Macropinocytosis Is the Entry Mechanism of Amphotropic Murine Leukemia Virus

The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined.We show here that A-MLV is internalized not by caveolae or other pinocytic mechanisms but by macropinocytosis. Thus, A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, and NIH 3T3 cells knocked down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH 3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymer-izing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional expression of dominant positive or negative mutants of Rac1, PAK1, and RhoG, to increase or decrease macropinocytosis, similarly corre-lated with an augmented or inhibited infection with A-MLV, respectively. The same experimental perturbations affected the in-fection of viruses that use clathrin-coated-pit endocytosis or other pathways for entry only mildly or not at all. These data agree with immunofluorescence studies and cryo-immunogold labeling for electronmicroscopy, which demonstrate the presence of A-MLV in protrusion-rich areas of the cell surface and in cortical fluid phase (dextran)-filled macropinosomes, which also ac-count for up to a half of the cellular uptake of the cell surface-binding lectin concanavalin A.We conclude that A-MLV use mac-ropinocytosis as the predominant entry portal into cells. IMPORTANCE Binding and entry of virus particles into mammalian cells are the first steps of infection. Understanding how pathogens and tox