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Enveloped viruses acquire membrane !by budding!
alphaviruses influenza virus
influenza virusalphaviruses
(e.g., Sinbdis virus)
HANA
RNP M
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Enveloped viruses penetrate cells
by fusion of viral and cellular membranes
Influenza: enters through endosomes,depends on low pH of endosome
to initiate fusion process
HIV: can fuse at cell surface, depends
on receptor (CD4) and co-receptorto initiate fusion process
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What is membrane fusion?
two bilayers hemifusion fusion pore
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Sequence of events in viral membrane fusion
Prefusion Extended
intermediate
Collapse of
intermediateHemifusion Fusion pore
(postfusion)
Cell
Virus
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Envelope glycoprotein
F TM
Receptor binding domain Fusion molecule
Orthomyxoviridae
Influenza A
Retroviridae
HIV-1
Filoviridae
Ebola
"Class 1" viral fusion proteins
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500 !
(X 1 million = golf ball)!
Electron micrograph!of influenza virus!
(X 5 million = golf tee)!hemagglutinin (HA) neuraminidase (NA)!
Influenza virus particle!
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hemagglutinin (HA): !three functions
!1. receptor binding!
2. antigenic variation!3. fusion
!
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hemagglutinin (HA): !synthesized as
precursor:!
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Influenza virus hemagglutinin
viral membrane
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Influenza virus hemagglutinin
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sialic-acid binding site
Influenza virus hemagglutinin
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HA2 N-
HA2 C- -N HA1
-C HA1
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Low-pH triggered conformational change
HA trimer: pH 7 pH
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HA0 post-translationalcleavage
neutral pH
conformation
metastablelow pH
conformation
energy
HA undergoes two irreversible changes ...
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...the second change catalyzes membrane
fusion
cell
virus
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Fusion mechanism
A. Cleaveprecursor (prime)B. Localizevirus to cell
(by receptor binding)C. Triggerrefolding
(by co-receptor, low pH, etc.)1.Exposefusion peptide2.Insertfusion peptide into
target membrane
3. Fold backto bring togethertarget and viral membranes
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trigger expose insert fold back
...the second change catalyzes membrane
fusion
cell
virus
cap
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Similarity among post-fusion structures
low pHinfluenza
eHA2
SIV gp41(NMR)
ebola GP2HTLV-1gp21
TM anchors andfusion peptides
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Gal McGill
molecularmovies.com
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Floyd et al, 2008
Single-virion fusion measurements
fluorescein
pH sensor
100 nm
ganglioside
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Histograms of times to hemifusion (lipid mixing)
and pore formation (content mixing)
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OR N independent parallel steps
Two-parameter kinetic scheme: N, k
N sequential steps
A X1 X2 XN-1 Hk k k k k
A Hk
A Hk
A Hk
....
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Best fits to observed histograms
N=3 (time to hemifusion)
interpret as number of HA
trimers required to create afusion pore
N=1 (exponential)
(hemifusion decay: time from
hemifusion to pore formationfor individual particles -- single
rate-limiting step)
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pH dependence
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pH dependence
N
k
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Influenza HA fusion
H+
Native HA1 open HA2 extended HA2 collapseH+
~20 sec
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Mutations here
accelerate
fusion
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Influenza HA fusion
H+
Native HA1 open HA2 extended HA2 collapseH+
~20 sec
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