Secretory full-length human prosaposin (PSAP) inhibits SARS-CoV-2 infection through facilitating the release of S1 subunit of spike protein.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has profoundly impacted global public health and the economy. Systematic screening of host factors influencing viral infection is critical for understanding virus-host interactions and for developing host-targeted antiviral strategies. Here, we conducted a host cDNA overexpression screen to identify host factors involved in regulating SARS-CoV-2 replication. This approach identified prosaposin (PSAP), a precursor of lysosomal saposin activators (saposin A, B, C, and D), as an efficient host restriction factor that blocks SARS-CoV-2 entry. We demonstrate that PSAP binds with high affinity to the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. Secreted PSAP efficiently inhibits infection by both SARS-CoV-2 pseudovirus and authentic virus. Mechanistically, PSAP binding facilitates the release of the S1 subunit from the S protein. Molecular docking analysis further revealed that PSAP interacts with the non-receptor-binding motif (non-RBM) region of the domain (RBD). These results suggest that PSAP inhibits SARS-CoV-2 entry through a mechanism analogous to that of neutralizing antibodies. Our findings highlight PSAP as a novel host restriction factor against SARS-CoV-2 infection, offering new insights into potential therapeutic strategies for combating SARS-CoV-2. IMPORTANCE: The systematic identification of host factors that modulate SARS-CoV-2 infection is critical for elucidating the mechanisms of virus-host interaction and for advancing the development of novel host-directed therapeutic interventions. In this study, we identify the human protein prosaposin (PSAP) as a novel and potent innate restriction factor that effectively blocks SARS-CoV-2 infection. By binding with high affinity to the spike protein's receptor-binding domain (RBD) at a unique site, PSAP neutralizes the virus, thereby preventing cellular entry. Consequently, this discovery establishes a foundation for a novel host-directed therapeutic strategy. The development of pharmacologic agents that recapitulate PSAP's action could yield a new class of antivirals that neutralize SARS-CoV-2 by mechanistically disrupting spike protein integrity, offering a complementary approach to conventional antibody therapies.