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 (VLPs) 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 will study this concentration-dependent change and its possible role in assembly. We will also perform analogous experiments with a peptide from MLV Gag, in which the sequence at the CA-NC border is radically different from that in HIV-1. b. Template-directed in vitro particle assembly In collaboration with Dr. Bogdan Dragnea, we have found that HIV-1 Gag assembles more uniformly on nanoparticle """"""""templates"""""""" than out of free solution. We will use this phenomenon to improve the resolution of cryoelectron microscopy (cryo-EM) images of particles. 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 Losche, we will analyze the binding of HIV-1 MA and Gag to artificial membranes of defined composition under well-controlled experimental conditions. Patent 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]

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National Cancer Institute (NCI)
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Barros, Marilia; Heinrich, Frank; Datta, Siddhartha A K et al. (2016) Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation. J Virol 90:4544-55
Dick, Robert A; Datta, Siddhartha A K; Nanda, Hirsh et al. (2015) Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein. J Virol 89:10371-82
Grohman, Jacob K; Gorelick, Robert J; Kottegoda, Sumith et al. (2014) An immature retroviral RNA genome resembles a kinetically trapped intermediate state. J Virol 88:6061-8
Munro, James B; Nath, Abhinav; Färber, Michael et al. (2014) A conformational transition observed in single HIV-1 Gag molecules during in vitro assembly of virus-like particles. J Virol 88:3577-85
Stavrou, Spyridon; Nitta, Takayuki; Kotla, Swathi et al. (2013) Murine leukemia virus glycosylated Gag blocks apolipoprotein B editing complex 3 and cytosolic sensor access to the reverse transcription complex. Proc Natl Acad Sci U S A 110:9078-83
Rein, Alan (2013) Murine leukemia virus p12 functions include hitchhiking into the nucleus. Proc Natl Acad Sci U S A 110:9195-6
O'Carroll, Ina P; Soheilian, Ferri; Kamata, Anne et al. (2013) Elements in HIV-1 Gag contributing to virus particle assembly. Virus Res 171:341-5
O'Carroll, Ina P; Crist, Rachael M; Mirro, Jane et al. (2012) Functional redundancy in HIV-1 viral particle assembly. J Virol 86:12991-6
Jones, Christopher P; Datta, Siddhartha A K; Rein, Alan et al. (2011) Matrix domain modulates HIV-1 Gag's nucleic acid chaperone activity via inositol phosphate binding. J Virol 85:1594-603
Datta, Siddhartha A K; Temeselew, Lakew G; Crist, Rachael M et al. (2011) On the role of the SP1 domain in HIV-1 particle assembly: a molecular switch? J Virol 85:4111-21

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