Monday, 11 June 2012

Mechanisms of viral membrane fusion and its function by Eckert


Conformational Changes on Fusion Activation
  • low pH activates membrane fusion potential of HA
  • Low pH induces irreversible conform change in HA
  • HIV-1 Env changes conform in response to CD4 binding, its fusion activation signal
  • Expose to fusion-activating conditions (lower pH for HA and CD4 exposure for Env)
          ---> both proteins become more hydrophobic
          --- HA forms aggregates
          --- can bind detergents and liposomes
          --- both proteins can bind bis-ANS, a hydrophobic fluorescent dye
  • HA and Env undergo changes in proteolytic suscpetibility
  • Interaction between surface and TM subunits alter
  • Electron microscopy shows HA changes shape
  • Globular head domains on top of mol partly dissoc
  • CD4 cause dissoc of gp120 from gp41 - called shedding
  • Mutations affect infectivity and glycoprotein-mediated membrane fusion
  • but does not alter proteolytic processing or surface expression
  • Mutations may disrupt fusion active state but not native form
  • Epitope accessibility of protein is altered
  • When HA is exposed to low pH, recognition of most epitopes is altered
  • change in antigenicity
  • struc rearrangements thruout mol
  • Monoclonal abs reactive with tip of native HA cannot immunoppt HA after low pH induced conform changes
  • Abs spec for epitopes exposed in low pH activated HA eg abs raised against amino terminal fusion peptide cannot immunoppt native HA. 
  • In HIV-1, gp120 undergoes changes in antigenicity early in fusion 
  • After receptor binding V3 loop on gp 120 recognised
  • epitopes on gp 41 more accessible
  • After binding CD4 novel epitopes are revealed on surface of gp 120
  • allow recognition of neutralising abs
  • Neutralising abs eg 17b block binding of gp 120/CD4 complexes to coreceptor
  • CD4 binding exposes hidden coreceptor binding site
  • After exposure to CD4 HIV-1 can fuse with cells expressing a coreceptor and lacking CD4
Spring-loaded model
  • A region of HA2 (54-81) has a high propensity for forming a coiled coil
  • In native x-ray crystal structure most of region maintains an extended loop conform
  • forms coiled coil in solution
  • Proposed in transitino from prefusogenic to fusognice low-pH-activated str conform of residues change from loop to coiled coil
  • Extend central trimeric coil
  • propel fusion peptide to opposite end of mol
  • let it interact with host-cell membrane
  • Spring-loaded mechanism
  • Confirmed by x-ray crystal str
  • in fusogenic str, residues 55-75 form an extension of central coiled coil of native str
  • Conform change expected to move fusion peptide 100 Angstrom from its position

Figure 2 Spring-loaded mechanism for viral membrane fusion  
  • In the native conformation of influenza hemagglutinin (HA) (left), the HA1 subunits (yellow balls) occupy the distal end of the structure, 
  • atop a trimeric coiled coil region of HA2 (blue). 
  • The fusion peptides (green) are buried in the core of the HA2 structure. 
  • On induction of low pH, fusion-activating conformational changes occur (right).
  •  The noncovalent interactions between HA1 and HA2 weaken and the loop regions of HA2 (red) “spring” into helical conformations,
  • extending the central trimeric coiled coil 
  • propelling the fusion peptides to the top of the structure to interact with the target membrane in the native conformation. 
  • Mutations that reduce the helical propensity of the spring-loaded region reduce membrane fusion 
  • With prolines at both positions 55 and 71, HA maintains surface expression and proteolytic maturation, 
  • yet no fusion activity occurs.
  • How is a shift in pH able to cause such major conformational changes? 
  • Evidence suggests the native prefusogenic conformation of HA is metastable, separated from the stable fusogenic state by a kinetic barrier 
  • The pH change serves to destabilize the native state, making it easier to overcome the kinetic barrier. 
  • low pH per se is not required for HA-mediated fusion. 
  • At neutral pH, high temperature or a chemical denaturant can activate the spring-loaded conformational change of HA and, therefore, HA-mediated membrane fusion 
High-Resolution Structures of Fusion-Active Conformations
  • HIV-1 gp41 ectodomain has a trimeric helical subdomain
  • made of 2 discontinuous peptides, N51 and C43
  • stable
  • unfolds only at extreme temp
  • Peptides from these 2 regions are called N and C peptides 
  • originate from amino- and carboxy-terminals of gp41 ectodomain
  • N-peptides form a central trimeic coiled coil
  • helical C-peptides bind outside of coiled coil, packed antiparallel to helices in N-peptide coiled coil core.
  • Str is trimer-of-hairpins
  • in trimer, 3 helical C-peptides bind outside of coiled-coil core of N peptides in antiparallel manner
  • each C-peptide binds conserved hydrophobic groove formed by 2  N peptides.
  • low-pH activated HA2 has trimer of hairpins
  • significant helix to loop change between native and low pH activated HA. 
  • 6 Cterminal residues near base of central trimeric coiled coil of native str become a loop in low-pH activated str
  • reverse direction of C terminal end of coiled coil
  • N and Cterminal regions of ectofomain come together at same end of folded str
  • Trimer of hairpins of HIV-1 gp41 and influenza HA thought to be fusion active conform.
  • Trimer of hairpins is stable and irreversible
  • Native state of HA is easily altered by low pH, heat and chemical denaturants
  • Spring-loaded change
  • displace fusion peptide from buried region to exposed region on amino terminus of trimeric coiled coil core
  • Proline substitutions in spring-loaded region of HA ablate fusion
  • When influenza is exposed to low pH without target membrane, it is inactivated
  • Trimer of hairpins may define fusion active conform of viral envelope glycoproteins that occus concomitantly or after membrane fusion
gp41-Derived Inhibitory Peptides

