membrane fusion Flashcards
What are the problems of fusion of viral and cellular membranes? how are they overcome?
- Fusion should only take place at target membrane –> viral receptor binding protein. partial proteolytic uncovering of fusion peptide iby only locally expressed proteases, pH dependent fusion
- membrane are biological barriers (energetic problem) –> conformational change in fusion proteins draws target membrane into close proximity, viral fusion peptide destabilizes lipid bilayer
- newly formed intracellukar virions have to be protected against fusion –> viral fusion peptide inaccessible
How can the viral fusion peptide be made inaccessible so that newly formed virions do not fuse with the membrane?
viral fusion peptide inaccessible due to either
1- fusion part
2- protein conformation
3- helper protein
Name the first steps of a viral infection
- association with cell membrane/receptor binding
- entry through target memebrane
- uncoating
name virus entry strategies
- receptor mediated endocytosis
- receptor mediated signaling
How does receptor mediated endocytosis work as viral entry strategy work?
- virus binds toi receptor on membrane surface
- aggregation of receptor
- uptake into early endosome
- pH dependent/independent fusion of virus and early/late endsome
how does receptor mediated signaling as viral entry strategy worK?
- virus binds to receptor
- pH independent fusion at the membrane
- uptake of virus into cell
Name the sites of virus particle fusion/penetration and give example viruses
- receptor mediated signaling (fusion directly at PM): Murine leukemia virus, Epstein-Barr virus, HIV, Herpes
- early endosome: HCV, VSV, HIV
- late endosome/lysosome: Influenza, SARS, Dengue
- escape from ER: SV40 polyomavirus
Name receptors involved in receptor mediated endocytosis in HCV
- LDL
- scavenger
- Claudin 1
- Occludin
- CD81
What is apoptotic mimicry?
a virus acquires host cell phosphatidylserine and incorporates it into the viral membrane –> PS as receptor binding molecule, no protein needed –> use of PS receptors –> internalization via macropinocytosis or clathrin-mediated uptake
How can viruses acquire PS?
- budding from lipid rafts
- from ER sheets
- budding from ER lumen
Which viruses use which endocytotic pathway?
~Adeno, arena, flavi, coxsackie b, sv40, echovirus1
- MAcropinocytosis: Adenovirus
- clathrin independent: Arenavirus
- clathrin mediated: flavivirus
- caveolar: Coxsackie B
- cholesterol dependent: SV40
- Dynamin-2 dependent: Echovirus1
describe the entry of SV40 (non- enveloped)
- endocytosis into caveolae
- fusion with caveosome. no pH shift
- long transport in vesicles into ER (actin, Rho GTPase and microtubuli dependent)
- structural rearrangement of the capsi in the reducing milieu of the ER
- penetration into cytoplasm, ERAD pathway
- import into nucleus by NPC and NLS in VP2/3
What is important for membrane fusion between vesicles in animal cells?
- SNAREs –> driving force: hydrophobic and ionic interactions in the formation of helix bundles
- resulting conformational change brings membranes into close proximity
describe the process of fusion of viruses and animal cells
- binding to receptor leads to conformational change in fusion protein
- hydrophobic fusion peptide becomes exposed and inserts intself into target membrane
- fusion protein trimerizes –> conformational change (super coil)
- membranes are brought in close proximity and fuse
Name the three classes of viral fusion proteins and examples
class I: Influenza Hemaglutinin, coronavirus spike
class II: Flavivirus E
class III: Rhabdovirus G
explain the mechanism behind viral fusion proteins
interaction with a target cell –> triggers exothermic fusogeni conformational change of the fusion protein –> irreversible transition from metastable activated prefusion form to its lowest energy, postfusion conformation
Explain class I fusion protein hemaglkuttinin of infleunza
- HA0 is elongated conformation (trimer)
- proteolytic cleavage of HA0 by extracellular ptotease into HA1 and HA2
- HA2 is an pH dependent metastable conformational state
- receptor binding leads to uptake into vesicle (endosome)
- acidification
- triggering step: conformational change towards hairpin structure –> HA shortened
- approach between membrane
- membrane fusion
is a pH required for influenza HA and coronavirus spike protein (fusion proteins class I)?
for influenza yes
for sars no
How many cleavage events are necessary for the fusion proteins spike of sars?
2
describe the mechanism of class I fusion proteins
- receptor binding or pH change
- extension of fusion protein
- insertion of fusion protein into host cell membrane (PM or endosomal), transmmebnrane anchoring into viral memebrane essential
- formation of local patches
- conformational changes of fusion proteins (shoertening)
- fusion
Explain HIV fusion
- attachment/receptor binding via gp 120
- coreceptor binding
- gp120 release
- fusion mediated by exposed gp41
–> gp41 zipping: helical HR2 domain folds back and associates with helical HR1 domain –> increased intimacy
What is the mode of action of neutralizing antibodies in influenza virus?
inhibition of fusion
Explain the fusion of viral and cellular membranes in class II fusion proteins (fp)
- receptor binding leads to uptake into endosome
- fp lays flat on membrane of virus and is shielded
- low pH makes the fusion protein accessible
- lateral rearangements into trimers
- insertion of fp into target membrane
- conformation change in and between e domains of fp leads to membrane approach
- hemifusion
- fusion
Explain the role of helper proteins in class II fusion proteins
- co-translationalassociation with helper protein
- helper protein block fusion activity to protect virus from premature fuison
- proteolytic cleavage of helper protein is required for fusion activity
- due to pH in secretory pathway, helper virus stays associated even after proteolysis for further protection
- peptide released in extracellular milieu
What is the trick behind capturing a flavivirus pre fusion intermeidate of fusion protein
antibodiy blocks formation of fusion active E trimers
How can the virion radius be widened?
pH dependently
describe the maturation of flaviviurs particles, especially pr
pr= protection from fusion during budding/ shields fusion loop
via secretory pathway
- ER: immature, trimer, prM-E bound
- TGN: immature, dimer, prM-E cleaved by furin into pr and M, but pr still associated to fusion protein
- extracellular: mature, dimer, pr dissociates (pH dependent)
What leads to a 1000 fold increase in infectivity in dengue virus (cell culture)
furin treatment
How can dengue virus be neutralized by neutralizinmg antibodies?
locking E protein dimers
What are the characteristics of class III fusion proteins?
