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LF213: Virology > Lecture 3. Introduction to Viral Replication - Influenza Virus Replication 2 > Flashcards

Lecture 3. Introduction to Viral Replication - Influenza Virus Replication 2 Flashcards

1
Q

When haemagglutinin is synthesised in an infected cell, what is it synthesised as?

A

Single point peptide with two subunits

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2
Q

Why is the cleavage of HA into two subunits required?

A

Required for pH-dependent conformational change that releases the fusion peptide

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3
Q

What cleaves the HA into two subunits?

A

Host proteases (acidification does not cause cleavage)

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4
Q

What are the three stages in influenza virus mRNA synthesis?

A

Cleavage (cap-snatching)
Initiation
Elongation

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5
Q

What happens in the cleavage stage of influenza virus mRNA synthesis?

A

More viral proteins need to be created in the genome segments for the life cycle to continue
Viral polymerase cleaves mRNA 10-13 nucleotides down from the cap and uses it as a primer to initiate synthesis of own mRNA

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6
Q

What happens in the initiation stage of influenza virus mRNA synthesis?

A

Influenza uses it’s -ve sense genome strand as a template, adding nucleotides that are complimentary to the the -ve sense strand to the primer

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7
Q

What happens in the elongation stage of influenza virus mRNA synthesis?

A

Complimentary nucleotides bind to the primer 10-13 nucleotides down from the cap

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8
Q

What does the polymerase basic protein 1 (PB1) contain?

A

Both binding sites for the viral genome RNA (5’ and 3’)
Polymerase active site

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9
Q

What does the polymerase basic protein 2 (PB2) contain?

A

Capped binding site

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10
Q

What is the full process of influenza virus mRNA synthesis?

A
  1. Nucleoprotein must be associated with the genome RNA
  2. Polymerase basic protein 1 (PB1) binds 5’ end of genome
  3. PB2 binds cap of host mRNA
  4. PB1 binds the 3’ end of genome and aligns this with the host mRNA at position 10-13
  5. If alignment is correct PA cleaves the mRNA
  6. PB1 elongates the mRNA using the short capped primer
  7. Polyadenylation occurs 17-22 bases from the 5’ end of the genome at a poly U tract
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11
Q

What does viral mRNA synthesis depend on?

A

Cellular mRNA synthesis by RNA Pol II, to supply capped primers

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12
Q

In influenza, where is viral mRNA synthesised from?

A

All 8 vRNP segments

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13
Q

In what direction do we write -ve sense?

A

From 3’ to 5’ (as opposed to the usual 5’ to 3’)

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14
Q

What are the differences in the -ve sense virion RNA when it becomes +ve sense mRNA?

A

Original sequence at 3’ end is replaced by a poly A tail (AAAA…)
Addition of a cap 10-13 nucleotides per cellular message at 5’ end

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15
Q

What is the antigenome (cRNA) of virion RNA?

A

An exact copy of the virion RNA but +ve sense instead of -ve sense (in influenza)

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16
Q

What are the qualities of cRNA?

A

cRNA is neither capped nor polyadenylated
cRNA (and vRNA) synthesis requires viral protein synthesis

17
Q

What is the role of cRNA?

A

To act as a template to make more copies of the virion RNA

18
Q

Why is cRNA unstable and how can it become stable?

A

Unstable due to no capping and not being polyadenylated
Becomes stable when wrapped up in nucleoprotein

19
Q

In influenza, how much RNA is mRNA and how much is cRNA?

A

mRNA = 95%
cRNA = 5%

20
Q

When is cRNA not degraded?

A

When it is encapsidated into a cRNP by Pol and NP

21
Q

What does nucleoprotein binding to cRNA allow?

A

Anti-termination at the poly(A) site

22
Q

What does cRNA not promote?

A

Cap-primed initiation and no poly(A) site - only vRNA is made

23
Q

What is the timeline of influenza RNA synthesis?

A
  1. mRNA is synthesised, exported to cytoplasm
  2. Viral proteins synthesised; Pol and NP back into nucleus
  3. cRNA stabilised as cRNP
  4. Many copies of vRNA can be made from each cRNP
  5. vRNA stabilised as vRNP, exported to cytoplasm for assembly
24
Q

What does segment 2 encode and how?

A

PB1 (subunit of polymerase); PB1-F2 (virulence factor)
Single mRNA; proteins translated from different start codons, in different reading frames
Leaky scanning

25
Q

What does segment 3 encode and how?

A

PA (endonuclease subunit of polymerase); PA-X (host cell shut off)
Single mRNA; proteins share common N-terminus; ribosomal frame-shifting gives different C-termini

26
Q

What does segment 7 encode and how?

A

M1 (matrix protein of virus); M2 (ion channel); M3 (unknown)
Splicing to give 3 different mRNAs, each coding for one protein

27
Q

What does segment 8 encode and how?

A

NS1 (accessory protein, antagonises interferon response); NS2 (nuclear export)
Splicing to give 2 different mRNAs, each coding for one protein

28
Q

What is the mechanism that allows segment 2 to encode for two proteins?

A

Leaky scanning
Some ribosomes miss the original start codon and bind later on in the sequence to a second start codon (first start codon has a weaker initiation context)
Some ribosomes scan past the PB1 start codon, and initiate translation at the PB1-F2 start codon

29
Q

What is the sequence for a strong initiation context?

A

A/G CC AUG G

30
Q

What is the mechanism that allows segment 3 to encode for two proteins?

A

Ribosomal frame-shifting (shift forward by one nucleotide)
Something has to cause a pause in translation and must be over a ‘slippery’ sequence
Pause has to be long enough for frame shift to occur
99% ribosomes translate PA; 1% frameshift to produce PA-X fusion protein

31
Q

What is PB1-F2?

A

PB1-F2 found in 75% of IAV strains (not essential)
Strains lacking PB1-F2 less virulent
PB1-F2 localises to mitochondria; pro-apoptotic
Causes more inflammation in host

32
Q

What is PA-X?

A

PA-X found in 99% of IAV strains
Endonuclease domain of PA, but doesn’t bind polymerase
Non-specific mRNA cleavage protein – host cell shut off of gene expression
Dampens host antiviral response (less inflammation)

33
Q

What two molecules may govern virulence and host response in influenza?

A

PB1-F2 and PA-X

34
Q

How is mRNA generated from influenza virus segment 7?

A

M1 (matrix) translated from unspliced mRNA
M2 (ion channel) and M3 (unknown function) translated from spliced
mRNAs; different 5’ splice sites, same 3’ splice site

35
Q

How long is M3 mRNA?

A

9 amino acids

36
Q

How is mRNA generated from influenza virus segment 8?

A

NS1 (non-structural protein 1) translated from unspliced mRNA
NS2 (nuclear export protein) translated from spliced mRNA

37
Q

What is NS1?

A

Multifunctional accessory protein

38
Q

What is NS2?

A

Required for vRNPs to be exported from the nucleus

LF213: Virology (14 decks)

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