  • gp41-derived peptides are inhibitory
  • C-peptides are much more potent than N peptides
  • Proposed that C-peptides bind to or near predicated helical region downstream from fusion peptide, which corresponds to N peptide region
  • Inhibit infection in dominant -ve manner
  • Supported by inhibitory activity of C peptide decreased when in presence of equal amount of N peptide. 
  • Mutant C peptides that destabilise formation of trimer of hairpins have weakened antiviral potency
  • Hydrophobic binding surface on N peptide coiled coil core to which C peptides bind is highly conserved between HIV-1 and SIV
  • SIV C-peptide inhibits HIV-1 mediated fusion
  • Viruses that develop resistance to C peptides have substituted residues in N-peptide regonis
  • Epitope-tagged C peptide can immunoppt gp41. 
  • C peptides inhibite formation of trimer of hairpins in dominant negative manner 
  • by binding N peptide region of gp 41

 Transition fusion intermediate

  • Proposed transient intmt in fusion profess
  • formed after receptor binds
  • before formation of trimer of hairpins
  • In intmt, N-peptide region is exposed
  • vulnerable to binding by synthetic C peptides
  • proposed C peptide-inhibited gp 41 is irreversibly inactivated

Existence of prehairpin ntmt is supported by:
  • C peptides must be present during exposure to host cell to be effective
  • If virus is preincubated with C peptide
          --- then C-peptide is removed
          --- before addition of target membrane
          ---> infection is not inhibited
  • Epitope-tagged C peptide can immunoppt gp41
          --- only after exposure to CD4
          ---> a receptor mediated conform change required to expose Npeptide region



Figure 4 Model of the prehairpin intermediate and inhibitors. 

  • After HIV-1 envelope protein, Env binds CD4 and co receptor
          --- transient intmt formed
          --- gp41 spans both viral and cell membranes
  • N peptide region of gp 41 exposed and vulnerable to inhibitors
  • Npeptide trimeric coiled coil has 3 grooves
          --- can be bound by C peptides
  • has 3 prominent hydrophobic pockets at base
  • can be targeted by potential small mol entry inhibitors eg D peptides
  • Binding C or D peptides to transient intmt ---> irreversible inactivation of membrane fusion



Figure 5 Schematic representation of the current working model for viral membrane
fusion. 

  • Native state of fusion protein: most of exposed surface area composed of surface subunit
  • Much of TM subunit inc fusion peptide is not exposed
  • After fusion-activating conditions
         --- conform changes
         --- free fusion peptide from unexposed location
  • For HA occurs via springloaded mech
  • In HIV1 conform changes cause formation of transient prehairpin intmt
  • This intmt likely to exist in HA-mediated fusion
  • Prehairpin intmt spans 2 membranes
          --- TM region in viral membrane
          --- fusion peptide in host cell membrane
  • N-peptide coiled coil and probably Cpeptide reigon is exposed
          --- vulnerable to inhibitory mols esp HIV 1
  • Prehairpin intmt of HIV 1 constrains gp 41
          --- N and C regions cannot interact 
  • Without inhibitors, prehairpin intmt resolves into trimer of hairpins
           --- membrane fusion occurs




