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. 2006 Feb 10:3:12.
doi: 10.1186/1742-4690-3-12.

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

Laurent Houzet  1 , Bernard Gay, Zakia Morichaud, Laurence Briant, Marylène Mougel
Affiliations

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

Laurent Houzet et al. Retrovirology. .

Abstract

Background: Murine Leukemia Virus (MLV) assembly has been long thought to occur exclusively at the plasma membrane. Current models of retroviral particle assembly describe the recruitment of the host vacuolar protein sorting machinery to the cell surface to induce the budding of new particles. Previous fluorescence microscopy study reported the vesicular traffic of the MLV components (Gag, Env and RNA). Here, electron microscopy (EM) associated with immunolabeling approaches were used to go deeply into the assembly of the "prototypic" MLV in chronically infected NIH3T3 cells.

Results: Beside the virus budding events seen at the cell surface of infected cells, we observed that intracellular budding events could also occur inside the intracellular vacuoles in which many VLPs accumulated. EM in situ hybridization and immunolabeling analyses confirmed that these latter were MLV particles. Similar intracellular particles were detected in cells expressing MLV Gag alone. Compartments containing the MLV particles were identified as late endosomes using Lamp1 endosomal/lysosomal marker and BSA-gold pulse-chase experiments. In addition, infectious activity was detected in lysates of infected cells.

Conclusion: Altogether, our results showed that assembly of MLV could occur in part in intracellular compartments of infected murine cells and participate in the production of infectious viruses. These observations suggested that MLV budding could present similarities with the particular intracellular budding of HIV in infected macrophages.

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Figures

Figure 1
Figure 1
Electron microscopy analysis of VLPs assembly in vacuoles of MLV-infected NIH3T3 cells. EM analysis of epon embedded NIH3T3 cells chronically infected with MLV. A) Virus budding at the plasma membrane. B) Numerous mature (arrows) and immature (arrowheads) particles inside the intracellular endosomes. C) Budding particle into a vacuole.
Figure 2
Figure 2
Immunoelectron microscopy analysis of Gag distribution in MLV-infected cells and progeny viruses. Gag was detected by immunogold labeling in lowicryl embedded sections of MLV-infected cells. A) Extracellular virus particle (black arrow) released from the plasma membrane (black arrowhead) labeled with 5 nm gold particles. B) VLPs (black arrows) present in intracellular vacuoles (white arrows) were labeled with similar intensity as extracellular viruses. C) Magnification of intravacuolar Gag-labeled VLPs (arrows). Weak labeling was also observed on the vacuolar delimiting membrane (arrowhead).
Figure 3
Figure 3
Detection of the viral RNA genome in intracellular VLPs. The MLV genomic RNA was specifically detected by EM in situ hybridization and was visualized by 10 nm gold particles. VLPs (black arrows) inside the intracellular vacuoles (white arrows) were labeled (arrowheads) with the specific antisense probe (A), while no signal was detected with the control sense riboprobe (B).
Figure 4
Figure 4
VLP-containing vacuoles and their VLPs are positive for Lamp-1. Lamp1 was detected in lowicryl embedded sections by immunogold labeling (5nm gold particles). A) Low labeling was observed on the periphery of MLV-VLPs containing vacuoles and on other intravacuolar components (white arrowheads). Gold particles could sometimes be found on individual intracellular MLV-VLP (black arrowhead). B) Magnification of the boxed area in A showed a Lamp1 positive MLV-VLP.
Figure 5
Figure 5
Specific labeling of lysosomal compartments by pulses of BSA-gold. Representative pictures of infected cells incubated with the BSA-gold. The BSA-gold accumulated in VLP-free lysosomes (arrowhead) (A). VLPs (large arrow) and budding event (little arrow) were shown in unlabeled vacuole (B). Absence of colocalization of VLP (arrow) and BSA-gold (arrowhead) (C).
Figure 7
Figure 7
Gag alone can promote intracellular VLPs formation. Gag was detected by immunogold labeling in lowicryl embedded sections of Fly packaging cells which expressed the Gag and Pol proteins only. A) Extracellular VLP (arrow) released from the plasma membrane (arrowhead) labeled with 5 nm gold particles. B) VLPs (arrows) present in intracellular vacuole (arrowhead).
Figure 6
Figure 6
Infectivity of intracellular particles released by freeze-thaw and sonication treatment. FIA was used to quantitate infectious particles present in the cell lyzed by freeze-thaw and sonication (cell lysate) or in the last wash of cells left intact (control). A) One typical FFU labeled with anti-Env antibody and detected in FIA. Insert: magnification of the boxed area showing infected (arrow) and non infected (arrowhead) Dunni cells. B) Results of the FIA expressed as the total number of infectious FFU detected in the total lysate of 5 × 106 cells. Lysis and infectivity experiments were performed at least 3 times and each infection test was performed in triplicate. Bars, the standard error of the mean of each series.
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