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J. Biol. Chem., Vol. 280, Issue 18, 17664-17670, May 6, 2005

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Assembly of Human Immunodeficiency Virus Precursor Gag Proteins^*

Doug Huseby, Robin Lid Barklis, Ayna Alfadhli, and Eric Barklis

From the Vollum Institute and Department of Microbiology, Oregon Health and Science University, Portland, Oregon 97201-3098

To investigate the mechanism by which human immunodeficiency virus (HIV) precursor Gag (PrGag) proteins assemble to form immature virus particles, we examined the in vitro assembly of MACANC proteins, composed of the PrGag matrix, capsid, and nucleocapsid domains. In the absence of other components, MACANC proteins assembled efficiently at physiological temperature but inefficiently at lower temperatures. However, the addition of RNA reduced the temperature sensitivity of assembly reactions. Assembly of MACANC proteins also was affected by pH because the proteins preferentially formed tubes at pH 6.0, whereas spheres were obtained at pH 8.0. Because neither tubes nor spheres were amenable to analysis of protein-protein contacts, we also examined the membrane-bound assemblies of MACANC proteins. Interestingly, MACANC proteins organized on membranes in tightly packed hexameric rings. The observed hexamer spacing of 79.7 Å is consistent with the notion that more PrGag proteins assemble into virions than are needed to provide capsid proteins for mature virus cores. Our data are also consistent with a model for PrGag contacts in immature virions where capsid hexamers are tightly packed, where nucleocapsid domains align beneath capsid C-terminal domains, and where matrix domains form trimers at the nexus of three neighbor hexamers.

Received for publication, November 1, 2004 , and in revised form, February 15, 2005.

^* This work was supported by NIGMS, National Institutes of Health Grant GM060170 (to E. B.) and American Foundation for AIDS Research (amfAR) Postdoctoral Fellowship Grant 106523-35-RFNT (to A. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Mail Code L220, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97201-3098. Tel.: 503-494-8098; Fax: 503-494-6862; E-mail: barklis{at}ohsu.edu.

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