Saturday, 26 May 2012

Lecture material; Viral membrane fusion by Stephen C Harrison

  • Fusion of 2 bilayer membranes is thermodynamically favourable
  • High kinetic barrier.
  • Fusogens lower barrier
  • Viral proteins lower barrier by using free enerby liberated during protein conform. change to draw membranes together.
  • Hemifusion state: apposed proximal leaflets of 2 bilayers but not distal leaflets have merged is obligatory intermediate. 
  • Evidence suggests viral fusion proteins lower kinetic barriers and catalyse membrane fusion. 

(a) The protein in the pre-fusion conformation, with its fusion peptide or loop (light green) sequestered. The representation is purely schematic, and various features of specific proteins are not incorporated—for example, the displacement of the N-terminal fragment of proteins that are cleaved from a precursor or the dimer-to-trimer rearrangement on the surface of flaviviruses. (b) Extended intermediate. The protein opens up, extending the fusion peptide or loop to interact with the target bilayer. The part of the protein that bears the fusion peptide forms a trimer cluster. (c) Collapse of the extended intermediate: a C-terminal segment of the protein folds back along the outside of the trimer core. The segments from the three subunits fold back independently, so that at any point in the process they can extend to different distances along the trimer axis, and the entire trimer can bow outward, away from the deforming membrane. (d) Hemifusion. When collapse of the intermediate has proceeded far enough to bring the two bilayers into contact, the apposed, proximal leaflets merge into a hemifusion stalk. (e) Fusion pore formation. As the hemifused bilayers open into a fusion pore, the final zipping up of the C-terminal ectodomain segments snaps the refolded trimer into its fully symmetric, post-fusion conformation, preventing the pore from resealing.
Step 1
  • Protein opens up
  • forms a bridge between 2 bilayers
  • All viral fusion proteins studied have 2 membrane-interacting elements:
  1. a C-terminal TM anchor that holds protein in viral membrane
  2. a hydrophobic patch (fusion peptide or fusion loop_ that interacts with target membrane. 
  • All are trimeric in fusion-active state.
  • When ligand binds (eg protons, as mech responds to low pH of endosome, cell or viral protein ligand)
  • fusion protein undergoes conform change
  • extend each subunit towards target membrane
  • yield contact between membrane and fusion peptide or loop
  • many fusion proteins are C-terminal frags of larger precursor (eg HA2 fram of influenza virus hemaglutinin or gp41 frag of HIV Env)
  • To initiate fusion they must shed N-terminal frag
  • N-terminal frag has receptor-binding domain (HA1 or gp120)
  • Extended state is pre-hairpin intermediate
  • Collapse into folded back conform.
  • HIV-1 gp41 intermediate has half life of many minutes
  • for others may be few seconds

.Step 2
  • The bridge collapses. 
  • fusion peptide or loop in target membrane and C-terminal TM anchor in viral membrane come together.
  • Collapse distorts 2 bilayers 
  • into nipplelike conformation.
Step 3
  • Distortion of membranes lowers energy barrier between separated and hemifused membranes 
  • hemifused stalk forms

 Step 4
  • Hemifusion stalk opens to form a transient fusion pore.
  • Final conformational step in protein refolding renders open state irreversible
  • Pore expands.
  • With some fusion proteins, pore may flicker open and close
  • Steps 3 and 4 probably need concerted action of more than one fusion protein trimer.
  • 3 viral fusion proteins from influenza, dengue, and vesicular stomatitis viruses are from class I, II and III viral fusion proteins. 

Fig 2: Free energy changes during fusion of bilayers. The relative heights of the various barriers are arbitrary. Fusion proteins accelerate the process by coupling traversal of these barriers to energetically favorable conformational changes.   

