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


CONCLUSIONS
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.
Laboratoire Infections Rétrovirales et Signalisation Cellulaire, CNRS UMR5121, UMI, IFR122, Institut de Biologie, Montpellier, France. lhouzet@univ-montp1.fr
Laurent
L
Houzet
Bernard
B
Gay
Zakia
Z
Morichaud
Laurence
L
Briant
Marylène
M
Mougel
D016475:3T3 Cells; D000818:Animals; D020285:Cryoelectron Microscopy; D017403:In Situ Hybridization; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000472:pathogenicity* / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D008954:Models, Biological; D012367:RNA, Viral / Q000235:genetics; D012192:Retroviridae Infections / Q000473:pathology / Q000821:virology; D014412:Tumor Virus Infections / Q000473:pathology / Q000821:virology
D012367:RNA, Viral