Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association

Andrawiss, Mariam; Takeuchi, Yasuhiro; Hewlett, Lindsay; Collins, Mary

doi:10.1128/jvi.77.21.11651-11660.2003

Cited by 35 publications

(43 citation statements)

References 53 publications

(20 reference statements)

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“…CA is not tolerant to large insertions, probably because of its "cage"-like structure. The constructs MA.GFP, GFP.p12, NC.GFP, and IN.GFP may serve as tools for virus trafficking studies (38)(39)(40)(41)(42)(43)(44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

Protein transduction from retroviral Gag precursors

Voelkel¹,

Galla²,

Maetzig³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

123

118

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Retroviral particles assemble a few thousand units of the Gag polyproteins. Proteolytic cleavage mediated by the retroviral protease forms the bioactive retroviral protein subunits before cell entry. We hypothesized that this process could be exploited for targeted, transient, and dose-controlled transduction of nonretroviral proteins into cultured cells. We demonstrate that gammaretroviral particles tolerate the incorporation of foreign protein at several positions of their Gag or Gag-Pol precursors. Receptor-mediated and thus potentially cell-specific uptake of engineered particles occurred within minutes after cell contact. Dose and kinetics of nonretroviral protein delivery were dependent upon the location within the polyprotein precursor. Proteins containing nuclear localization signals were incorporated into retroviral particles, and the proteins of interest were released from the precursor by the retroviral protease, recognizing engineered target sites. In contrast to integration-defective lentiviral vectors, protein transduction by retroviral polyprotein precursors was completely transient, as protein transducing retrovirus-like particles could be produced that did not transduce genes into target cells. Alternatively, bifunctional protein-delivering particle preparations were generated that maintained their ability to serve as vectors for retroviral transgenes. We show the potential of this approach for targeted genome engineering of induced pluripotent stem cells by delivering the site-specific DNA recombinase, Flp. Protein transduction of Flp after proteolytic release from the matrix position of Gag allowed excision of a lentivirally transduced cassette that concomitantly expresses the canonical reprogramming transcription factors (Oct4, Klf4, Sox2, c-Myc) and a fluorescent marker gene, thus generating induced pluripotent stem cells that are free of lentivirally transduced reprogramming genes.Flp recombinase | murine leukemia virus | pluripotent cells | protein transfer

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Section: Discussionmentioning

confidence: 99%

Protein transduction from retroviral Gag precursors

Voelkel¹,

Galla²,

Maetzig³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

123

118

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“…It had previously been found that when creating pseudovirus with an MLV core containing a Gag-GFP chimera with a cleavage site downstream of NC still intact (see Materials and Methods), GFP alone was predominantly cleaved from a Gag-GFP. There were only minor amounts of unprocessed Gag-GFP and of NC-GFP (Andrawiss et al, 2003). Because GFP is slightly smaller than NC-GFP, this alternate Gag-GFP chimera may be somewhat preferable for future fusion experiments.…”

Section: The Pseudoviral-cell Fusion Systemmentioning

confidence: 99%

“…The Gag-GFP chimera was constructed by inserting GFP in place of Pol, and this eliminated a viral protease cleavage site downstream of NC (Sherer et al, 2003). As a consequence, GFP was associated with NC within the virions, whereas GFP should have been unassociated if the cleavage site were still present (Andrawiss et al, 2003). HIV-1 Rev was expressed from a pCDNA3 plasmid provided by Dr. R. Doms (University of Pennsylvania, Philadelphia, PA).…”

