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Retrovirus Assembly and Maturation – Rockville

Alan Rein

1 Collaborator(s)

Funding source

National Cancer Institute (NIH)
We have continued our analysis of assembly and maturation of HIV-1 and murine leukemia virus (MLV) particles. Specifically, we have characterized the properties of the HIV-1 Gag protein, the fundamental building block of the HIV-1 virus particle. We found that the Gag protein is in monomer-dimer equilibrium in solution. We have continued our analysis of assembly and maturation of HIV-1 and murine leukemia virus (MLV) particles. Specifically, we have characterized the properties of the HIV-1 Gag protein, the fundamental building block of the HIV-1 virus particle. We found that the Gag protein is in monomer-dimer equilibrium in solution. We identified the interface responsible for the dimerization; it is a site previously described as the site at which the HIV-1 capsid protein (which forms a portion of Gag) dimerizes. We also found that inositol hexakisphosphate (IP6), which modulates the nature of the virus-like particles assembled from Gag when nucleic acid is added, causes a shift from monomer-dimer to monomer-trimer equilibrium. Several lines of evidence indicate that when both nucleic acid and IP6 are present, the nucleic acid binds to the C-terminal, nucleocapsid (NC) domain of the protein while the IP6 binds to the N-terminal matrix (MA) domain. _____By mutating the dimer interface in Gag, we generated a protein which remains monomeric in solution. We then used 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, to characterize the conformation of this protein. All of the data indicate that the protein is folded over in solution, with its ends near each other in 3-dimensional space. In contrast, the protein is known to be a highly extended rod in assembled virus particles; thus, it must undergo a major conformational change when it assembles. _____We have analyzed the assembly properties of chimeric proteins in which the C-terminal domains of HIV-1 Gag, including the NC domain, is replaced by a leucine-zipper domain. These proteins assemble into virus-like particles in vivo; these particles are morphologically almost identical to those assembled from wild-type Gag, but appear to contain no RNA. These proteins can coassemble with wild-type Gag. When purified from bacteria, these chimeric proteins are oligomeric, but do not assemble unless a cofactor is added; the cofactors can be either RNA or inositol phosphates, and probably function to neutralize positive charges in the MA and/or CA domains. Normal assembly by wild-type Gag presumably also requires these neutralizing cofactors, but this requirement was not apparent in experiments with wild-type Gag because RNA is also required for NC binding in the wild-type case. _____We are performing a similar analysis of the MLV Gag protein. Remarkably, we find that the properties of this protein are quite different from those of HIV-1 Gag. Thus, it does not oligomerize in solution, and is not folded over in solution. Rather, it appears to be an extended rod, with approximately the same dimensions in solution as in the assembled virus particle. _____Recent Accomplishments and Current Research: _____a. Analysis of the function of the SP1 domain in HIV-1 assembly. Proper HIV-1 particle assembly is extremely sensitive to changes in SP1. We found that an 18-residue peptide representing SP1 adopts an alpha-helical conformation when it is at high concentration, but not at low concentration. We proposed that SP1 is a "switch" that changes conformation when Gag oligomerizes on RNA. We have studied the associative properties of SP1 in small protein constructs; the results indicate that SP1-SP1 interactions can contribute to the assembly of HIV-1 immature particles. We will study the properties of the corresponding region of Moloney murine leukemia virus (MLV) Gag, in which the sequence at the capsid-nucleocapsid (CA-NC) border is radically different from that in HIV-1. _____b. Template-directed in vitro particle assembly. In collaboration with Drs. Bogdan Dragnea and John Briggs, we have found that HIV-1 Gag assembles more uniformly on nanoparticle "templates" than out of free solution. We are using this phenomenon to improve the resolution of cryoelectron microscopy (cryo-EM) images of particles and analyze the packing of Gag molecules on the curved surface of the particle. We will also determine whether mutants that fail to assemble correctly in vivo can form spherical particles in this novel experimental situation. _____c. Characterization of MLV Gag protein. We found that MLV Gag protein has a far weaker tendency to dimerize than HIV-1 Gag. Also unlike HIV-1 Gag, MLV Gag is a rod in solution, with approximately the same dimensions as it has in a virus-like particle (VLP). Short proline runs in matrix (MA) and p12, and the "electric wire" near the end of CA, contribute to its rigidity. _____d. Analysis of conformation of HIV-1 Gag within mammalian cells. We have shown that HIV-1 Gag is folded over in solution, but extends when it assembles into full-size VLPs or when preferred ligands for both the MA and NC domains are present. We will determine cellular locations of the folded and extended forms by fluorescence resonance energy transfer (FRET). _____e. Binding of HIV-1 MA and Gag proteins to artificial membranes. In collaboration with Drs. Hirsh Nanda and Matthias Loesche, we are continuing to analyze the binding of HIV-1 MA and Gag to artificial membranes of defined composition under well-controlled experimental conditions. _____Patents Linked to Project: U.S. Patent #6,001,555: "Method for Identifying and Using Compounds That Inactivate HIV-1 and Other Retroviruses by Attaching Highly Conserved Zinc Fingers in the Viral Nucleocapsid Protein"; issued December 14, 1999; Louis E. Henderson, Larry O. Arthur, William G. Rice, and Alan Rein. Describes compounds that inactivate retroviruses, including HIV-1, by reaction with internal viral components. The inactivated particles have completely native outer surfaces and are useful for many immunological experiments, as well as for conceivable vaccine use. _____[Corresponds to Rein Project 1 in the October 2011 site visit report of the HIV Drug Resistance Program (renamed HIV Dynamics and Replication Program in 2015)]

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