Monday, 4 June 2012

Intracellular and viral membrane fusion by Sollner


Viral fusion machinery

  • In case of viral fusion, the fusogenic machinery is exclusively provided by the virus. 
  • Two distinct classes of fusion proteins have emerged, class I (e.g. influenza, Ebola, HIV, and measles) and class II (e.g. tick-borne encephalitis, dengue and yellow fever), which differ on the structural level, but follow a mechanistically similar reaction cascade.

Class I viral fusion proteins

  • HA, class I prototype exists in prefusion state as a trimer with a central triplestranded coiled coil
  • HA is synthesised as large precurosr
  • Proteolytic cleavage generates an N terminal hydrophobic fusion peptide
          --- buried in pocket in prefusion trimer
  • Ha binding its receptor( sialic acid) on host cell membrane ---> attach virus to host cell
  • endocytosis triggers extension of central coiled at N terminus
  • expel fusion peptide which inserts into cell membrane
  • pH changes
  • Refolding at C terminus generates an antiparallel coiled coil
  • lets linker region which connects TM domain to rest of protein pack into a groove
          --- between adj a helices in newly generated extended trimeric stem
  • bring viral and cell membranes in close proximity ---> membrane fusion

Class II viral fusion proteins

  • Dengue and tick-borne encephalitis virus (TBEV) anvelope glycoprotein E and SEmilki Forest virus (SFV) exist in prefusion state as dimers
  • Monomer of class II fusion proteins contains 3 domains
  • each adopts b-sheet folds
  • Instead of NTerminal fusion peptide, an internal fusion loop is at tip of domain II
          --- shielded by intermolecular interactions
  • Dengue virus and TBEV glycoproteins form reversible antiparallel homodimers
  • SFV glycoprotein E1 forms a heterodimer with E2
          --- binds host cell receptor 



  • Dimers dissoc at low pH ---> expose fusion loops
  • Fusion loops insert into host cell membrane
  • triggers irreversible trimerisation of domain II
  • bring fusion loops together
  • create fusion patch 
          --- stable membrane anchor
  • Fusion loops in class II do not penetrate lipid bilayer
  • deform and destabilise membrane locally
  • Trimerisation generates binding site for linker (Stem) to Cterminal TMD
  • 9-aa linker segment which is not ordered in monomeric prefusion E1 ectodomain at neutral pH
  • becomes ordered in postfusion trimer at low pH
  • Domain III folds back ---> moves stem with its TMD closer to fusion loops
  • Bring viral and cell membranes into close proximity ---> fusion


 Cellular membrane fusion machinery

  • In contrast to viral fusion, the intracellular fusion 
    machinery is split in two matching halves, which are
    1. prelocalized to the compartments that will later fuse
  • Consists of v-SNAREs on vesicles on cognate t-SNAREs on target
  • SNARE s= SNAP receptor
  • SNARE family consists of compmt spec type II membrane proteins
  • Cognate v and t SNAREs drive fusion of liposomes and entire cells
  • when separately reconstituted into liposomes or translocated onto cell surface
  • SNAREs are core fusion machinery along secretory and endocytic pathway
  • At structural level a fully assembled (postfusion or cis) v/tSNARE complex consists of a 4 helix bundle
  • v SNARE contributes 1 helix
  • t SNARE contributes 3 helices
  • SHARES use assembly of coil coiled str to drive fusion
  • In prefusion state, separate v and t SNAREs are unstructured or only partly folded
  • SNARE complex formation starts with assembly of t SNAREs
          --- adopt trimer coiled coil fold in its N terminal membrane distal end
          --- still unstructured in its membrane-proximal region
  • Partially asssembled t SNARE provides template for binding and folding of unstructured v SNARE
  • SNAREpin forms (trans v/t SNARE complex)
  • bridge 2 membranes
  • SNAREpin assembly progresses towards TMDs
  • bring 2 membranes into close apposition
  • membranes fuse
  • Viral fusion proteins are used once in lifetime for fusion to enter host cell
  • SNAREs are recycled for further rounds of IC transport
  • Cis v/t SNARE complexes are extremely stable
  • become substrate for cytosolic SNAPs (soluble NSF attachment protreins)and hexameric ATPAse NSF
  • NSF hydrolyses ATPase
  • dissambles SNARe complexes
  • recycle individual SNAREs
  • coonvert SNAREs to high energy state
  • SNAREs are spec recognised by coat proteins
  • bud transport vesicles
  • Vesicle coat proteins may select fusion-competent conformation of SNAREs
  • Direct coupling of vesicle budding and spec SNARE packaging ensures production of fusion-competent vesicles


