Conformational changes of Gag HIV-1 on a tethered bilayer provide insights into viral assembly

Hirsh Nanda, Susan Krueger

NIST Center for Neutron Research, Gaithersburg, MD, USA

Frank Heinrich, Mathias Loesche

Carnegie Mellon, Pittsburgh, PA, USA

Siddhartha .A.K. Datta, Alan Rein

National Cancer Institute, Frederick, MD, USA

Viral Life Cycle

• HIV rapidly develops drug resistance

• Patients are given multi-drug treatments, targeting several stages of the viral life cycle

• No anti-virals target assemblyGag protein mediates viral formation

• Binds other viral components• Self-assembles on the cellular

membrane

Matrix Domain of Gag Binds the Membrane

- - - - -+ + + Matrix (MA): Targets the

membrane - basic residues interact favorably with charged lipids - myristate group inserts in bilayer

Capsid (CA): Lateral organization of Gag proteins on the membrane surface

Nucleocapsid (NC): Binds viral RNA, brings viral genome into assembling particle

PNAS (2006) v103(8) p2641

Viral Membrane:• 20% anionic lipids • Enriched in cholesterol• Specialized lipids such as PIP / celluar targeting of MA

Conformational Variability in Gag

SANS Shows Gag is Compact in Solution (Rg 35-38Å) Gag Bound to Single Stranded DNA is also compact (Rg 43-45Å)

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Purified Gag with Yeast tRNA forms Particles too Small for a Virion (shell thickness ~70Å)

280 Å

~200 ÅPNAS 95: 7299, 1998

Immature Virion Shows Gag Extended on the Membrane

1100 Å

• Tether anchors membrane to substrate

• PEO spacer decouples bilayer from gold surface

• Fluid bilayer is highly stable• exchange of aqueous phase• proteins and other molecules can access outer leaflet

Tethered Bilayer Membranes (tBLM)Bio-mimetic environment for studying protein-lipid interactions

silicon

chromiumgold

bulk solvent

Ternary Lipid Mixture:• 30% anionic DMPS necessary to promote MA binding: lacks the myristate

• 70% DMPC & small amounts of cholesterol aided in forming complete bilayers

Basic residue patch (blue) suggests a putative binding orientation

Matrix Potein: Side View

4

6

0.1

2

4

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1

2

4

6

10

Fresnel normalized reflectivity,

R/RF

0.250.200.150.100.05

momentum transfer, Qz (Å-1)

2

1

0

-1

Difference withneat bilayer

neat bilayer 1 µM Gag MA 10 µM Gag MA

Reflectivity Profile Shows Changes withIncreasing Concentration of MA

Comparison of Scattering Length Density fits to Reflectivity using a Box Model and Atomistic Structures

Matrix Binding to a Membrane Mimic

MALys+ Arg+

Full Length Gag on the Membrane

• Gag Bound at 1uM• Bound Gag incubated w/ TG14 (14 base DNA sequence with nM affinity at NC)

• High Salt Rinse (500mM NaCl)• Protein modeled using a multi-box model. Fitting

for all measurements and contrasts was conducted simultaneously. D2O

CM4

H2O

-0.5

0

.5

1

1.5

2

2.5

nSLD,

ρ, (10-6 Å-2)

0 50 100 150 200 250 300 , , ( )Distance from Au Surface z Å

-0.5

0

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1.5

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2.5

nSLD,

ρ, (10-6 Å-2)

0 50 100 150 200 250 300 , , ( )Distance from Au Surface z Å

-0.5

0

.5

1

1.5

2

2.5

nSLD,

ρ, (10-6 Å-2)

0 50 100 150 200 250 300 , , ( )Distance from Au Surface z Å

-0.5

0

.5

1

1.5

2

2.5

nSLD,

ρ, (10-6 Å-2)

0 50 100 150 200 250 300 , , ( )Distance from Au Surface z Å

Autether

lipidheads

lipidtails

pure bilayerGag foldedGag + TG14Gag + 500mMNaCl Rinse

(1) Gag bound to the membrane adopts a compact structure (red).

(2) Introducing single stranded TG7 DNA causes an extended profiile (blue).

(3) Partial recovery of compact Gag is possible by a high salt buffer (green).

Gag Extension on the Membrane

Model for Gag Extension

RNA

MA

CA

Gag

NC

- - - - - -

- Gag is folded on the membrane or bound to viral RNA through favorable electrostatic interactions.

- However cross-linking of Gag on the membrane surface by the RNA strand causes extension.

ConclusionsMA-myr domain can target an anionic membrane with anorientation equivalent to the native MA protein

Investigate role of membrane composition and other co-factors

(ex PIP-lipids) in Gag assembly

Conformational changes in gag are observed when bound to TG14 cross linking DNA

Suggests Gag extension requires both the membrane and the cross-linking nucleic acid

Neutralized NC domain mutants to show role in binding to

charged lipid

Acknowledgements

Joseph CurtisChuck MajkrzakJoe Dura

Sidd Shenoy