Amphotropic murine leukemia virus is preferentially attached to cholesterol-rich microdomains after binding to mouse fibroblasts

Abstract

Background: We 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.

Results: Here, 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.

Conclusion: The 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.

Figure 1

A-MLV binds preferentially to large rafts. A) NIH3T3 cells were incubated with GagYFP A-MLV (green) for 3 hours, fixed, and GM1 was stained with fluorescently labeled CTX (red). B) NIH3T3 cells were incubated with GagYFP A-MLV (green) for 3 hours and fixed. The cells were permeabilized with Triton X-100 and cav-1 was stained (red). C) NIH3T3 cells stably expressing cav-1 mRed fusion protein (red) were incubated with GagYFP A-MLV (green) for 3 hours and fixed. Clusters of viral particles as those found bound to large rafts in A are labelled with arrows. All pictures were taken using confocal microscopy.

Figure 2

Large rafts are present in NIH3T3 cells independent of A-MLV binding. A), and B) NIH3T3 cells were incubated with GagYFP A-MLV (green) for 30 min, fixed, and stained for GM1 with fluorescently labeled CTX (red). Clusters of viral particles bound to large rafts are labelled with arrows in B). C), and D) NIH3T3 were incubated with VSV (green) for 30 min, fixed, and stained for GM1 with fluorescently labeled CTX (red). All images were taken using confocal microscopy. A) and C) are merged images, B) and D) show only GagYFP A-MLV or GagYFP VSV particles from A) and C), respectively.

Figure 3

A-MLV binding to large rafts is independent of extraction of plasma membrane cholesterol. NIH3T3 cells were treated with 10 mM MBCD, washed, and incubated with GagYFP A-MLV (green) for 30 min. Subsequently, the cells were washed, fixed, and stained for GM1 using fluorescently labeled CTX (red). Images were taken using confocal microscopy.