Introduction to Viral Replication + Influenza (2-4)

Question 1

How to do viruses synthesise proteins?

Answer 1

Viruses use host cell ribosome and translation machinery to synthesise their proteins from mRNA
→ but, different viruses have different genomes so they have different strategies for genome replication and mRNA synthesis
→ almost all viruses carry their own polymerases

Question 2

What are the different types of virus genomes?

Answer 2

DNA → double-stranded - linear, circular, gapped
→ single-stranded - +ve or -ve

RNA → double stranded - segmented, non-segmented
→ single-stranded - +ve, -ve, ambisense

Question 3

Why do some viruses need their own polymerases?

Answer 3

Our (host) polymerases are DNA-dependant
→ RNA viruses use RNA as a template so need RNA-dependant polymerases

Question 4

How are viruses classified with the Baltimore system?

Answer 4

Class 1 → dsDNA
Class 2 → ssDNA
Class 3 → dsRNA
Class 4 → +ve ssRNA
Class 5 → -ve ssRNA
Class 6 → +ve ssRNA RT
Class 7 → gapped dsDNA

have different mechanisms to produce mRNA

Question 5

What type of virus is influenza?

Answer 5

Class 5 orthomyxovirus
→ segmented genome

Question 6

What are some if the most prevalent influenza pandemics?

Answer 6

1918 → Spanish flu, 25,000,000 deaths
1956 → Asian flu, 70,000 deaths
1968 → Hong Kong flu, 34,000 deaths
2009 → Swine flu (H1N1), +450 deaths

? → H5N1 again flu, +250 deaths since 2003
→ mutation might make it highly transmissible in humans

Question 7

What are the proteins present on the surface of an influenza particle?

Answer 7

HA → haemagglutinin - present as a trimer
NA → neuraminidase - present as a tetramer

Question 8

How is influenza genome material structured?

Answer 8

8 segments of -ve sense ssRNA
→ wrapped in nucleoprotein with a copy of viral polymerase attached - ribonucleoprotein (RNP)
→ each segment forms helix with pan handle structure

Question 9

What proteins make up the influenza viral polymerase?

Answer 9

3 polymerase subunits

PB1 → polymerase basic protein 1
PB2 → polymerase basic protein 2
PA → polymerase acidic protein

Question 10

What do the different influenza genome segments encode?

Answer 10

from largest - smallest

1 → PB2
2 → PB1, PB1-F2
3 → PA, PA-X
4 → HA
5 → NP
6 → NA
7 → M1, M2
8 → NS1, NS2

Question 11

What is the different between human and avian influenza strain receptors?

Answer 11

Human strains (e.g. H1N1) → binds sialic acid linked to galactose via α(2,6) in human respiratory epithelium
Avain strains (e.g. H5N1) → binds sialic acid linked to galactose via α(2,3) in duck guy epithelium

→ sialic acid points in a different direction - leads to difference in tropism

Question 12

How does influenza enter human cells?

Answer 12

HA trimer (on virus membrane) binds to sialic acid on human endosomal membrane - endocytosis
→ low pH changes the conformation of HA to expose the fusion peptide - so it can be inserted into the membrane
→ H+ ions enter virus via M2 - acidification causes further conformational change - α helices fold bringing membranes close together
→ fusion of membranes and release of vRNPs from M1

Question 13

What occurs to influenza contents after entry into human cells?

Answer 13

Fusion of viral and endosome membranes allows the contents of virus to be inserted into cytoplasm - then move to nucleus
→ the nucleoproteins on vRNPs have nuclear localisation signal - allows it to interact with nuclear pore complex
→ vRNPs imported into nucleus - influenza replicates in nucleus

Question 14

What are the stages in influenza virus mRNA synthesis?

Answer 14

1. Cleavage (cap-snatching) → to use host cell translation machinery efficiently needs to make capped mRNA - steals cap from cellular mRNAs in nucleus
   → cleaves host mRNA downstream from cap
2. Initiation → uses cap as a primer to initiate synthesis on its own mRNA
3. Elongation → mRNA synthesised, polyadenylation occurs at 17-22 bases from the 5’ end of the genome

Question 15

Why does influenza mRNA synthesis need to occur before further parts of the viral life cycle?

Answer 15

No further parts of the life cycle can occur until viral proteins are synthesised
→ particularly NP nucleoprotein and polymerase subunits
→ mRNA synthesis is the first thing to occur once it enters nucleus

Question 16

What is cap-snatching in influenza mRNA synthesis?

Answer 16

Stealing caps from host cell mRNA

1 → PB1 (on viral polymerase) binds 5’ end of genome
2 → PB2 binds cap of host mRNA
3 → PB1 binds 3’ end of genome and aligns this with the host mRNA at position 10-13
4 → If the alignment is correct PA cleaves the mRNA

→ therefore viral mRNA synthesis depends on cellular mRNA synthesis by RNA Pol II to supply capped primers

Question 17

What are the 3 types of RNA found in influenza virus infected cells?

