Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry

African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na⁺/H⁺ ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed.Many animal viruses have evolved to exploit endocytosis to gain entry into host cells after initial attachment of virions to specific cell surface receptors. To date, a number of different routes of endocytosis used by viruses have been characterized, including clathrin-mediated endocytosis, uptake via caveolae/lipid rafts, macropinocytosis, phagocytosis, and other routes that are currently poorly understood.In recent years, viruses have also been used as tools to study cellular endocytosis and membrane trafficking at the molecular level, with there being special interest in the regulation of the diverse routes (31), since examples of viruses using each route can be found (reviewed in references 26, 31, and 38). The clathrin-mediated endocytic route has been the most extensively studied at the molecular level, and it has been shown to be used by diverse mammalian enveloped viruses, such as vesicular stomatitis virus (42), Semliki Forest virus (19), and West Nile virus (11), to infect cells. Influenza virus and HIV-1 also can use this pathway as an alternative route of entry (12, 39). Clathrin is assembled on the inside face of the plasma membrane to form a characteristic coated pit (CCP). During this process, clathrin also interacts with a number of essential molecules, including Eps15, adapter protein AP2, and dynamin GTPase (9). Additionally, clathrin-mediated endocytosis also provides endocytic vesicles as an acidified environment for those viruses that require a low-pH step during the first stages of infection to initiate capsid destabilization and genome uncoating. On the other hand, the lipid raft/caveola-based route is generally used by those acid-independent viruses. Recently, macropinocytosis is generating growing interest, since it has been demonstrated to be induced by some viruses from diverse families, such as vaccinia virus and adenovirus serotype 3 (5, 29), to gain entry into cells.In this study, we have focused on the entry of African swine fever virus (ASFV), a large enveloped DNA virus with a genomic composition similar to that of poxviruses, although the virion structure and morphology resemble those of iridoviruses. At present, it is the sole member of the newly created family Asfarviridae (16, 43). It is the etiologic agent responsible for a highly lethal and hemorrhagic disease affecting domestic swine, which often results in important economic losses in affected countries because of the high rate of mortality associated with this illness and the lack of an effective vaccine.Early studies of the entry of BA71V, a Vero cell-adapted ASFV strain, into host cells showed that this internalization of virus particles is a temperature-, energy-, and low-pH-dependent process, since it does not occur at 4°C or in the presence of inhibitors of cellular respiration or lysosomotropic agents (2, 44). All these features are consistent with a receptor-mediated endocytosis mechanism of entry. However, there are still numerous questions to be answered. One of them is the nature of the cellular receptor(s) that mediates ASFV entry, which remains largely unknown, although a correlation between cell susceptibility to infection and expression of porcine CD163 on the surface of swine monocytes/macrophages has been reported (36). In regard to the viral components involved in this initial step, the p12 and p54 proteins were shown to play a role during attachment to the cell surface and p30 during internalization, as inferred from previous studies with neutralizing antibodies against p30 and p54 (17) and blockage of infection after saturation of virus binding sites with recombinant p12 (6). Early electron microscopy (EM) studies (2, 45) revealed that attachment of ASFV virions to the cell surface often occurs in coated pits; however, their later presence inside coated vesicles is not fully clear. After attachment, virions are detected inside endosomes, where fusion with the viral membrane takes place. The ASFV cycle continues with the transport of viral cores via retrograde transport along microtubules to reach a perinuclear area, known as the viral factory, where replication occurs (4).In recent times, knowledge about different endocytic pathways and their regulatory molecules has notably increased, and the development of molecular tools to study these processes is becoming increasingly precise (38). In the present study, we examined the ASFV infection using a variety of chemical inhibitors and dominant negative molecules to disrupt different endocytic pathways. Our results confirmed a major role for dynamin-dependent and clathrin-mediated endocytosis during the first stages of ASFV infection, with no significant differences in the behavior of the two ASFV strains and the two cell lines analyzed.