of genetic immunity in plants and invertebrates 1) 2) 3) 4)
1) Production of 21 nucleotide siRNA.
2) Contains a perfectly-duplexed 19bp double helix.
3) 3’ 2-nucleotide single-stranded tail.
4) This is a siRNA, which binds to complementary RNA transcripts
What triggers RNA silencing pathways?
21-27 nucleotide small RNAs
Does RNAi induction from long dsRNA occur in chordates?
No
pri-mRNA
80 nucleotide RNA stem-loop structure.
Used to form miRNA.
Drosha
Protein in cells that recognises pri-mRNA hairpin loop structure and cleaves it from surrounding RNA sequence.
This sequence goes on to form miRNA
shRNA
Short hairpin RNA
RNA-induced transcriptional silencing
Epigenetic method of controlling gene activity.
siRNA guides methylation of DNA, heterochromatin formation and silencing of genes.
Name for process of epigenetically-silencing DNA genes using siRNA
RNA-induced transcriptional silencing
Method of silencing DNA viruses
RNA-induced transcriptional silencing.
Use siRNA to induce epigenetic silencing of DNA (heterochromatin formation)
Example of dsRNA immunity in mammals
dsRNA activates PKR and OAS (formation of 2’5’ adenylic acid, RNase L activation)
Do mammals detect long or short dsRNA for immunity?
Long.
Imperfectly-complexed short RNAs
miRNAs
Function of mammalian miRNAs
Arrest mRNA translation.
When there’s a high level of duplex, degrades target mRNA.
Can maintain viral latency.
What is miRNA?
22bp imperfectly-duplexed RNA
Precursor of miRNA
priRNA
Why is establishing the target of miRNA hard?
G-U wobble pairing
When does mRNA degradation occur with siRNA?
When a high level of duplex is formed with siRNA
Viruses that encode siRNAs
DNA viruses.
Herpes encodes a lot.
Virus that humans encode a microRNA against
Primate foamy virus (retrovirus)
Virus that uses human miRNA to its adavantage
Hepatitis C
Why might hep C be restricted to hepatocytes?
Hepatocytes express mir-122 miRNA, which enhances hep C protein expression
Adenoviral RNA that affects host
VA1
Adenoviral VA1 effects
1)
2)
3)
1) Inhibits PKR action
2) Blocks nuclear export of pre-miRNAs
3) Inhibits dicer
SV40 miRNA use
Targets T proteins to promote switch from early to late gene expression and reduce change of CTL detection and host-cell killing
HIV TAT protein
1)
2)
3)
1) Inhibits the TAR-RNA binding protein (TRBP), a dicer cofactor
2) Inhibits PKR
3) Translational enhancer of HIV transcripts
Host factor needed for function of VA1
RNA polymerase III
APOBEC3 effect on HIV
Incorporated into virion, induces lethal G->A mutations into DNA after reverse-transcription
Which HIV protein inhibits APOBEC3?
vif
Ways that RNA silencing can be induced in plants and invertebrates
1)
2)
3)
1) Long viral dsRNA
2) Self mRNA transcripts with a short hairpin loop (shRNA)
3) Self mRNA transcripts with a stem-loop, imperfectly-duplexed structure (miRNA)
