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 assembly Gag 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) Purified Gag with Yeast tRNA forms Particles too Small for a Virion (shell thickness ~70Å) 280 Å ~200 Å PNAS 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 chromium gold 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 Reflectivity Profile Shows Changes with Increasing Concentration of MA Comparison of Scattering Length Density fits to Reflectivity using a Box Model and Atomistic Structures Width in the Free-Form fit profile represent 95% Confidence Intervals Matrix Binding to a Membrane Mimic MA Lys+ 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. D2OD2O CM4 H2OH2O

Au tether lipid heads lipid tails pure bilayer Gag folded Gag + TG14 Gag + 500mM NaCl 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.

Conclusions MA -myr domain can target an anionic membrane with an orientation 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 Curtis Chuck Majkrzak Joe Dura Sidd Shenoy