Section: Cells and Reagentsmentioning

confidence: 99%

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Markosyan

Cohen

Melikyan

2005

MBoC

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A method has been developed to follow fusion of individual pseudotyped virus expressing HIV-1 Env to cells by time-resolved fluorescence microscopy. Viral envelopes were labeled with a fluorescent lipid dye (DiD) and virus content was rendered visible by incorporating a Gag-GFP chimera. The Gag-GFP is naturally cleaved to the much smaller NC-GFP fragment in the mature virions. NC-GFP was readily released upon permeabilization of the viral envelope, whereas the capsid was retained. The NC-GFP thus provides a relatively small and mobile aqueous marker to follow viral content transfer. In fusion experiments, virions were bound to cells at low temperature, and fusion was synchronously triggered by a temperature jump. DiD transferred from virions to cells without a significant lag after the temperature jump. Some virions released DiD but retained NC-GFP. Surprisingly, the fraction of lipid mixing events yielding NC-GFP transfer was dependent on the type of target cell: of three infectable cell lines, only one permitted NC-GFP transfer within minutes of raising temperature. NC-GFP release did not correlate with the level of CD4 or coreceptor expression in the target cells. The data indicate that fusion pores formed by HIV-1 Env can remain small for a relatively long time before they enlarge. INTRODUCTIONIn the process of membrane fusion, two continuities are created: separate membranes join and distinct aqueous compartments become one. To identify the mechanisms of membrane fusion, both continuities should be followed and the temporal order in which they occur identified. In the case of the HIV-1 fusion protein Env, mechanistic studies have largely relied on fusion of cells expressing the protein to cells expressing CD4 and cognate chemokine receptors (e.g., Munoz-Barroso et al., 1998;Melikyan et al., 2000;Reeves et al., 2002;Abrahamyan et al., 2003;Gallo et al., 2003;Markosyan et al., 2003). But monitoring lipid dye spread in HIV-1 Env-mediated cell-cell fusion has not proved to be very useful in following membrane continuity, for several reasons. The dye does not reliably move between cells, and when it does move, the time course is slow, in large part because the dye segregates nonuniformly over the cell membrane. Also, the dye enters intracellular pools, making it difficult to follow transfer between fused membranes. Furthermore, in cell-cell fusion, the two bound cells are in contact over an appreciable area, so fusion could potentially occur at many sites (Frolov et al., 2000;Leikina and Chernomordik, 2000); the sites of origin for the observed lipid and aqueous dye spread could well be different.By studying fusion of viral particles to cells, these problems can be overcome. The small size of virus minimizes the area of contact and accurately reflects the biological situation. Also, membrane retrieval processes that internalize lipid dye are absent in virions, and the viral envelope is simpler than a cell membrane. But until recently, suitable assays to follow both lipid and contents mixing and to time-order the...

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“…However, several independent lines of evidence indicate that Gag-Gag interactions occur in the cytoplasm prior to reaching the plasma membrane. Complementation experiments reveal that budding can be restored when Gag mutants defective in membrane binding are coexpressed with wild-type Gag (2,4,5,35). Moreover, cytoplasmic extracts from cells expressing Gag contain higher-order Gag assembly intermediates that increase in size in a stepwise fashion, suggesting an ordered process of dimerization, oligomerization, and multimerization (29).…”

mentioning

confidence: 97%

Intermolecular Interactions between Retroviral Gag Proteins in the Nucleus

Kenney¹,

Lochmann

Schmid

et al. 2008

J Virol

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The retroviral Gag polyprotein directs virus particle assembly, resulting in the release of virions from the plasma membranes of infected cells. The earliest steps in assembly, those immediately following Gag synthesis, are very poorly understood. For Rous sarcoma virus (RSV), Gag proteins are synthesized in the cytoplasm and then undergo transient nuclear trafficking before returning to the cytoplasm for transport to the plasma membrane. Thus, RSV provides a useful model to study the initial steps in assembly because the early and later stages are spatially separated by the nuclear envelope. We previously described mutants of RSV Gag that are defective in nuclear export, thereby isolating these "trapped" Gag proteins at an early assembly step. Using the nuclear export mutants, we asked whether Gag protein-protein interactions occur within the nucleus. Complementation experiments revealed that the wild-type Gag protein could partially rescue export-defective Gag mutants into virus-like particles (VLPs). Additionally, the export mutants had a trans-dominant negative effect on wild-type Gag, interfering with its release into VLPs. Confocal imaging of wild-type and mutant Gag proteins bearing different fluorescent tags suggested that complementation between Gag proteins occurred in the nucleus. Additional evidence for nuclear Gag-Gag interactions was obtained using fluorescence resonance energy transfer, and we found that the formation of intranuclear Gag complexes was dependent on the NC domain. Bimolecular fluorescence complementation allowed the direct visualization of intranuclear Gag-Gag dimers. Together, these experimental results strongly suggest that RSV Gag proteins are capable of interacting within the nucleus.

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Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association

Cited by 35 publications

References 53 publications

Protein transduction from retroviral Gag precursors

Protein transduction from retroviral Gag precursors

Time-resolved Imaging of HIV-1 Env-mediated Lipid and Content Mixing between a Single Virion and Cell Membrane

Intermolecular Interactions between Retroviral Gag Proteins in the Nucleus

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