African swine fever virus pMGF110-9L plays a critical role in the integrated stress response and viral pathogenesis.

African swine fever virus (ASFV) is the causative agent of ASF, a highly contagious and frequently fatal hemorrhagic disease in domestic swine and wild boars, posing a significant threat to the global pig industry. To ensure successful replication, ASFV may modulate cellular stress mechanisms to hijack the host translation machinery and promote viral factory formation to favor viral protein synthesis and counteract host defenses. However, the interplay between ASFV replication and cellular stress responses has not yet been elucidated, hindering the development of vaccines or therapeutic options. In this study, we dissected how ASFV manipulates the integrated stress response (ISR) to facilitate viral replication and identified the virulence-related factor pMGF110-9L as the strongest activator of ATF4 expression, a key factor downstream of eIF2α phosphorylation during ISR activation. We characterized pMGF110-9L as an endoplasmic reticulum (ER)-localized membrane protein, the expression of which profoundly reorganized the cellular compartments, leading to ER swelling and aggregation. Further proteomic analyses and biochemical experiments revealed that pMGF110-9L is crucial for ASFV-induced ER stress and promotes the activation of the PERK-eIF2α-ATF4, IRE1α-XBP1, and ATF6 branches of the unfolded protein response. Specifically, we demonstrated that pMGF110-9L activates the ISR through the PERK/PKR-eIF2α pathway, leading to viral takeover of the protein synthesis machinery, accompanied by global protein synthesis shutdown and stress granule formation. Furthermore, the transmembrane domain of pMGF110-9L is indispensable for targeting the ER and subsequent stress responses. Overall, these findings inform novel insights into ASFV-host interactions and define a previously uncharacterized role for pMGF110-9L in ASFV pathogenesis.IMPORTANCEAfrican swine fever (ASF), caused by African swine fever virus (ASFV), poses a catastrophic threat to global pork production. The complex pathogenesis and immunoevasion strategies of ASFV have not been clearly elucidated, raising safety concerns regarding modified live virus (MLV) vaccines. Therefore, a better understanding of how ASFV manipulates host responses to establish intracellular conditions favorable for viral replication could provide essential insights into ASFV-host interactions and enable the development of safer and more effective MLV vaccines. In this study, we comprehensively elucidated the relationship between ASFV-encoded proteins and the activation of the integrated stress response. We identified pMGF110-9L as a prominent virulence factor involved in viral takeover of the protein synthesis machinery, accompanied by endoplasmic reticulum (ER) stress, ER-associated aggresome formation, halted host protein translation, and stress granule formation. Importantly, the transmembrane domain of pMGF110-9L was critical for triggering cellular stress mechanisms. This study advances our understanding of ASFV pathogenesis and highlights pMGF110-9L as a potential target for MLV vaccine development.