Systematic functional screening of 3,835 pathogen effector ORFs from viruses, bacteria, and parasites across multiple host pathways reveals that pathogen effectors function as evolved perturbagens with both pathway-specific and pleiotropic activities, uncovering novel mechanisms including HHV6A U14 as a p53 sequestration factor, HHV7 U21 as a dual-function inhibitor of MHC-I and STING through separable domains, and adenoviral 13.6K proteins as a recently evolved family of TAP inhibitors that suppress antigen presentation.

Pathogens deploy effector proteins to exploit host cell biology, and most pathogen open reading frames (ORFs) are rapidly evolving and lack functional annotation. We developed the eORFeome, a scalable functional genomics platform encompassing 3,835 effector ORFs from diverse viruses, bacteria, and parasites. High-throughput barcoded screens across NFκB, apoptosis, p53, cGAS–STING and MHC-I pathways revealed functions for hundreds of uncharacterized eORFs, unexpected new activities for known effectors, and distinct pathway-specific functions encoded by single ORFs. Illustrating the power of the approach, we identify HHV6A U14 as a p53 antagonist, HHV7 U21 as a dual-function STING antagonist and MHC-I antigen display inhibitor, and adenoviral 13.6K/i-leader protein as a de novo evolved TAP inhibitor that suppresses MHC-I display. These results establish a general framework for systematic effector annotation, uncover new mechanisms of host–pathogen interaction across kingdoms, and highlight pathogen effectors as a versatile toolkit for rewiring and probing human cellular pathways.