Influenza virus hemagglutinin
  • Core of HA1 is a sialic acid-binding domain borne on stalk formed by HA2
  • Central feature of stalk is a 3-chain, α-helical coiled coil.
  • HA0 and its cleaved product, prefusion HA1-HA2 are identical ins overall structure.
  • Cleavage occus in trans Golgi network and after viral budding
  • Cleavage causes modest local rearrangement
  • newly generate N terminus of HA2 inserts into a pocket along threefold axis.
  • Buries fusion peptide. 
  • Pocket is created when C termini of HA2 coiled-coil helices splay apart
  • 3 helices diverge from threefold axis and from each other like tripod (fig 3a)
  • Sialic acid on glycoproteins or glycolipds is influenza virus receptor
  • HA1 bears binding site
  • Shallow pocket exposed on its outward-facing surface. 
  • Plasma membrane recycles regularly through endocytosis
  • virus-receptor complex may not need a spec endocytic signal to reach an endosome.
  • HA1:HA2 trimer encounters low p ---> undergoes conform. rearrangement ---> HA1 separates from HA2 (3a to 3b) except for a residual disulphide tether
  • Latter turns inside out.
  • In HA2 folding, loop to helix transition 
  • In region connecting fusion peptide to central coiled coil (3b to 3c)
  • C terminal of molecule reorients so it zips up alongside extended coil (3c to 3d)
  • Correspond to formation of extended intermediate and collapse into a conform that brings together fusion peptide and TM anchor.
  • Loop to helix transition in N terminal part of HA2 augments central coiled coil at N terminal end (3c)
  • C terminal part of protein reorients ---> break central helices where they splay apart
  • at site from which fusion protein withdrew earlier in fusion process.
  • Mutations affect stability of trimer interfaces that break during rearrangement
  • alter threshold pH for fusion
  • Conserved ionisable residues (2 Asp and a His) in vicinity of buried fusion protein may contribute to trigger
  • Fusion peptide is thought to form an amphipathic helix
  • When fusion is complete, pp chain segment C terminal to fusion peptide and membrane-proximal segment of HA2 interact
  • Postfusion comform of HA2 : residues connecting fusion peptides to coiled voil and residues N terminal to TM anchors cap the N termini of 3 central helices
  • Extended intermediate collapses
  • It must bend outwards away from nascent hemifusion stalk
  • let 2 membranes come together. 
  • Each HA2 chain can complete refolding independently of other 2
  • Loss of 3 fold symmetry
  • Forming cap restores global 3 fold symmetry (3e)
  • If TM helices pass completely thru bilayer, may need presence of aqueous channel - a committed fusion pore

Figure 3 - Influenza virus hemagglutinin: proposed sequence of fusogenic conformational changes.

Activating and initiating

  • Priming is result of proteolysis
  • Either of fusion protein (influenza hemagglutinin or retroviral Env) or an accompanying protein e.g. flavivirus and alphavirus.
  • For these viruses, priming occurs during transport of immature glycoprotein to cell surface
  • either before assmebly of virus aprticle by budding at cell surface
  • or after formation of an immature particle by budding thru an internal membrane
  • Glycoproteins of other viruses eg Ebola and SARS coronavirus need to be cleaved byendosomal cathepsins B or L during cell entry rather than during maturation 
  • Primed fusion protein is metastable in prefusion state.
  • Covalent peptide bond in fusion protein, or chaperone restrains initial folded conform of precursor.
  • After covalent restraint is gone high kinetic barrier separates primed from postfusion conform.
  • To lower barrier can be proton binding for viruses that evolved to detect low pH endosomal env
  • or binding of a coreceptor in HIV-1.
  • For herpesviruses and paramyxoviruses, trigger is altered lateral contact with other viral surface protein
  • Association with ligand alters free energy profile
  • so rearrangement to postfusion state is rapid.
  • Free energy liberated by rearrangement can overcome barrier to merging 2 membrane bilayers.
  • Extended intermediate forms.