A common fusion mechanism

  • SNARE-mediated and viral fusion is driven by protein folding
  • fusion protein precursors seem to exist in kinetically stable high energy state not requiring external energy at fusion stage
  • Protein folding releases energy
  • overcome repulsive forces
          --- keep membranes from converging
Step 1:
  • Initial tethering step
  • Provide first contact between virus and host cell
  • or transport vesicle and IC Target membrane or plasma membrane
  • contribute targeting spec
  • eg. pww5, tether protein at cis side of Colgi binds t-SNARE components syntaxin 5 and GOD-28
  • stimulate SNARE complex assembly

Step 2:

  • fusion machinery activate ---> docking
  • dusion machinery links 2 opposing membranes
  • Triggered by tethering or subsequent changes in env
          --- lower pH 
          ---> structural change in fusion proteins
  • for viral fusion fusion loops/peptide insert into host cell membrane
  • In IC fusion this is skipped
  • both halves are already stably anchored in membranes
  • v-SNAREs and t-SNAREs are activated by regulatory reaction inc sasembly of t SNARE complex
Step 3:
  • Protein folding bring membranes in close proximity
  • On viral fusion needs folding back reaction and interaction of TMD linker with a binding site that emerged from trimerisation or trimer extension
  • In cell fusion, assembled trimeric t-SNARE provides folding template for v-SNARE
  • Zipperlike fashion towards membrane
Step 4 
  • Fusion may involve formation of hemifusion intmts
  • Viral hemifusion intmts may be trapped be alterations in TMD
           --- shorten or replace TMD of HA with a GPI anchor
  • TMD with polar amino acids at C terminus favours full fusion
  • Unlike viral fusion proteins SNAREs have membrane anchors that span entire membrane
          --- contain polar or charged residues at C termini
  • Replace TMDs with phopholipids or short chain isoprenoids ---> block fusion
Step 5
  • Fusion pore formation and dilation
  • Viral and cell fusion proceed thru fusion pore intmt
  • SFV E1 ectodomains (which lack part of stem and TMD) insert in cooperative manner
          --- in membranes
          --- form rings of 5 or 5 homotrimers
  • Intertrimer contacts provided by membrane distal ectodomain heads and fusion loops
  • oligomeristion may cause local nipple deformation in host cell membrane
  • pull it closer to viral membrane
  • facilitate hemifusion

Exocytic fusion pore architecture

  • Viral fusion pores are transient intmts
  • fusion pores in regulated exocytosis can form stable intmts
  • opening and closing is tightly controlled
  • Exocytic fusion pores have initial inner diameter of 1-2 nm
  • Initial opening can be reversible
  • followed by full pore dilation
  • vesicular membrane completely incorpd into target membrane
  • At neuronal synpase, Ca2+  influx triggers neurotransmitter release in 100 msec
  • fusion pore must be in preassembled closed state
  • ready to open
  • SNAREpins are partly assembled at neuronal synapse
  • couples SNAREs to fusion pore
  • component of neuronal t-SNARE syntaxin 1 is integral part of exocytic fusion pose


Regulation of exocytic fusion pores

  • during regulated exocytosis fusion pore opening is tightly controlled
  • Calcium is key regulator
  • SNAREs must be coupled to a calcium sensing machinery
  • Synaptotagmins regulate fusion pore opening
  • Each member of synaptotagmin family has an N terminal TMD
  • followed by tandem C2 domain, C2A and C2B
          --- calcium binding motifs


  • Calcium binding properties of C2A and C2B important in choice between transient fusion pore opening and complete fusion pore dilation
  • Tandem C2 domains interact in calcium dependent manner with SNAREs and acid phospholipids


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