RPSA enhances African swine fever virus entry via caveola-mediated endocytic pathway

African swine fever virus (ASFV) poses a severe threat to the global pig industry, yet the precise mechanisms of its entry into host cells are not fully understood. In this study, we identified the host ribosomal protein SA (RPSA), a protein located in lipid rafts, as a critical viral receptor that facilitates ASFV entry. Immunoprecipitation-mass spectrometry (IP-MS) screening results identified RPSA as a binding partner of the ASFV capsid protein pE120R, which was further confirmed by co-immunoprecipitation (Co-IP) and immunofluorescence assays (IFA) in ASFV-infected cells. Moreover, knockdown of RPSA expression in porcine alveolar macrophages (PAMs) by siRNA significantly reduced ASFV attachment and internalization. In contrast, ectopic overexpression of RPSA in permissive CV-1 cells enhanced the attachment and internalization of ASFV virions. Additionally, we observed that both pharmacological inhibition of caveola-mediated endocytosis (CavME) with nystatin and genetic knockdown of caveolin-1 (CAV1) decreased caveola-mediated ASFV virions endocytosis and internalization. Mechanistically, RPSA interacts with CAV1, a major structural protein of caveolae. Domain mapping showed that pE120R mainly binds to the LR2 and LR3 domains of RPSA, while RPSA interacts with CAV1 via its 16B domain, forming a pE120R-RPSA-CAV1 complex that facilitates ASFV entry. Overall, these results demonstrate that RPSA works as a potential host receptor linking ASFV pE120R to the CAV1-mediated endocytic pathway, facilitating viral entry through caveola-mediated endocytosis. These findings advance our understanding of ASFV entry mechanism and offer a potential target for antiviral strategies against ASFV. IMPORTANCE: African swine fever threatens the global pig industry, yet its entry route remains unclear. Here, host ribosomal protein SA (RPSA) was identified as a binding partner of ASFV capsid protein pE120R. Knockdown of RPSA expression using siRNA in porcine alveolar macrophages reduced ASFV attachment and internalization, whereas ectopic overexpression in CV-1 cells enhanced the uptake of ASFV virions. Pharmacological blockade of caveola-mediated endocytosis (CavME) with nystatin or genetic silencing of caveolin-1 (CAV1) similarly impaired infection, indicating that ASFV entry into its target cells is CavME dependent. Domain mapping showed that pE120R binds LR2 and LR3 of RPSA, while RPSA uses its 16B domain to recruit CAV1, assembling a pE120R-RPSA-CAV1 entry complex. Thus, RPSA bridges ASFV to the CAV1-driven caveolar pathway. These findings define a novel receptor-mediated entry mechanism for ASFV virions and reveal that RPSA-CAV1 pathway may be an actionable antiviral target.