A Common Fusion Mechanism

  • In cells infected by either virus, viral envelope glycoprotein is expressed as an unprocessed precursor
          --- unable to fuse
  • Precursor is proteolytically processed
          --- lock protein into metastable state
  • HA0 unprocessed precursor differs from native processed form in 18 residues surrounding cleavage site
  • In precursor, residues are exposed to solvent and folded as an extended uncleaved loop
  • Cleavage ---> newly created C terminus of HA1 and N terminus of HA 2 separate
  • Fusion peptide is deeply buried in an interior region at base of HA
         --- primed for low pH-activated str rearrangement
  • Cleavage may lock fusion glycoprotein into a metastable state
  • block from its most stable fold by a kinetic barrier
  • When HA2 is expressed in absence of HA1, it adopts the fusogenic str
  • Processed protein exposed to fusion-activating conditions
  • overcomes kinetic barrier of native state
  • initiate conform changes necessary for fusion
  • In influenza, HA1 domains lose trimeric contacts
  • Fusion peptide released from buried position
  • propelled in spring-loaded way to N terminus of central trimeric coiled coil
  • Exposed ---> fusion peptide can interact with host cell membrane.
  • In HIV1 binding of gp 120 to CD4 ---> conform change in gp 120
          ---> allow attachment to coreceptor
          ==== conform changes in gp 120 and gp41
          --->weaken their interaction
  • transient prehairpin intmt of gp41 formed
          --- free buried fusion peptide
          --- interact with host cell membrane
          --- expose N peptide region

  • influenza HA may go thru transient intmt state
          --- after springloaded mech
          --- before hairpin formation
  • In absence of traget membrane HA is quickly inactivated after acidification
  • In inactive state, fusion peptide interacts with viral membrane
          --- form fusogenic str anchored in viral membrane
  • In both viruses, after fusion peptide inserts into target membrane
          --- TM subunit spans 2 membranes
          --- of virus and cell
  • In fusogenic str, both termini of TM subunit ectodomain are at same end of folded mol
  • very stable
  • most thermodynamically favourable form of mol
  • brings 2 membrane proximal regions close together
  • Energy gained overcome unfavourable process of bringing together 2 phosphlipid membranes
  • Both TM regions of fusion glycoprotein (fusion peptide and tM helix) occupy same membrane as viral and cell membranes become one
  • Fusion between influenza and host cell membranes involve a flickering pore
           --- expands over time
           --- let release  of viral contents into cell
  • mulitple HA mols likely required to gather at fusion site to promote pore formation
  • proposed that pore formation occurs thru lipid intmt
          --- outer monolayers fuse first
          --- form a stalk between 2 enveloped netities
         --- pore forms when inner monolayers fuse


INHIBITING HIV-1 ENTRY

  • 2 viral targets are reverse transcriptase (transcribe HIV1 RNA genome to DNA) and protease (processes HIV1 Gag/Pol polyprotein and subsequent Gag protein) 
  • Cpeptides have inhibitory activity
  • feasible to target transient gp41 str that emerge during viral infection
  • HIV1 coreceptors were identified
  • str of gp 120 core bound to CD4 and an ab mimicking co receptor enabled discovery of mols that inhibit HIV 1 fr binding cell
  • Anti HIV1 mols that inhibit entry stop virus before it infects cell



Targeting the Transient Fusion Intermediate

  • gp41 transient intmt of viral entry possible target for inhibition
  • C peptides which bind intmt have some success
  • viral reduction
  • Disadvantage: size
          --- cannot enter orally
          --- must be injected
  • Expensive to produce long C peptide
  • Need a lot of peptide for antiviral effect
  • in gp41 there is a small pocket in conserved hydrophobic groove of N peptide trimeric coiled coil
  • 2 hydrohobic residues from C peptide, 2 W and an I bind pocket
  • Pocket can a target for drug
  • It is small
  • Ideal for binding by a small mol
  • Many residues lining pocket are critical for membrane fusion
  • C peptide inhibitory activity depends on its ability to bind pocket
  • Drugs that target pocket may elude resistant virus
  1. residues in pocket are highly conserved
  2. mrNA coding this region is ingegral part
  • Selective pressure not to mutate at both protein and RNA levels





High-Resolution View of gp120

  • primary seq of gp 120 made of 5 regions of high seq variability 
  • interspersed with 5 conserved regions
  • CD4 binding site is large 
  • at interface of all 3 gp120 domains
  • binding surface has conserved residues needed for CD 4 thru H bonding to main chain atoms
  • Fab frag binds side of bridging domain composed of part of 4th conserved domain and base of V1 and V2 stem
  • May be co-receptor binding site


Inhibiting the gp120/CD4 Interaction

  • Early efforts to inihibit HIV1 entry focused on inhibiting binding of envelope protein to CD4 on cell surface
          --- by competition with soluble version of CD4
  • Recent designs of sCD4-like mols eg Pro542
  • gp120 binding region of CD4 is fused to constant region on IgG2
  • Increased affinity for gp120
  • Decreased viral loads of HIV1 infected patients
  • Graft critical gp120 binding motifs from CD4 omto toxins of similar str
  • Toxin chimeras mimic CDr indeuced conform change in gp120
  • inhibit CD4 binding and viral infectivity






No comments:

Post a Comment