Lecture 15- Influenza

Question 1

The Influenza Virion

Answer 1

M1- the matrix protein:
Tightly bound to each protein segment, on the inside of the envelope. Structural role and also binds to segments to shut off replication in the late infection phase
NEP: Present in the virion, not associated with RNA segments; nuclear export protein
M2: ion channel. Embedded in lipid bilayer; has a pore to let ions flow in and out of virion.
RNA pol: associated with each of the 8 segments.
NA: cleaves sialic acid off of proteins
HA: virion attachment protein. Receptor is the carbohydrate sialic acid.

Question 2

PB2

Answer 2

transcription, cap snatching

Question 3

PB1

Answer 3

Rna polymerase

Question 4

PA

Answer 4

accessory protein, endonuclease

Question 5

HA

Answer 5

virion attachment protein

Question 6

NA

Answer 6

cleaves sialic acid off glycoproteins

Question 7

M1

Answer 7

matrix protein, from genome segment/mRNA 7

Question 8

M2

Answer 8

ion channel

made from spliced 7 mRNA

Question 9

NEP

Answer 9

(NS2) nuclear export protein

made from spliced 8 mRNA

Question 10

Influenza RNP

Answer 10

Ribonucleoprotein (RNP)

consists of genome segment associated with NP, PB1, PA, and PB2

Question 11

Adsorption and Entry

Answer 11

HA attaches to sialic acid on glycoproteins (sialic acid containing receptors)
enters by receptor mediated endocytosis
results in endosomal vesicle
low pH of endosome activates M2 ion channel resulting in flow of protons into the interior of virion. The resulting low pH of the virion core alters M1 and causes M1 to dissociate from RNP particles. so acidification of endosome occurs as well as membrane fusion and youre left with just segments attached to rna pol.
NP has nuclear targeting signal, which results in it getting brought to the nucleus.

Question 12

Hemagglutinin and receptor-mediated endocytosis

Answer 12

Synthesis and processing of HA:
HA0 is cleaved in the middle by golgi process. This creates HA1 and HA2, which are connected by a disulfide bond.
HA1 has a sialic acid binding pocket
HA2 has a fusion peptide on the N terminus (close to C term of HA1) which fuses envelope with endosomal membrane. Also has TM region on C terminus- trimerizes in the membrane of the golgi.
Conformational change of HA induced by low pH:
Neutral pH form of (HA)3
Sialic acid binding pocket up at the top, binds to sialic acid in the plasma membrane. Fusion peptide is tucked away, unneeded near viral envelope.
Upon acidification of endosome ( low pH form of (HA)3))
Sialic acid binding pockets tucked away, fusion peptide brought out to top. Fusion peptide protrudes: promotes fusion event to release nucleocapsids.

Question 13

Transcription vs Replication

Answer 13

Transcription requires PB1, PA, PB2
(PB1 = polymerase)
Have a segment with a UC at 3’ end and a 5’ end, NPs coating the - strand RNA.
Add PB1/PB2, PA complex
Add Cellular mRNA including 5’ cap
PB complex binds to 3’ end
PB2 recruits cellular mRNA that has 5’ cap (in nucleus)
PB2 captures end of the mRNA. PA has endonuclease activity: cleaves 10-13 nt from 5’ end by PA. generally after an A residue
PB2 continues to hold onto cap (PA just chills from now on)
A pairs with U at the very 3’ end of - RNA
PB1 uses this snatched cap to start synthesis!
addition of NTPs results in + viral mRNA (no NP)
Transcription stops about 15-22 nucleotides from 5’ end of template: sequence of seven U’s
Stutters to make poly A tail
wind up with + viral mRNA (no NP)
NP protein does not block access to template- actually helps!

Question 14

Replication: (requires PB1, PA, and NP)

Answer 14

• strand genome RNA coated with NP
  add on PB1/PB2/PA complex
  PA in the presence of NP protein, promotes initiation
  PA promotes initiation at 3’ end of template without the need for a primer
  requires NP to coat nascent chain. otherwise replication would abort and wait until there was enough NP
  wind up with + nascent antigenome coated with NP
  At the end: full length + antigenome coated with NP; no poly A
  distinguishing between this and transcription is NP protein, and PA (endonuclease vs promoting initiation)

Question 15

Assembly and release

Answer 15

M1 protein binds to RNPs and blocks both transcription and replication
NEP bound to segments as well, plus RNA pol
NEP protein binds to RNPs after M1.
leaves nucleus, M1 on inside of plasma membrane and HA and NA embedded in lipid bilayer
get budding.
NA removes sialic acid from NA, HA and plasma membrane proteins. Removes sialic acid from surrounding viruses too! This prevents clumping

Question 16

Mechanism of packaging for influenza

Answer 16

Principles of assembly for influenza:

1) Each virion contains one of each of the 8 different segments
2) 5’ and 3’ sequences in RNA segments required for proper packaging
3) viral proteins may interact with RNA ends to sequentially build the cylindrical bundle containing 8 segments.