Assembly of the Murine Leukemia Virus Is Directed towards Sites of Cell–Cell Contact

Jin, Jing; Sherer, Nathan M.; Heidecker, Gisela; Derse, David; Mothes, Walther

doi:10.1371/journal.pbio.1000163

Cited by 89 publications

(156 citation statements)

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“…Mutant MLV envelope expression vectors were generated by site-directed mutagenesis based on the Friend MLV envelope expression vector previously described (16,46). MLV Gag-GFP expression vector was made by fusing enhanced green fluorescent protein (eGFP) to C-terminal of Gag and deleting a large fragment of Pol in full-length Friend57 MLV expression vector pLRB303 (23,35).…”

Section: Methodsmentioning

confidence: 99%

“…HIV Gag-GFP expression vector and chimeric HIV Gag with MA swapped with MLV MA were described previously (17). HEK293 and XC cells were previously described (16).…”

Section: Methodsmentioning

confidence: 99%

“…HEK293 cells were transfected with indicated plasmids by FuGENE 6 and cocultured 4 h posttransfection with XC cells expressing mCherry or cyan fluorescent protein (CFP)-tagged mCAT-1 as previously described (16). The formation of MLV synapses was reproducibly observed 2 to 6 postinitiation of cocultures for MLV generated by the tripartite system (MLV GagPol, Gag-YFP, MLV LTR, and MLV Env) or 4 to 10 h postinitiation of cocultures for MLV generated using proviral pLRB303-derived constructs.…”

Section: Methodsmentioning

confidence: 99%

“…The formation of MLV synapses was reproducibly observed 2 to 6 postinitiation of cocultures for MLV generated by the tripartite system (MLV GagPol, Gag-YFP, MLV LTR, and MLV Env) or 4 to 10 h postinitiation of cocultures for MLV generated using proviral pLRB303-derived constructs. Imaging of fixed samples, as well as live-cell imaging, was performed using spinning disc confocal microscopy as described previously (16). The extent of polarization of assembly to the MLV synapse in fixed images was quantified using Volocity (Perkin-Elmer) as described in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Using the murine leukemia virus (MLV) as a model system, we demonstrated that the interaction between the viral Env glycoprotein (Env), expressed on the infected cell and the viral receptor, expressed on the uninfected target cell, leads to the establishment of cell-cell contacts (32,45). Importantly, following the establishment of cellcell adhesion, assembly was polarized toward the cell-cell contact (16). These data suggested that the infected cell can "sense" the uninfected cell and direct the viral components needed to assemble viruses toward the uninfected cell.…”

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confidence: 99%

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Viral Determinants of Polarized Assembly for the Murine Leukemia Virus

Jin

Mothes

2011

J Virol

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Retrovirus transmission via direct cell-cell contact is more efficient than diffusion through the extracellular milieu. This is believed to be due to the ability of viruses to efficiently coordinate several steps of the retroviral life cycle at cell-cell contact sites (D. Viruses exploit and manipulate cell-cell contacts for efficient spreading (12,18,32,37,43). In the case of the human immunodeficiency virus (HIV) and the human T cell lymphoma virus these cell-cell contacts share features with immunological synapses and have been designated infectious or virological synapses (13,14,19,29,41). Using the murine leukemia virus (MLV) as a model system, we demonstrated that the interaction between the viral Env glycoprotein (Env), expressed on the infected cell and the viral receptor, expressed on the uninfected target cell, leads to the establishment of cell-cell contacts (32, 45). Importantly, following the establishment of cellcell adhesion, assembly was polarized toward the cell-cell contact (16). These data suggested that the infected cell can "sense" the uninfected cell and direct the viral components needed to assemble viruses toward the uninfected cell. Sensing of the target cell turned out to be mediated by the accumulation of Env and receptor at the cell-cell interface and depending on the presence of a cytoplasmic tail (C-tail) of Env, MLV Gag was recruited toward the site of cell-cell contact. Here, we identify a tyrosine residue within the C-tail of Env and matrix within Gag as the viral determinants responsible for this process.C MATERIALS AND METHODSPlasmids and reagents. Mutant MLV envelope expression vectors were generated by site-directed mutagenesis based on the Friend MLV envelope expression vector previously described (16,46). MLV Gag-GFP expression vector was made by fusing enhanced green fluorescent protein (eGFP) to C-terminal of Gag and deleting a large fragment of Pol in full-length Friend57 MLV expression vector pLRB303 (23,35). Individual domains of MLV Gag-GFP were deleted or replaced by a standard overlapping PCR approach. MLV Gag-YFP expression vector was previously described (46). Chimeric MLV Gag with MA swapped with HIV MA was kindly provided by Marc Johnson, University of Missouri. HIV Gag-GFP expression vector and chimeric HIV Gag with MA swapped with MLV MA were described previously (17). HEK293 and XC cells were previously described (16).Visualizing virus cell-to-cell transmission. HEK293 cells were transfected with indicated plasmids by FuGENE 6 and cocultured 4 h posttransfection with XC cells expressing mCherry or cyan fluorescent protein (CFP)-tagged mCAT-1 as previously described (16). The formation of MLV synapses was reproducibly observed 2 to 6 postinitiation of cocultures for MLV generated by the tripartite system (MLV GagPol, Gag-YFP, MLV LTR, and MLV Env) or 4 to 10 h postinitiation of cocultures for MLV generated using proviral pLRB303-derived constructs. Imaging of fixed samples, as well as live-cell imaging, was performed using spinning disc confocal microscopy as ...

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Section: Methodsmentioning

confidence: 99%

“…HIV Gag-GFP expression vector and chimeric HIV Gag with MA swapped with MLV MA were described previously (17). HEK293 and XC cells were previously described (16).…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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