CONFORMATIONAL CHANGES OF GAG HIV-1 ON A TETHERED BILAYER MEASURED BY NEUTRON REFLECTIVITY PROVIDES INSIGHTS INTO VIRAL PARTICLE ASSEMBLY

 

Hirsh Nanda1, Sidartha A.K. Datta2, Frank Heinrich1, Alan Rein2 and Susan Krueger1

1National Center for Neutron Research, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD 20899, USA. 2HIV Drug Resistance Program-National Cancer Institute, Frederick, MD 21702, USA

 

Formation of the HIV-1 is mediated by the Gag polyprotein at the cytoplasmic membrane surface of the infected host cell. Individual Gag molecules contain several domains connected by flexible linkers. Early cryo-EM data showed Gag in the immature virus as elongated rods radial from the membrane with one termini tightly bound to the viral genome [Current Biology, 1997 (7) p.729]. However, in vitro assembly with purified Gag incubated with nucleic acids, forms virus particles significantly smaller in radius than the natural virion [J. Virology, 1999 (73) p.2270]. Furthermore, solution measurements using SANS and other techniques suggest a compact structure for Gag [J. Mol. Biol. 2007 (365) p. 812]. These studies indicate large conformational changes in the Gag protein must occur concomitant with virus assembly. The dimension of Gag bound to the bilayer interface was determined at high resolution by neutron reflectometry. The bio-mimetic environment for observing Gag association consisted of a supported membrane attached to a gold surface via a PEO tether. The membrane was a ternary composition of DMPS:DMPC:Cholesterol lipids capturing key characteristics of the viral lipodome. The Gag protein bound to the lipid membrane was shown to adopt a folded conformation of ~75. Upon addition of a 14 base pair DNA oligo (TGx7), a significantly thicker protein layer of ~200 was observed. A high salt buffer rinse reversed the conformational change. These results suggest a mechanism by which Gag extension is possible only once bound to the plasma membrane and in the presence of the viral genome. This provides a picture consistent with earlier in vivo and solution studies. A detailed understanding of the viral particle assembly process may elucidate susceptible points providing opportunities to inhibit proper virus formation.

 

Additional Information:

Presenter: Hirsh Nanda

Mentor: Susan Krueger

Division: NIST Center for Neutron Research (610)

Office: NIST Center for Neutron Research (235), Room E130.2

Mail Stop: 6102

Phone: (301) 975-6734

Fax: (301) 921-9847

E-Mail: hirsh@nist.gov

 

Hirsh Nanda is not a member of Sigma Chi