African swine fever virus MGF_110-3L and MGF_110-4L signal TLR2-mediated lethal inflammation.

African swine fever virus (ASFV) is a lethal pathogen that triggers uncontrolled cytokine storms and severe immunopathology. However, the viral factors responsible for systemic inflammation remain unclear. Here, we show that the ASFV-encoded proteins MGF_110-3L and MGF_110-4L are secreted via the conventional ER-Golgi pathway. Single-cell RNA sequencing of porcine PBMCs revealed that MGF_110-3L preferentially activates inflammatory responses in monocytic cells. Mechanistically, both proteins bind Toll-like receptor 2 (TLR2) and signal through TLR2/TLR1 and TLR2/TLR6 heterocomplexes, with the co-receptor CD14 enhancing ligand recognition and signal amplification. These interactions activate MyD88-dependent NF-κB signaling, leading to robust induction of proinflammatory cytokines. ASFV strains lacking either MGF_110-3L or MGF_110-4L cause attenuated cytokine responses in vitro and impaired inflammatory pathology in pigs. Together, these findings establish MGF_110-3L and MGF_110-4L as secreted virulence factors that subvert innate immune recognition and drive lethal inflammation, highlighting their potential as targets for antiviral and vaccine development.