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. [Corresponds to Rein Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]

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National Cancer Institute (NCI)
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Comas-Garcia, Mauricio; Datta, Siddhartha Ak; Baker, Laura et al. (2017) Dissection of specific binding of HIV-1 Gag to the 'packaging signal' in viral RNA. Elife 6:
Saxena, Pooja; He, Li; Malyutin, Andrey et al. (2016) Virus Matryoshka: A Bacteriophage Particle-Guided Molecular Assembly Approach to a Monodisperse Model of the Immature Human Immunodeficiency Virus. Small 12:5862-5872
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 90:4544-4555
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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
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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

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