Abstract
BackgroundWe have recently shown that amphotropic murine leukemia virus (A-MLV) can enter the mouse fibroblast cell line NIH3T3 via caveola-dependent endocytosis. But due to the size and omega-like shape of caveolae it is possible that A-MLV initially binds cells outside of caveolae. Rafts have been suggested to be pre-caveolae and we here investigate whether A-MLV initially binds to its receptor Pit2, a sodium-dependent phosphate transporter, in rafts or caveolae or outside these cholesterol-rich microdomains.ResultsHere, we show that a high amount of cell-bound A-MLV was attached to large rafts of NIH3T3 at the time of investigation. These large rafts were not enriched in caveolin-1, a major structural component of caveolae. In addition, they are rather of natural occurrence in NIH3T3 cells than a result of patching of smaller rafts by A-MLV. Thus cells incubated in parallel with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped MLV particles showed the same pattern of large rafts as cells incubated with A-MLV, but VSV-G pseudotyped MLV particles did not show any preference to attach to these large microdomains.ConclusionThe high concentration of A-MLV particles bound to large rafts of NIH3T3 cells suggests a role of these microdomains in early A-MLV binding events.
Highlights
We have recently shown that amphotropic murine leukemia virus (A-MLV) can enter the mouse fibroblast cell line NIH3T3 via caveola-dependent endocytosis
NIH3T3 cells were incubated for 3 hours at 37°C with fluorescently labeled A-MLV (GagYFP A-MLV) containing a nucleocapsid protein fused with yellow fluorescence protein (YFP) [12]
As GM1 is a general marker for cholesterol-rich microdomains, we investigated if these regions of preferred A-MLV binding were enriched in caveolin-1, a major structural protein of caveolae
Summary
We have recently shown that amphotropic murine leukemia virus (A-MLV) can enter the mouse fibroblast cell line NIH3T3 via caveola-dependent endocytosis. We have previously shown that A-MLV entry is closely associated with cholesterol-rich microdomains like rafts and caveolae [1] and that A-MLV envelope protein is associated with rafts in infected cells suggesting a possible role of rafts in A-MLV assembly [2] It has been shown for other viruses that rafts and/or caveolae are important for their entry and assembly [3,4,5,6,7,8]; has caveola-mediated entry been shown for, e.g., SV40 [4], echovirus 1 [7], and human coronavirus 229E [8]. The unique lipid composition of rafts and caveolae leads to the specific incorporation or exclusion of proteins in these domains thereby creating distinct microenvironments for cellular processes [10,11]
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