ALIX and TSG101 are essential for cellular entry and replication of two porcine alphacoronaviruses

Alphacoronaviruses are the primary coronaviruses responsible for causing severe economic losses in the pig industry with the potential to cause human outbreaks. Currently, extensive studies have reported the essential role of endosomal sorting and transport complexes (ESCRT) in the life cycle of enveloped viruses. However, very little information is available about which ESCRT components are crucial for alphacoronaviruses infection. By using RNA interference in combination with Co-immunoprecipitation, as well as fluorescence and electron microscopy approaches, we have dissected the role of ALIX and TSG101 for two porcine alphacoronavirus cellular entry and replication. Results show that infection by two porcine alphacoronaviruses, including porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV), is dramatically decreased in ALIX- or TSG101-depleted cells. Furthermore, PEDV entry significantly increases the interaction of ALIX with caveolin-1 (CAV1) and RAB7, which are crucial for viral endocytosis and lysosomal transport, however, does not require TSG101. Interestingly, PEAV not only relies on ALIX to regulate viral endocytosis and lysosomal transport, but also requires TSG101 to regulate macropinocytosis. Besides, ALIX and TSG101 are recruited to the replication sites of PEDV and PEAV where they become localized within the endoplasmic reticulum and virus-induced double-membrane vesicles. PEDV and PEAV replication were significantly inhibited by depletion of ALIX and TSG101 in Vero cells or primary jejunal epithelial cells, indicating that ALIX and TSG101 are crucial for PEDV and PEAV replication. Collectively, these data highlight the dual role of ALIX and TSG101 in the entry and replication of two porcine alphacoronaviruses. Thus, ESCRT proteins could serve as therapeutic targets against two porcine alphacoronaviruses infection. Alphacoronaviruses are one of the main coronaviruses that cause infections in humans and animals. However, effective antivirals to protect against alphacoronavirus infections are lacking. It is, therefore, necessary to better understand the interactions between these viruses and host cells to inform the development of improved preventatives and therapeutics. Here we assessed the role of the ESCRT system-responsible for viral lifecycle-in the common porcine epidemic diarrhea virus (PEDV) and the emerging highly pathogenic porcine enteric alphacoronavirus (PEAV). In particular, the roles of ALIX, and TSG101 were evaluated in the entry and replication of PEDV and PEAV. The results indicate that ALIX and TSG101 regulate viral infection by inducing host cell-mediated endocytosis and altering viral transport dynamics in the endosome/lysosomal system. In addition, both ESCRT proteins have important roles in mediating the formation of viral replication organelles. Hence, ALIX and TSG101 may represent host proteins that can act as targets of antiviral therapies to protect animals and humans against alphacoronavirus infection.