Endocytosis and a low-pH step are required for productive entry of equine infectious anemia virus

Recently, it has become evident that entry of some retroviruses into host cells is dependent upon a vesicle-localized, low-pH step. The entry mechanism of equine infectious anemia virus (EIAV) has yet to be examined. Here, we demonstrate that wild-type strains of EIAV require a low-pH step for productive entry. Lysosomotropic agents that inhibit the acidification of internal vesicles inhibited productive entry of EIAV. The presence of ammonium chloride (30 mM), monensin (30 mu M), or bafilomycin A (50 nM) in the medium dramatically decreased the number of EIAV antigen-positive cells. We found that a low-pH step was required for ELAV infection of tissue culture cell lines as well as primary cells, such as endothelial cells and monocyte-derived macrophages. The ammonium chloride treatment did not reduce virion stability, nor did the treatment prevent virion binding to cells. Consistent with a requirement for a low-pH step, virion infectivity was enhanced more than threefold by brief low-pH treatment following binding of viral particles to permissive cells. A superinfecting variant strain of EIAV, vNU-1c, did not require a low-pH step for productive infection of fibroblasts. However, lysosomotropic agents were inhibitory to vNU-1c infection in the other cell types that vNU-1c infected but did not superinfect, indicating that the entry pathway used by vNU-1c for superinfection abrogates the need for the low-pH step.