Lecture 16 - Nucleic Acid-Activated Viral Defence Pathways

Question 1

How can ssRNA fold?

Answer 1

By having self-complementary sections (self-annealing)

Question 2

How can ssRNA form structures even when it hasn't got self-complementary sections?
1)
2)
3)
4)
5)

Answer 2

1) Wobble G-U pairs
2) Sheared G-A pairs
3) U - H20 - C pairs
4) Reverse Hoogsteen AU
5) Base triples

Question 3

Base triples

Answer 3

Where 3 G bind together

Question 4

RNA structure that can cleave RNA

Answer 4

P4-P6 ribozyme

Question 5

Chordate response to detection of viral nucleic acids

Answer 5

Protein-mediated response, leading to IFN expression

Question 6

Invertebrate response to detection of viral nucleic acids

Answer 6

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

Question 7

How are viral dsRNAs degraded for loading into RISC?

Answer 7

By dicer family proteins

Question 8

viRNA

Answer 8

Virus-derived interfering RNA

Question 9

Which organisms use viRNA to protect against viral infection?

Answer 9

Arthropods, nematodes, plants

Question 10

miRNA

Answer 10

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.

Question 11

Is siRNA naturally-occurring in humans?

Answer 11

No

Question 12

shRNA

Answer 12

Short-hairpin RNA.

Artificial, designed to be a dicer substrate that produces a functional siRNA

Question 13

How was a plant immune system first observed?

Answer 13

Plant infected with tobacco ringspot virus showed a decline in symptoms on the upper leaves of the plant (further from the site of infection)

Question 14

Plant immune system

Answer 14

Post-transcriptional gene silencing (RNA-based)

Question 15

Invertebrate immune system

Answer 15

RNA interference

Question 16

How does RNA-induced silencing complex (RISC) interfere with viral RNA?
1)
2)
3)
4)
5)

Answer 16

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

Question 17

Common pathway of genetic immunity in plants and invertebrates
1)
2)
3)
4)

Answer 17

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

Question 18

What triggers RNA silencing pathways?

Answer 18

21-27 nucleotide small RNAs

Question 19

Does RNAi induction from long dsRNA occur in chordates?

Answer 19

No

Question 20

pri-mRNA

Answer 20

80 nucleotide RNA stem-loop structure.

Used to form miRNA.

Question 21

Drosha

Answer 21

Protein in cells that recognises pri-mRNA hairpin loop structure and cleaves it from surrounding RNA sequence.

This sequence goes on to form miRNA

Question 22

shRNA

Answer 22

Short hairpin RNA

Question 23

RNA-induced transcriptional silencing

Answer 23

Epigenetic method of controlling gene activity.

siRNA guides methylation of DNA, heterochromatin formation and silencing of genes.

Question 24

Name for process of epigenetically-silencing DNA genes using siRNA

Answer 24

RNA-induced transcriptional silencing

Question 25

Method of silencing DNA viruses

Answer 25

RNA-induced transcriptional silencing. | Use siRNA to induce epigenetic silencing of DNA (heterochromatin formation)

Question 26

Example of dsRNA immunity in mammals

Answer 26

dsRNA activates PKR and OAS (formation of 2'5' adenylic acid, RNase L activation)

Question 27

Do mammals detect long or short dsRNA for immunity?

Answer 27

Long.

Question 28

Imperfectly-complexed short RNAs

Answer 28

miRNAs

Question 29

Function of mammalian miRNAs

Answer 29

Arrest mRNA translation. When there's a high level of duplex, degrades target mRNA. Can maintain viral latency.

Question 30

What is miRNA?

Answer 30

22bp imperfectly-duplexed RNA

Question 31

Precursor of miRNA

Answer 31

priRNA

Question 32

Why is establishing the target of miRNA hard?

Answer 32

G-U wobble pairing

Question 33

When does mRNA degradation occur with siRNA?

Answer 33

When a high level of duplex is formed with siRNA

Question 34

Viruses that encode siRNAs

Answer 34

DNA viruses. | Herpes encodes a lot.

Question 35

Virus that humans encode a microRNA against

Answer 35

Primate foamy virus (retrovirus)

Question 36

Virus that uses human miRNA to its adavantage

Answer 36

Hepatitis C

Question 37

Why might hep C be restricted to hepatocytes?

Answer 37

Hepatocytes express mir-122 miRNA, which enhances hep C protein expression

Question 38

Adenoviral RNA that affects host

Answer 38

VA1

Question 39

Adenoviral VA1 effects 1) 2) 3)

Answer 39

1) Inhibits PKR action 2) Blocks nuclear export of pre-miRNAs 3) Inhibits dicer

Question 40

SV40 miRNA use

Answer 40

Targets T proteins to promote switch from early to late gene expression and reduce change of CTL detection and host-cell killing

Question 41

HIV TAT protein 1) 2) 3)

Answer 41

1) Inhibits the TAR-RNA binding protein (TRBP), a dicer cofactor 2) Inhibits PKR 3) Translational enhancer of HIV transcripts

Question 42

Host factor needed for function of VA1

Answer 42

RNA polymerase III

Question 43

APOBEC3 effect on HIV

Answer 43

Incorporated into virion, induces lethal G->A mutations into DNA after reverse-transcription

Question 44

Which HIV protein inhibits APOBEC3?

Answer 44

vif

Question 45

Ways that RNA silencing can be induced in plants and invertebrates 1) 2) 3)

Answer 45

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)