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October 17, 2006

Molecular Componentsof HIV

RNA

Protein

Lipid

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HIV Life Cycle and the Properties of Membranes

1. Difference between viruses and cellsa. HIV anatomy and genome

2. Overview of HIV life cycle and targets for therapeuticsa. Host cell recognitionb. Entry into host cell

3. Membrane structure and propertiesa. Phospholipidsb. Lipid properties and behavior in aqueous solutionc. Membrane fluidityd. How we know: FRAP and green fluorescent proteine. Influence of fatty acid structure on fluidityf. Influence of cholesterol on membrane properties

Alberts 221-222, 365-388;McMurry 744-767, 808-810

Lecture Readings

The Anatomy of HIV

HIV viralparticle

capsid

viral RNA moleculecoated with

structural protein

glycoproteins

membrane

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Cells Can Replicate AutonomouslyBut Viruses Cannot

WHY?

HIV Genome and Proteins

HIV genome

HIV viralparticle

capsid

viral RNA moleculecoated with

structural protein

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HIV Life Cycle

1 2 3 4

recognition of host cell

entry intocell

viralamplification/

replication

viral assemblyand exit from cellHIV

host cellnucleus

1 2 3 4

recognition of host cell

entry intocell

viralamplification/

replication

viral assemblyand exit from cellHIV

host cell

HIV Targets for Therapeutics

reverse transcriptaseinhibitors

fusioninhibitors

protease inhibitors

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Recognition of Appropriate Host Cell

macrophageT-cell

HIV

CD4

chemokine receptor

chemokine

membrane

nucleus

CHEMOKINERECEPTOR

BINDING

Recognition of Appropriate Host Cell

HIV

membrane

gp41

gp120CD4

FREE VIRUS

host cell membranechemokine

receptor

CD4ATTACHMENT

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Entry into Host Cell

membrane

membrane

glycoproteinsgp120gp41

host cell

diameter >10 µm

diameter~100 nm

Cell Membranes

cytoplasm

outside of cell

plasmamembrane

electron micrograph

~10 nm long

(covalent bond ~ 0.1 nm)

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Phospholipids: Phosphatidylcholine

saturated unsaturated

O

O

OPO

O

N

O

O

cis double bond

O

Hydrophobic Effect

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Packing of Lipids in Aqueous Solutions

conical

cylindrical

Spontaneous Closure to Form a Sealed Compartment

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Phospholipid Mobility

leaflet

leaflet

Animation: Membranes are Dynamic!

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How Do We Know?: An Experiment

fluorescence recovery afterphotobleaching (FRAP)

fluorescentmolecule

protein inmembrane

cell

Green Fluorescent Protein (GFP) Fusions

fluorescentproteins CFP

GFPYFP

jellyfish

cell

nucleus

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FRAP With Membrane Protein-GFP Fusion

• Tm (melting temperature) is a phase transition, a changefrom a more rigid solid-like state to a fluid-like state

Measures of Membrane Fluidity: Melting Temperature

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unsaturated fatty acid chains

Membrane Composition Influences MembraneFluidity: Fatty Acid Structure

saturated fatty acid chains

lower Tm higher Tm

cis double

bond

oleic acid

OH

O O

OH

stearic acid

17 carbons

olive oil candle wax

Membrane Composition Influences MembraneFluidity: Cholesterol Content

HO

H

H

H

H

H

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Cholesterol Influences Membrane Fluidity

Summary of Main Points

• HIV needs host cells to replicate because its genome does not encodeall of the proteins required for living systems

• HIV recognizes host cells by interacting with specific protein receptorsfound on the surface of those cell types

• Cell membranes are bilayers composed of amphipathic phospholipidscontaining charged head groups and hydrophobic tails

• The hydrophobic effect drives the packing of lipids into structures whichminimize exposed hydrophobic groups

• Membranes are fluid because phospholipids and proteins can move inthe plane of the bilayer; fatty acid structure and cholesterol contentinfluence fluidity