The life cycle of influenza

1. Virus binds sialic acid receptor via HA
2. Endocytosed
3. Low pH induced membrane fusion via HA 
4. Segments released into cytoplasm
5. Segments imported into the nucleus
6. RNA/RNP synthesis
7. RNA/RNP export
8. Translation of NP, PB1, PB2, PA
9. Virus assembly at the plasma membrane
10. Release

• Mediated by hemagglutin protein (HA) 
• HA recognises sialic acid on surface of epithelial cells
  Tends to associate with ciliated epithelial cells in the respiratory tracts. These cells are found lining the surface of mainly the LRT but also the URT.
• A common addition to galactose to cause sialic acid production

Sialic acid can be linked to galactose in 2 ways:
- alpha 2-3 or alpha 2-6 linkages

In general a given isolate of influenza virus can infect only one type of sialic acid linkage

• Human influenza viruses infect mainly cells with alpha 2-6 linkages- in the respiratory tract,
• Avian influenza viruses infect mainly cells with alpha 2-3 linkages, in the gut 

HA is initially synthesised as HA0 in cells.
HA assembles into a trimer
HA0 cleaved to HA1, HA2, disulphide linked
Cleavage caused by intra and extracellular proteases
HA1 forms a globular head
HA2 forms a long stable stem via 6 helix bundle 
The receptor binding site is located in the globular head HA1 domain 

Can change the specificity of sialic acid binding can be switched through single residues changes e.g Glu190 from alpha 2-6 to alpha 2-3 (Mutating 1 AA)

• Virus enters into an endosome
• It escapes by fusion of the viral and endosomal membrane 
• Fusion of the membranes allows the RNP segments to be released
• Fusion is mediated by the HA protein and its fusion peptide

• The fusion peptide is buried in the middle of the HA0
• To be active, the fusion peptide must be positioned at a free polypeptide amino-terminus 
• This is achieved by the HA1/HA2 cleavage event 

The pH5 in the endosome triggers a radical conformational change
The 2 helices in HA2 become aligned 
This flips the fusion peptide upwards
The fusion peptide inserts into the endosome membrane 
FURTHER pH-INDUCED CHANGES BRINGS 2 MEMBRANES TOGETHER

The HA protein possesses 2 critical functions:
1) Virus attachment to host cell via sialic acid 
2) Virus entry into cell interior by fusion of viral and endosome membranes

After fusion, segments are still contained within the matrix protein layer
RNP release from the matrix is mediated by M2 protein 
4 copies of M2 form a tetramer- M2- viroporin 
M2 allows influx of H+ ions into the virion 

The matrix protein layer breaks down to allow segments to be released ino the cell

• All influenza virus RNA synthesis occurs in the nucleus
• RNP's must move from cytoplasm into the nucleus 
• RNP are too big to diffuse

There is an active process for RNP import
- Individual NP monomers have 2 import singals 
- Only NLS1 is exposed on form RNP's
NLS1 recruits importin alpha 
This recruits importin beta 
RNP/importin complex binds pore 
RNP's enter nucleus 

RNA synthesis can be divided into 2 processes:

1. Transcription 
-ive sense input genome forms a positive sense mRNA 
This generates a coding positive sense mRNA for translation into viral proteins 

2. Replication
Replicates negative sense input genome from positive 
This amplifies the number of negative stranded genome segments 

The template for both these activities is the RNP= An RNA segment associated with 4 viral proteins

• Structure solved using crystallography 
• Shows position of all 2229 amino acids 
• Shows how 3 subunits interact 
  - Shows 3 holes leading to the centre of the polymerase
  - RNA template entry
  - rNTP entry
  - RNA product exi 
• Dimension will only allow the entry/exit of naked RNA- not RNP
• Polymerase activity is v.well understood

Results in the generation of 1 RNA transcripts per segment (2 of these transcripts spliced using host component) 
Mature mRNAs resemble cellular RNA's, 5' capped and 3' tailed.
mRNA then exported to the cytoplasm
Translated into protein using cellular translation machinary to make viral proteins

1) RdRp PB2 binds a cellular mRNA in the nucleus and PA steals its 5' cap 
2) RdRp and capped oligonucleotides binds to negative stranded RNA genome 
3) RdRp PB1 uses capped RNA to begin transcription, gets incorporated
4) RdRp moves along template, PB1 generates an RNA transcript with 5' cap and 3' poly(A)tail- mRNA is not encapsidated

Once mRNA's are made, they are exported for translation 
Some proteins stay in the cytoplasm for assembly
- HA, NA, M2, NS1
Some are importd into nucleus to make more RNP's
The import is signalled by NLS sequences
- NP, PB1, PB2, PA, M 

Replication then begins

4 stages
1. RdRp binds 3' end of the RNA segment, no primer is need 
2. RdRp travels along the segment 3'-5' generating a complimentary copy
3. NP wraps RNA as it is made- so replication products are RNP's
4. Cycles of replication allow amplification of genomes 

REQUIREMENT FOR NP MEANS REPLICATION CANNOT PRECEDE TRANSCRIPTION

Cycles of replication allow copies of all 8 segments to build up in the nucleus 

Involves the binding of 2 different viral proteins -M1 and NS2
RNPs 1st associate with multiple copies of M1 protein in the nucleus 
- NS2 binds M 
- NS2 binding hides import signal on M
- NS2 has an export signal- NO import signal 

NS2 binding over rides all other import signals
This occurs as the NLS on the M1 protein forms the NS2 binding site
The export signal engages host cell export machinary 
- Mediated through a protein called CRM1
- CRM1 binds RanGTP and exists the NCP 
- The RNPs then enter the cytoplasm where they must reach their final destination for virus assembly. 
RNPs must reach the apical surface plasma membrane-along with all the 3 envelope proteins 
HA, NA and M2 traffic via secretory pathway 
RNPs traffic on recycling endosomes
- Achieved via RabII
- M1 associates with cytoplasmic tails of HA, NA, M2

Interaction is via the cytoplasmic tails 
Current model is that M1 acts as a master recruiter
Evidence: 
- M1 binds RNPs
- M1 binds HA, NA, M2
- M1 essential for particle formation 

The virus particles must posses all 8 segments to be infectious
Random grabbing of segments results in only 1:4000 having all 8.
In reality, almost all particles have 8.
Strongly suggests segments are selected 

Mechanism

• RNP segments probably interact with each other
• 2 models
  - each segment specifically interacts with 1 or 2 others
  - master segment interacts with other 7

• Newly released virus needs to infect new cells
• It can do this via HA and the sialic acid receptor 
• But HA on released viruses can also bind to sialic acid on the old cells 
• This prevents the virus spreading

Solved problem using NA
- NA is abundant on virus envelope- Its on a long stalk 
- NA cleaves sialic acid from previously infected cell
- This makes sure that only new cells can be infected

Because of this critical activity NA is essential for inactivity and is an important therapeutic target.