The extended intermediate

  • Post fusion conform of gp41 ectodomain is a trimer of hairpins
  • both prongs of hairpin are a-helices. 
  • Approximately 50 residues immediately C-terminal to the fusion peptide (designated HR1, where "HR" stands for "heptad repeats") form a central, three-chain coiled coil.
  •  A loop that contains a conserved disulfide bond connects the HR1 segment to a second heptad-repeat element, HR2, which forms an outer-layer alpha-helix. 
  • Peptides from this outer layer can inhibit the fusion process
  • The mechanism involves association of the peptide with the inner core,
  • preventing transition to the post-fusion conformation.
  •  HIV fusion occurs at the cell surface, and one such inhibitory peptide (T-20, or enfuvirtide) is a clinically useful drug 
  • Mutations conferring resistance to T-20 can occur at various positions in the envelope protein, including residues in gp120 
  •  Some of the mutations in HR1 that reduce T-20 binding also retard fusion and enhance sensitivity to antibodies targeting the membrane-proximal region of gp41 
How many fusion-protein trimers contribute to formation of a fusion pore?
  • The energy barrier that must be overcome en route to a hemifusion stalk is thought to be about approx40–50 kcal mol-1 
  • 15-20 fusion proteins are estimated for HIV. 
  • In a mechanism of cooperativity, lateral contacts form between adj trimers in a ring round fusion pore site.
  • Proposed for alphavirus. 
  • Lateral contacts couple conform. change of one trimer to that in another, like allosteric regulation of miultisubunit enzymes. 
  • Other mechanism caused by response of 2 membranes to distortions necessary to promote fusion. 
  • 2 properties of bilayer membranes cause them to resist collapse of extended intermediate.

  1. Energy of bending in bilayer membranes into nipple like configuration.
  2. Hydration force creates barrier when apposing membranes come closer than  10–20 Å 
  • All proteins in fusion event bridge same pair of membranes
  • So behavious of one extended intermediate is not independent of behaviour of neighbouring one
  • They are coupled by deformation energies of 2 bilayers they connect.
  • Deforming a planar membrane into a bed creates a cap with positive curvature and a flared reigion joining it to planar membrane of the membrane. 
  • Fusion loops and peptides insert only partway into outer leaflet of traget membrane
  • displace lipid head groups laterally
  • favours curvature of leaflet. 
Does the membrane-proximal segment of the ectodomain have a mechanistic role?

  • The membrane-proximal segments (10–15 residues) of many fusion-protein ectodomains have characteristic hydrophobic and often relatively tryptophan-rich sequences.
  • At the end of the fusion-inducing conformational change, these segments are apposed to the merged membrane and could in principle interact with the membrane or the fusion peptides/loops (or both).
What structural rearrangements lower the kinetic barrier between hemifusion and fusion-pore formation? 

  • If influenza virus hemagglutinin is liked to  a glycosyl phosphatidylinositol anchor or to a truncated protein anchor that does not completely traverse the membrane, the fusion process halts at hemifusion and proceeds forward very slowly
  • Collapse of extended intermediate may not be enough to drive fusion process to completion.
  • At hemifusion stage, a hemagglutinin-induced fusion pore can flicker open and close.
  • Central helix in influenza HA2 (fig 3e) caps
  • shows formation of structure with tight hydrophobic and hydrogen-bonding interactions among aa
  • brings about concerted closure of conformational transition
  • draw near ends of TM helices together
  • cytoplasmic segments on opposite side of membrane pass into or thru nascent fusion pore.
  • Segments in aqueous channel of fusoin pore prevent its resealing
  • formation of cap on HA2 helical bundle when zipping up is complete makes pore formation irreversible.
  • cap formation couples to a translocation, into or through the pore, of material from the cytoplasmic face of the bilayer.

Fusion inhibitors

  • Ligands can retard or block viral entry if they bind selectively to any conformation of the fusion protein that precedes merger of the two bilayers.
  • Some neutralising antibodies block viral infection by inhibiting fusion
  • Some small molecules bind HIV gp 120 in prefusion conformation (as in HIV-1)
  • raise barrier to initial steps of fusion sequence
  • Site for molecules may be pocket in gp 120 
  • closes up in initial CD4-induced conformation transition
  • Productive receptor binding must expel compound from site and incur inhibitor-spec free energy cost.
  • A similar site on dengue virus E is a pocket that disappears when protein starts to rearrange 

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