The avian leukosis virus (ALV) entry mechanism is controversial, with evidence for and against a low-pH requirement for viral fusion. To further address this question, we tested the entry of human immunodeficiency virus type 1 (HIV-1) pseudotyped with the envelope protein of subgroup B ALV (ALV-B) in the presence of three different lysosomotropic agents. These lysosomotropic agents were able to block the entry of wild-type and pseudotyped ALV-B in two different cell lines, strongly suggesting that ALV-B requires a low-pH step for entry. ALV-B and pH-dependent Semliki Forest virus (SFV) entered cells with slower uptake kinetics than HIV-1, which is pH independent. These slow uptake rates support the theory that ALV-B utilizes endocytic pathways to enter cells. Using immunofluorescence and electron microscopy analysis, we visualized the colocalization of virus particles with the endosomal marker transferrin and demonstrated virus particles in clathrin-coated vesicles and endosome-like structures. Surprisingly, a low-pH treatment did not overcome the inhibition of ALV-B entry by lysosomotropic agents. This indicates that, in contrast to SFV, ALV-B is unable to fuse at the cellular surface, even at a low pH. Taken together, our findings suggest that endocytosis and a subsequent low-pH step are critical for successful ALV-B infection.
Viruses exploit different cellular processes to infect and replicate in target cells. Endocytosis, an essential cell function, is used by several viruses to gain entry into target cells (41).Transport of virus particles into acidic compartments is a prerequisite for the fusion of enveloped pH-dependent viruses, e.g., alphaviruses (Semliki Forest virus [SFV] and Sindbis virus [25,26,35,37,39,54] . Low pH triggers a conformational change of the viral envelope protein, from a fusion-inactive to a fusion-active state, which leads to the exposure of the hydrophobic fusion peptide and allows lipid mixing between the viral and cellular membranes (19,22,55). Viral fusion in endosomes results in the release of the viral nucleocapsid into the cytosolic compartment.Viral endocytosis is required in some instances for reasons other than pH dependence (40). For example, forced fusion of SFV with the plasma membrane by acid treatment renders a productive infection in baby hamster kidney (BHK-21) cells but not in Chinese hamster ovary (CHO) cells. This indicates the presence of a postentry barrier in CHO cells that is overcome by endocytosis of virus particles (36). Similarly, the entry mechanism of ecotropic murine leukemia virus (e-MLV) is cell type specific. e-MLV fuses directly at the cell surface in rat XC sarcoma cells but requires endocytosis in NIH 3T3 cells (29). In addition, human immunodeficiency virus type 1 (HIV-1) macropinocytosis plays an important role in macrophage infection, demonstrating the use of an endocytic pathway of a pH-independent virus (34). This indicates that the endocytic pathway might overcome cell-specific restrictions in viral entry for reasons other than pH dependenc...
