Activation and effector mechanisms of bacterial reverse transcriptase-mediated antiviral immunity

The prokaryotic type 2 defense-associated reverse transcriptase (DRT2) system mediate antiviral defense by catalyzing rolling-circle reverse transcription of a noncoding RNA (ncRNA) and producing the toxic Neo protein that arrests bacterial growth. However, the mechanisms underlying DRT2 activation and effector function remain unknown. Here, we identified two distinct activation mechanisms: (1) binding of phage-encoded single-stranded DNA-binding or annealing proteins to DRT2-produced cDNA; and (2) phage-induced increase of intracellular dNTP levels via phage-encoded ribonucleotide reductase NrdAB. Once activated, the produced Neo protein targets the bacterial membrane, inducing membrane depolarization and growth inhibition. Cryo-EM structures of the DRT2–ncRNA complex in its arrested and dGTP-bound active states provide mechanistic insights into rolling-circle cDNA synthesis and template jumping. These insights allow us to reprogram the DRT2 system with an engineered ncRNA to produce user-defined double-stranded DNA in vivo, independent of phage infection, highlighting its potential for genome engineering.