Effects of Blocking Individual Maturation Cleavages in Murine Leukemia Virus Gag

Oshima, Masamichi; Muriaux, Delphine; Mirro, Jane; Nagashima, Kunio; Dryden, Kelly A.; Yeager, Mark; Rein, Alan

doi:10.1128/jvi.78.3.1411-1420.2004

Cited by 35 publications

(39 citation statements)

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“…As well, there are several examples of HIV Gag mutants that form tubes, cones, and spherical cores, including deletions in the p2/SP1 spacer (19,21,22,30). Together with these findings, our data favor the interpretation that Gag molecules are capable of forming either cylindrical or spherical particles, and the regions flanking CA (p10 in RSV, p12 in Moloney MLV, and MA and SP1 in HIV) serve as "conformational switches" that provide the flexibility to allow the intermolecular rearrangements that are needed for capsid assembly (21,32,35,49,52).…”

Section: Discussionsupporting

confidence: 69%

“…These domains are insufficient, however, to determine the normal organization of the mature virus core. Additional sequences, including CA and its flanking regions, are also necessary (21,32,35,49,52).In addition to these defined assembly domains, independent subcellular trafficking signals that provide important functions in the virus life cycle have been identified in Gag proteins. In the Gag protein of Rous sarcoma virus (RSV), there are two distinct nuclear localization signals (NLSs) within the MA and NC domains and a nuclear export signal (NES) in the p10 cleavage product located between p2 and CA ( Fig.…”

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confidence: 99%

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Overlapping Roles of the Rous Sarcoma Virus Gag p10 Domain in Nuclear Export and Virion Core Morphology

Scheifele¹,

Kenney

Cairns

et al. 2007

J Virol

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Nucleocytoplasmic shuttling of the Rous sarcoma virus (RSV) Gag polyprotein is an integral step in virus particle assembly. A nuclear export signal (NES) was previously identified within the p10 domain of RSV Gag. Gag mutants containing deletions of the p10 NES or mutations of critical hydrophobic residues at positions 219, 222, 225, or 229 become trapped within the nucleus and exhibit defects in the efficiency of virus particle release. To investigate other potential roles for Gag nuclear trafficking in RSV replication, we created viruses bearing NES mutant Gag proteins. Viruses carrying p10 mutations produced low levels of particles, as anticipated, and those particles that were released were noninfectious. The p10 mutant viruses contained approximately normal amounts of Gag, Gag-Pol, and Env proteins and genomic viral RNA (vRNA), but several major structural defects were found. Thin-section transmission electron microscopy revealed that the mature particles appeared misshapen, while the viral cores were cylindrical, horseshoe-shaped, or fragmented, with some particles containing multiple small, electron-dense aggregates. Immature virus-like particles produced by the expression of Gag proteins bearing p10 mutations were also aberrant, with both spherical and tubular filamentous particles produced. Interestingly, the secondary structure of the encapsidated vRNA was altered; although dimeric vRNA was predominant, there was an additional high-molecular-weight fraction. Together, these results indicate that the p10 NES domain of Gag is critical for virus replication and that it plays overlapping roles required for the nuclear shuttling of Gag and for the maintenance of proper virion core morphology.Retroviral assembly is directed by the viral Gag polyprotein precursor. Despite containing very little sequence homology, all Gag proteins share three canonical domains, the MA (matrix), CA (capsid), and NC (nucleocapsid) domains, as well as additional regions that vary for each retrovirus. Immature virus particles contain unprocessed Gag and Gag-Pol fusion proteins arranged in a concentric array just inside the viral envelope, with the NC domain of Gag toward the center, bound to the dimeric, positive-sense viral RNA (vRNA) genome (4, 18, 51). The cleavage of Gag during the maturation process occurs during or immediately after budding, leading to a dramatic change in morphology, with the appearance of an electrondense core near the particle center. This dense core is comprised of vRNA coated by the NC protein, together forming the ribonucleoprotein (RNP) complex. The RNP is enclosed by a shell composed of CA multimers forming the virion core, which is in turn surrounded by the MA protein in association with the lipid envelope (reviewed in references 45 and 46). The molecular events that govern RNP and core formation during virus assembly and maturation are still not well understood.Functional mapping of the Gag polyprotein identified three discrete motifs, known as assembly domains, that coordinate virion formation and...

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Section: Discussionsupporting

confidence: 69%

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confidence: 99%

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Overlapping Roles of the Rous Sarcoma Virus Gag p10 Domain in Nuclear Export and Virion Core Morphology

Scheifele¹,

Kenney

Cairns

et al. 2007

J Virol

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“…A recent comparison of the region corresponding to SP1 in immature lattices of HIV, RSV, and MPMV reveals somewhat different geometries (24). However, in all retroviruses tested, including avian leukosis virus (37,38), bovine immunodeficiency virus (33), and murine leukemia virus (MLV) (16,53), as well as HIV-1, changes at or near the C terminus of the CA domain are extremely detrimental for correct particle assembly. In MLV, which does not contain a spacer between CA and NC, the C-terminal end of CA consists of an extraordinary run of charged residues, termed the "charged assembly helix" or "electric wire" motif (16).…”

Section: Discussionmentioning

confidence: 99%

On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

Datta

Temeselew

Crist

et al. 2011

J Virol

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115

156

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Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an ␣-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.

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“…By analogy with thermal melting studies, 99,106,107 the equilibrium melting force (F m ) may be shown to change with protein binding (ignoring double-strand binding) 23,77,99 ;…”

Section: Figurementioning

confidence: 99%

Mechanisms of DNA binding determined in optical tweezers experiments

McCauley¹,

Williams²

2006

Biopolymers

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The last decade has seen rapid development in single molecule manipulation of RNA and DNA. Measuring the response force for a particular manipulation has allowed the free energies of various nucleic acid structures and configurations to be determined. Optical tweezers represent a class of single molecule experiments that allows the energies and structural dynamics of DNA to be probed up to and beyond the transition from the double helix to its melted single strands. These experiments are capable of high force resolution over a wide dynamic range. Additionally, these investigations may be compared with results obtained when the nucleic acids are in the presence of proteins or other binding ligands. These ligands may bind into the major or minor groove of the double helix, intercalate between bases or associate with an already melted single strand of DNA. By varying solution conditions and the pulling dynamics, energetic and dynamic information may be deduced about the mechanisms of binding to nucleic acids, providing insight into the function of proteins and the utility of drug treatments. © 2006 Wiley Periodicals, Inc. Biopolymers 85:154–168, 2007.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Effects of Blocking Individual Maturation Cleavages in Murine Leukemia Virus Gag

Cited by 35 publications

References 50 publications

Overlapping Roles of the Rous Sarcoma Virus Gag p10 Domain in Nuclear Export and Virion Core Morphology

Overlapping Roles of the Rous Sarcoma Virus Gag p10 Domain in Nuclear Export and Virion Core Morphology

On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

Mechanisms of DNA binding determined in optical tweezers experiments

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