- trimeric in pre and post fusion state
- no cleavage event needed at all for conformational changes required for fusion
- no terminal fusion peptide but fusion loop
- reversible, pH depoendent conformational change due to reversible protonation of His residues
what is needed for fusion protein mediated fusion?
conformational change
which fusion protein classes need a pH change?
II and III definitely
in I pH change can trigger attachement in Infleunza, but SARS does not necessarily need ph Change (receptor mediated)
How do non enveloped viruses enter the cell?
- trafficking to membrane penetration site
- receptor binding
- conformational change
4a. exposure of hydrophic surfaces, membrane binding and disruption OR
4b. release of lytic factpors and membrane disruption - (a and b): membrane penetration
Name Viurs, trigger and membrane interaction for example viruses
- polio virus –> pvr (cell receptor)–> Vp1/4 channel for RNA injection
- reovirus –> endosomal cell protease (cathepsin B/L) –> conformational change to IVSP form (µ1N on surface)
–> autolysis –> µ1N released (soluable) for poreformation - Rotavirus: cell protease trypsin –> VP5 and hydrophobic loop exposed –> pore formation
- adenovirus –> low pH –> protein IIIa and protein VI released as soluble factors for pore formation
- polyomavirus –> cell chaperone ERp29 –> VP2/3 exposed —> interaction with ER membrane
what plays an essential role in membrane penetration of non-enveloped viruses?
peptides which can insert into target membrane
How can parvoviruses (non enveloped) escape from the endosome?
deploying of a phospholipase A2 domain located at the N-terminus of VP1
describe the reovirus coded cell-cell fusion machinery
=viral spreading via syncytia formation
they are reovirus fusion associated small transmembrane (FAST) proteins
- NSP (not in virion)
- only mediate fusion step
- active actin remodelling in cell required
- spatial proximity of the membrane due to cellular adhesion
How are viruses transported to the nucleus?
- via dynein and microtubuli (herpes virus, Adeno) –> use of nuclear pore complex or directly passing through nuclear membrane
How is adenovirus DNA genome released into the nucleus
- fiberprotein beinds to receptor
- interaction of penton base protein with integrin receptor on cell leads to endocytosis
- acid dependent partial disassembly
- capsid release and transport via microtubuli to nuclear poire compßlex
- binding to CAN/Nup124
- bindin g of cellular histone H1 to capsid leads by binding of importin7 and beta zo disassembly
- import of viral DNA into nucleus
how can parvoviruses lead to the break down of nuclear envelope?
by activating key anzymes of mitosis
HOw can feline calcivirus escape the endosome?
hydrophobic N termini of VP2 insert themself into the endosomal membrane and form a channel through which the genome may be released
How can herpes virus genome be inserted into the nucleus?
ATP driven packaging motor located at capsid vertex generates high internal capsid pressure
Match viruses and the membrane these viruses assemble and bud
PM, intermediate compartment, ER, golgi
PM: Alpha, retro, Rhabdo, Orthomyxo, Paramyxo
vesicular transport to PM and release via exocytosis:
- intermediate compartment: SARS
- ER: hepadna
- ER/golgi: flavivirus
which viral elements are essential for budding? Name one example virus
- type I: spike and NC dependent (alpha)
- type II: gag-protein (retro)
- type III: M and E protein (SARS)
- type IV: M and/or spike protein (ortho, paramyxo)
what is rewuired for influenza assembly and egress?
HA and M2
M2 is essential for pinch off
describe herpes assembly
- translation of viral proteins in cytoplams
- import via nuclear pores
- assemb ly of b-capsid with scaffold proteins
- viral protease cleaves scaffold and thus allows DNA entry
- nuclear agress complex formation (UL50 and UL53)
- binding and bending of nuclear membrane by complex
- molecular mimicry of cellular kinase to pass through nuclear membrane
- entry into golgi
- envelopment with golgi membrane (this temains)
- egress out of golgi via secretory vesicles
What is the vacuolar protein sorting pathway (VPS) and how can it be abused by viruses?
VPS= cellular budding process in the lysosomal degradation pathway for membrane proteins
–> machinery for vesicle budding already exists in cell (ESCRT) –> viurses mediate a conversion to a new function at the cell membrane
–> two steps:
1. formation of a budding pore
2. membrane fusion
this functions via Gag, that recruits ESCRT from the endosome top PM so budding happens out of the cell and not into MVB
What are the pulling and pushing forces of virus assembly and egress?
pulling: membrane microdomains, viral proteins
pushing: host cell proteins/machinery
How do alphaviurs, influenzavirus and vsv assemble?
alpha: budding without VPS pathway, integral membrane proteins as driving force
influenza: HA localized in lipöid rafts, high local conmcentration promotes budding. pinch off induced by M2
VSV: M protein can induce vesicle formation in liposomes
