Understanding the molecular mechanisms underlying virus particle assembly and maturation is of critical importance for several reasons. First, it can serve as a model for the broader problem of complex macromolecular assembly, with the particular virtue that the relevant structures are foreign to the cell but will be assembled in the cellular context, with the help of cellular constituents. Second, it offers many potential targets for antiviral therapy, which have not been exploited to date because we lack the detailed knowledge needed for the development of therapeutic strategies. _____In this project, we have continued our analysis of the assembly and maturation of HIV-1 particles. Specifically, we have characterized the properties of the HIV-1 Gag protein, the fundamental building block of the HIV-1 virus particle. Gag assembles in mammalian cells into the immature virus particle, containing several thousand Gag molecules. Similarly, recombinant HIV-1 Gag protein, purified from bacteria, can assemble into virus-like particles (VLPs) in a defined system in vitro; this shows that the protein will, upon interaction with a single-stranded nucleic acid (NA), engage in rather regular protein-protein interactions, forming a roughly spherical structure with a rather uniform radius of curvature. The arrangement of Gag molecules is the same in particles assembled in vitro and in cells, and many mutants of Gag have analogous effects in the two systems. We study assembly of this protein both in vitro and in mammalian cells, where Gag is obviously far more dilute than in vitro and is surrounded by myriad cellular constituents. While the structures in the assembled particle have been defined to a considerable degree, little is known about the steps by which a Gag molecule is converted from a free, soluble protein to a molecule ready to assemble and then to a constituent of the final, assembled structure. We are addressing the following questions: What is the structure of an HIV-1 Gag molecule, and how does this structure enable it to coassemble, with other Gag molecules and with nucleic acid, into immature retrovirus particles? If the structure of a Gag molecule in solution is different from that in an immature virion, what controls this difference? How is particle assembly triggered by binding to nucleic acid? How are Gag proteins and virions from different retroviral genera similar to each other and how do they differ? Do these differences affect the control of assembly? _____We have exploited the simplicity of the in vitro system to analyze the molecular changes accompanying HIV-1 Gag assembly. Using a wide variety of experimental approaches, including gel filtration, static and quasi-elastic light-scattering, sedimentation velocity analysis, and small-angle neutron scattering, as well as molecular modeling, we have characterized the conformation of the HIV-1 Gag protein. We found that when Gag molecules are brought into close proximity, they undergo a major conformational change that prepares them for assembly. All of the data indicate that HIV-1 Gag protein is folded over in solution, with its ends near each other in three-dimensional space, in striking contrast to its rod-like shape in immature particles. We have recently focused on conformational changes in the SP1 domain of Gag, a short linker between the capsid (CA) and nucleocapsid (NC) domains. Our data show that SP1 assumes an alpha-helical conformation when two or more molecules containing SP1 are brought into juxtaposition. This change occurs because SP1 can form an amphipathic helix; association of several such helices produces helical bundles in which hydrophobic residues face inward, shielded from the solvent. We proposed that this change in SP1 apparently leads to changes in the CA domain and exposes new interfaces for the Gag-Gag interactions that are needed to convert Gag to an assembly-ready protein. The data also suggest that the associative interactions between SP1 domains participate, along with those of CA domains, in immature particle assembly. In collaboration with Dr. Eric Freed (HIV Dynamics and Replication Program), we are testing the possibility that small SP1-containing helical bundles will bind maturation inhibitors such as bevirimat; if so, they would be extremely useful for in vitro screens for new inhibitors of HIV-1. _____We are collaborating in a detailed study of the structure and assembly of immature particles of murine leukemia virus (MLV, a gammaretrovirus). The final structure of the MLV particle shows significant differences from that of HIV-1. These differences may reflect the fact that MLV Gag has a far weaker tendency to dimerize than HIV-1 Gag, does not contain SP1, and appears to be converted to an assembly-ready state by a somewhat different pathway from HIV-1. Unlike HIV-1 Gag, MLV Gag is a rod in solution, with approximately the same dimensions as it has in a VLP. Short proline runs in the matrix and p12 domains, and the electric wire near the end of CA, contribute to rigidity of the MLV Gag structure. Analysis of these differences will shed light on the mechanistic principles underlying particle assembly.
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