Influenza Virus / Fusion Flashcards
Influenza virus
- Orthomyxoviridae
- Membrane envelope
- RNA (8 segments)
- 4 types: A, B, C and D
- Type A: Epidemics & Pandemics, Animals and humans (A, B and C)
Eight segments (HA, NA, M, NP, PA, PB1, PB2 and NS) in influenza A code for up to eleven viral proteins: hemagglutinin (HA), Neuraminidase (NA), nucleoprotein (NP), matrix proteins (M1 + M2), RNA polymerase (PA), polymerase-binding proteins (PB1 + PB2 + possibly PB1-F2) and non-structural proteins (NS1 + NS2).
Structure and Nomenclature
Virus Type / Geographical origin / Strain number / Year of isolation / Virus-Subtype
Helical symmetry
- Polymerase complex: PB1, PB2, PA
- Viral genomic RNA
- NP monomer
- Minor groove
- Major groove
Linkage of sialic acid
- Host species specificity of the infection is related to the type of linkage:
- bird: α2-3 linkage type
- pig: α2-6 and α2-3 linkage type
- human: α2-6 linkage type
Seasonal vs Pandemic
SEASONAL
* “Drift”
* New variant of old acquaintant
* Partialimmunity of host possible
* Usually,normal immune response
* Annoyingand every year again
PANDEMIC
* “Shift”
* New through zoonosis / reassortment
* Immunity of host unlikely
* Often overreaction of the immune system
* Unpredictable and fatal
Influenza and inflammation
- Cause of death: inflammatory response, organ failure
- 1918H1N1 in the mouse model(Kashetal.): Increased neutrophil inflammatory response, death receptor, interleukin (IL) 6, type I interferon, and TLR response
- Avian influenza H5N1 infected persons (DeJong et al.): Monocyte / macrophage chemoattractants (IP-10, MIG, and MCP-1), neutrophil chemoattractant IL-8 in lung tissue, and high plasma levels of IL-10, IL-6 & Interferon-g
influenza virus hemaglutinin
- synthesized on ER ribosomes
-> co-translationally inserted into the membrane - HA is cleaved (seasonal strains, airway tryptase Clara; highly pathogenic strains by Furin and PCs) into two chains, HA1 and HA2
-> held together by S-S bonds - trimers (HA1* HA2)3 form in the rough endoplasmic reticulum
-> are transported through the Golgi apparatus to the plasma membrane
-> functional part of molecule (HA1) outside, HA2 tails anchored in the membrane
-> progeny virus particles then bud from the infected cell’s plasma membrane
-> carry both neuraminidase (NA) and hemagglutinin (HA) - HA trimer is a membrane-bound protein -> difficult to study by crystallo- graphy -> cleave off membrane anchor by protease -> 47 residues long -> remain in the membrane
- soluble fragment: 3 complete HA1 chains SS-bonded to 3 HA2 chains lacking C-terminal fragment
RBD (receptor binding domain)
- monomeric subunit: long, fibrous stem-like region
- part of HA1 and all of HA2 forms stem (so-called spike proteins!)
- globular region formed by HA1
The receptor-binding-domain of the HA has a jelly-roll fold (8 beta-strands). Yellow sphere: receptor-binding site
HA1
-> N-terminus of HA1 at base of the stem, near to membrane
- first 63 residues (ochre) -> extended structure -> almost 100 A along the length of molecule -> part of stem
- global tips is 8-stranded jelly roll
- remaining 70 (red) residues return to stem
HA2
HA2 (blue) -> hairpin of two a-helices
- 2nd helix is 50 residues (76 Å) long
- bottom of stem: b-sheet, 5 antiparallel strands, central strand from HA1
- 20 residues at N-terminus of HA2 -> fusogenic peptide -> hydrophobic -> used by virus to initiate fusion with host cell membrane
Hemaglutinin trimer
- 135 Å long
- cross-section 15 - 40 Å
- 3 long HA2 helices form coiled- coil -> pack against each other over a length of 40 Å -> hydrophobic contacts and salt bridges
- head domains also form trimer contacts
How does HA induce membrane fusion?
- Originally, the HA had been crystallized at pH > 6 -> no indication of how membrane fusion could be initiated
-> soluble trimers aggregate at pH < 6 -> cannot be crystallized - But: monoclonal antibodies against specific epitopes on HA1 and HA2 -> massive conformational change -> HA becomes highly susceptible to proteolytic cleavage -> soluble fragment of low pH form -> residues 1 - 27 of HA1 and 38 - 175 of HA2
- the low-pH fragment is also a trimer
-> most of the secondary structure elements preserved
-> including the 3-helix coiled coil
-> 2 important conformational changes:
- loop region B in high pH structure (between helices A and C-D) changes into helix
- alpha-helical region at the middle of C-D of high-pH form changes into loop
-> helix A-B-C has 65 residues, 100 A long
- in the high-pH structure, only region C forms a coiled coil
- in the low-pH structure, A-B-C is a coiled coil
The fusogenic peptide
- in the high-pH structure -> attached to the N-terminus of helix A -> 100 Å away from receptor-binding site
- in the low-pH form -> N-terminus of helix A moves by 100 Å -> fusion peptide near receptor-binding domain (missing here due to proteolytic cleavage)
-> structure explains membrane fusion by HA
Spring-loaded mechanism for viral membrane fusion
- Prefusogenic conformation of HA (left) -> HA1 global domains occupy distal end of the structure, atop a trimeric coiled coil region of HA2 (blue). Fusion peptides (green) buried.
- Fusogenic conformation (low pH) -> interactions between HA1 und HA2 weaken -> loop regions of HA2
“spring“ into helical conformation -> extending trimeric coiled coil and propelling fusion peptide to the top of the structure -> interaction with target membran
Fusion snapshots
1 pre-fusion
2 Extended intermediate
3 Collapse of intermediate
4 Hemifusion
5 Fusion pore (post-fusion)