Answer 17

(+) mRNA → for protein synthesis (95%)
(-) virion RNA → genome segments that have entered
(+) cRNA anti-genome → exact copy of virion RNA to make more copies (can’t use mRNA as its modified)
→ cRNA is unstable unless wrapped in nucleoprotein, thus replication can’t occur until proteins made (i.e. mRNA)

Question 18

What is the role of NP in influenza vRNA synthesis?

Answer 18

NP protein binds to cRNA as soon as it is made to stabilise
→ prevents polyadenalation - allows anti-termination of the poly(A) site
→ cRNA has a different sequence - doesn’t promote cap-primes initiation and no poly(A) site (no mRNA) - only vRNA is made

Question 19

What is the order of RNA production for influenza?

Answer 19

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

Question 20

How are the genes on influenza gene segment 2 translated?

Answer 20

Leaky scanning
→ segment 2 codes for PB1 (polymerase) and PB1-F2 (virulence factor)
→ the proteins are translated from different start codons in different reading frames (different aa - different protein)

In order for a start codon AUG to be recognised it needs to be in a particular sequence context
→ if its surrounded by a strong imitation context ribosomes are highly likely to pause and start translating

Question 21

How are the genes on influenza segment 3 translated?

Answer 21

Ribosomal frame shifting
→ segment 3 codes for PA (endonuclease, polymerase) and PA-X (host cell shut off)
→ proteins share the same common N-terminus, ribosomal frame shifting gives different C-termini

Slippery sequence (repeated A/U) causes ribosome to pause and continue translating in new reading frame
→ e.g. prefers base pair with UUG over UUU

Question 22

How are the genes on influenza segment 7 translated?

Answer 22

mRNA splicing
→ segment 7 codes M1 (matrix), M2 (ion channel) and M3 (unknown)
→ splicing gives 3 different mRNAs, each coding for one protein
→ various splice sites produce mRNAs of different lengths

Question 23

How are the genes on influenza segment 8 translated?

Answer 23

mRNA splicing
→ segment codes NS1 (accessory protein) and NS2 (nuclear transport)
→ splicing gives 2 different mRNAs, each coding for one protein
→ NS1 translated from unspliced mRNA, NS2 from spliced

Question 24

What are the 3 functions of NS1 protein in influenza?

Answer 24

1. Inhibits nuclear export of cellular mRNA
2. Increases translation of viral mRNAs
3. Interferon antagonist

(manipulated environment to make it more favourable for the virus)

Question 25

How does influenza NS1 protein inhibit nuclear export of cellular mRNA?

Answer 25

NS1 prevents 3’ end processing of cellular mRNAs by
1. Binding the 30kDa component of the cellular poly-adenylation/cleave complex - preventing its binding to cellular poly(A) signal
→ therefore no cleavage of pre-mRNA
2. Binding poly(A) polymerase-binding protein and preventing its functional interaction with poly(A) polymerase
→ therefore no polyadenylation

Inhibits cleavage and polyadenylation
→ can’t be exported out of nucleus
→ less ribosome competition

Question 26

How does NS1 influenza protein increase translation of viral mRNAs?

Answer 26

NS1 binds to the 5’ end of viral mRNAs in the cytoplasm and recruits translation initiation factors (IF4G) - recruiting ribosomes
→ stimulating translation of own mRNAs

Question 27

How is NS1 influenza protein a interferon antagonist?

Answer 27

NS1 binds RIG-1 blocking sensing of viral RNA - preventing induction of IFN transcription

Inhibition of cellular mRNA blocks synthesis of IFN itself and IFN-induced proteins

NS1 binds to and locks activation of the IFN-inducible dsRNA-dependant kinase PKR

Question 28

How is influenza RNP exported out of nucleus?

Answer 28

Via CRM1
→ M1 and NS2 bind to RNP
→ reacts with CRM1 - export into nucleus

LMB → antibiotic that’s inhibits CRM1 function

Question 29

What is the function of neuraminidase for influenza?

Answer 29

Removes sialic acid from HA at cell surface during the export process
→ prevents particles getting stuck and new virus aggregating together

Question 30

What are influenza antivirals that inhibit neuraminidase?

Answer 30

Zanamivir and oseltamivir
→ analogues of sialic acid
→ bind and inhibit neuraminidase - effective at preventing viral replication
→ only work if early on - use as preventative

Question 31

How does influenza ensure 1 of each 8 gene segments is packaged into each viral particle?

Answer 31

Specific packaging (not random)
→ unique packaging signals for each experiment
→ evidence: you do see specific arrangements of RNPs