Equine Infectious Anemia Virus Entry Occurs through Clathrin-Mediated Endocytosis

Entry of wild-type lentivirus equine infectious anemia virus (EIAV) into cells requires a low-pH step. This low-pH constraint implicates endocytosis in EIAV entry. To identify the endocytic pathway involved in EIAV entry, we examined the entry requirements for EIAV into two different cells: equine dermal (ED) cells and primary equine endothelial cells. We investigated the entry mechanism of several strains of EIAV and found that both macrophage-tropic and tissue culture-adapted strains utilize clathrin-coated pits for entry. In contrast, a superinfecting strain of EIAV, EIAVvMA-1c, utilizes two mechanisms of entry. In cells such as ED cells that EIAVvMA-1c is able to superinfect, viral entry is pH independent and appears to be mediated by plasma membrane fusion, whereas in cells where no detectable superinfection occurs, EIAVvMA-1c entry that is low-pH dependent occurs through clathrin-coated pits in a manner similar to wild-type virus. Regardless of the mechanism of entry being utilized, the internalization kinetics of EIAV is rapid with 50% of cell-associated virions internalizing within 60 to 90 min. Cathepsin inhibitors did not prevent EIAV entry, suggesting that the low-pH step required by wild-type EIAV is not required to activate cellular cathepsins. Enveloped virus infection is initiated by the viral glycoprotein binding to its cellular receptor. The binding event either triggers membrane fusion at the plasma membrane or internalization of the virus into an endosome. For those viruses that are endocytosed, subsequent endosomal events lead to fusion of the viral membrane with the vesicle, releasing the core particle into the cytoplasm. Cells utilize several endocytosis mechanisms to take up nutrients from their environment, and viruses usurp these mechanisms for internalization. Defined pathways of endocytosis include clathrin-mediated endocytosis, caveolae-mediated endocytosis, nonclathrin- noncaveolaemediated endocytosis, and macropinocytosis (40). Many of these pathways traffic through acidic compartments. Viruses can take advantage of the pH decrease to stimulate events that trigger membrane fusion (1, 10, 65). Two mechanistically diverse examples of viral use of a low-pH step are influenza virus and Ebola virus. The vesicle-associated, low-pH environment initiates conformational changes in the influenza virus glycoprotein, leading to membrane fusion (65), whereas endosomal low-pH-activated proteases cleave the Ebola virus glycoprotein 1, allowing subsequent fusion events (14, 60). An evolution in the understanding of retroviral entry has occurred and now incorporates a role for endocytosis in the internalization of many retroviruses. A general model for mammalian retroviral entry was initially proposed 17 years ago (45); however, the specific requirements for internalization of only a few viruses had been closely examined at that time. The model proposed that a prototypic retrovirus enters cells at the plasma membrane through a pH-independent fusion event (44, 45). As mechanisms of entry of more retroviruses have been examined, numerous retroviruses have been determined to