Intracellular assembly and budding of the Murine Leukemia Virus in infected cells

Houzet, Laurent; Gay, Bernard; Morichaud, Zakia; Briant, Laurence; Mougel, Marylène

doi:10.1186/1742-4690-3-12

Cited by 23 publications

(13 citation statements)

References 34 publications

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“…Although intracellular particle assembly was initially thought to be a unique feature of macrophages, similar studies reporting localization of HIV-1 Gag and virions in intracellular CD63 + and/or Lamp1 + compartments in fibroblast, epithelial, and T-lymphocyte cell lines, as well as immunoprecipitation of extracellular virions with CD63 antibodies, have extended this notion, leading to the proposal that HIV-1 assembly is initiated on endosomal membranes in all cell types [8–13]. Moreover, several other works have reported the observation of murine leukemia virus and human T-cell leukemia virus type-1 particles in late endosomes [12,14–17], suggesting that assembly in late endosomes is a general feature of retroviruses.…”

Section: Introductionmentioning

confidence: 99%

Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly

et al. 2006

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Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag) to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM) and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes.

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Section: Introductionmentioning

confidence: 99%

Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly

et al. 2006

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“…However, approximately 8–17% of viruses do appear to have been labeled by the dyes. This does not come as a complete surprise since it has been previously documented that a small fraction of MLVs are released through the endosomal pathway rather than budding at the cell surface and these viral particles might take up dye more efficiently than virus budding at the cell surface 66 , 67 . This would suggest that these dual-stained retroviruses share even greater biochemical similarities to small EVs, which are mostly comprised of exosomes 17 .…”

Section: Discussionmentioning

confidence: 90%

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

Tang

Renner

Fritzsche

et al. 2017

Sci Rep

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Retroviruses and small EVs overlap in size, buoyant densities, refractive indices and share many cellderived surface markers making them virtually indistinguishable by standard biochemical methods. This poses a significant challenge when purifying retroviruses for downstream analyses or for phenotypic characterization studies of markers on individual virions given that EVs are a major contaminant of retroviral preparations. Nanoscale flow cytometry (NFC), also called flow virometry, is an adaptation of flow cytometry technology for the analysis of individual nanoparticles such as extracellular vesicles (EVs) and retroviruses. In this study we systematically optimized NFC parameters for the detection of retroviral particles in the range of 115-130 nm, including viral production, sample labeling, laser power and voltage settings. By using the retroviral envelope glycoprotein as a selection marker, and evaluating a number of fluorescent dyes and labeling methods, we demonstrate that it is possible to confidently distinguish retroviruses from small EVs by NFC. Our findings make it now possible to individually phenotype genetically modified retroviral particles that express a fluorescent envelope glycoprotein without removing EV contaminants from the sample.Retroviruses, such as the human immunodeficiency virus (HIV), are enveloped RNA viruses that range between 90-150 nm in diameter, depending on the species 1-3 . When nascent virions egress from infected cells, they bear contents of the cytosol (e.g., proteins, mRNAs, miRNAs), as well as a portion of the cell membrane embedded with surface receptors to form the viral envelope [4][5][6] . The phenotypic analysis of host-derived markers on the surface of individual viruses is of considerable interest, as it can provide information on the identity of the specific cell types that are infected in a host. However, a major hurdle in purifying retroviruses for single-particle characterization studies is the removal of EVs that are concomitantly released by the cells 7-11 . EV is a broad term that describes all particles with a membrane bilayer released from cells; these can include exosomes, microvesicles, and apoptotic vesicles [12][13][14][15][16] . Small EVs, that are in the size range of retroviruses constitute a major contaminant of virus preparations as they are biochemically and biophysically similar to retroviruses in terms of their refractive indices, buoyant densities, and surface markers 5,7,[17][18][19] . Additionally, EVs can also package retroviral proteins and RNAs that further complicate discrimination [20][21][22][23][24] .Nanoscale flow cytometry (NFC), also called flow virometry, is a new and powerful tool in the field of virology that enables the phenotypic analysis of the markers at the surface of individual virions 11,[25][26][27][28][29][30][31][32] . Virus populations can now be profiled and sorted in multi-parameter analyses, much in the same way as cells 25,27,30,32,33 . However, NFC analysis with current instrumentation can be challenging due to ...

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“…To obtain further clues of the different roles of CHMP1A and CHMP1B in MLV virus propagation, we performed infectivity studies. Previous studies demonstrated that maturation inside the immature MLV virion is required for infectivity [ 26 , 38 , 39 ]. Accordingly, the yield of extracellular virions might not be a mandatory indicator of virus infectivity.…”

Section: Resultsmentioning

confidence: 99%

ESCRT Requirements for Murine Leukemia Virus Release

Bartusch

Prange

2016

Viruses

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The Murine Leukemia Virus (MLV) is a gammaretrovirus that hijack host components of the endosomal sorting complex required for transport (ESCRT) for budding. To determine the minimal requirements for ESCRT factors in MLV viral and viral-like particles (VLP) release, an siRNA knockdown screen of ESCRT(-associated) proteins was performed in MLV-producing human cells. We found that MLV VLPs and virions primarily engage the ESCRT-I factor Tsg101 and marginally the ESCRT-associated adaptors Nedd4-1 and Alix to enter the ESCRT pathway. Conversely, the inactivation of ESCRT-II had no impact on VLP and virion egress. By analyzing the effects of individual ESCRT-III knockdowns, VLP and virion release was profoundly inhibited in CHMP2A- and CHMP4B-knockdown cells. In contrast, neither the CHMP2B and CHMP4A isoforms nor CHMP3, CHMP5, and CHMP6 were found to be essential. In case of CHMP1, we unexpectedly observed that the CHMP1A isoform was specifically required for virus budding, but dispensable for VLP release. Hence, MLV utilizes only a subset of ESCRT factors, and viral and viral-like particles differ in ESCRT-III factor requirements.

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Intracellular assembly and budding of the Murine Leukemia Virus in infected cells

Cited by 23 publications

References 34 publications

Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly

Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly

Single-Particle Discrimination of Retroviruses from Extracellular Vesicles by Nanoscale Flow Cytometry

ESCRT Requirements for Murine Leukemia Virus Release

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