harrison powerpoint pt2

7/31/2019 Harrison Powerpoint Pt2

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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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sialic-acid binding site



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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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harrison powerpoint pt2

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