Bacterial reverse transcriptase synthesizes long poly(A)-rich cDNA for antiphage defense.

Prokaryotic defense-associated reverse transcriptases (DRTs) were recently identified with antiviral functions; however, their functional mechanisms remain largely unexplored. Here we show that DRT9 forms a hexameric complex with its upstream noncoding RNA (ncRNA) to mediate antiphage defense by inducing cell growth arrest through abortive infection. Upon phage infection, the phage-encoded ribonucleotide reductase NrdAB complex increases intracellular deoxyadenosine triphosphate levels, activating DRT9 to synthesize long, polyadenylate [poly(A)]–rich single-stranded complementary DNA (cDNA), which likely sequesters the essential phage single-stranded DNA binding (SSB) protein and disrupts phage propagation. We further determined the cryo–electron microscopy structure of the DRT9-ncRNA hexamer complex, providing mechanistic insights into its cDNA synthesis. These findings highlight the diversity of RT-based antiviral defense mechanisms, expand our understanding of RT biological functions, and provide a structural basis for developing DRT9-based biotechnological tools. Editor's summary: Reverse transcriptases (RTs) are enzymes that synthesize complementary DNA (cDNA) from RNA templates, a process widely used in biotechnology. Defense-associated reverse transcriptases (DRTs) have emerged as a key player in bacterial antiviral immunity. Song et al. discovered an antiviral mechanism in which a bacterial RT, DRT9, senses phage infection–driven increases in intracellular dATP levels and responds by synthesizing poly(A)–rich single-stranded cDNA. These repetitive poly(A) cDNA molecules likely sequester phage single-stranded DNA–binding proteins, disrupting viral replication. This discovery expands the known functions of reverse transcription, drawing parallels to telomerase-mediated DNA elongation and revealing an unexpected role for cDNA in bacterial immune defense. —Di Jiang INTRODUCTION: Reverse transcriptases (RTs) synthesize complementary DNA (cDNA) from RNA templates and are widely used in biotechnology. Recent studies have identified diverse prokaryotic RTs involved in antiphage defense, including CRISPR-associated RTs, retrons, abortive infection (Abi) systems, and uncharacterized groups (UGs). A newly defined class, defense-associated RTs (DRTs), often co-occurs with noncoding RNAs (ncRNAs) and has been phylogenetically grouped into nine types (DRT1 to DRT9), suggesting diverse mechanistic functions. Although the DRT2 system has been mechanistically characterized as mediating phage resistance through rolling-circle reverse transcription of an ncRNA to de novo synthesize nearly endless open reading frame (neo) genes, revealing new biological functions of cDNA products, the mechanisms of other DRTs remain unclear. RATIONALE: The diversity of RT-produced cDNA, templated by RT-associated ncRNAs, plays a central role in antiviral defense, underscoring the mechanistic versatility of RTs in antiphage immunity. We investigated the Escherichia coli A178 DRT type 9 (DRT9) system, which comprises only two essential components: an upstream ncRNA and an RT. Both are required for antiphage defense. The ncRNA adopts a distinctive secondary structure, suggesting a specific mode of action compared with previously characterized systems. RESULTS: We show that DRT9 forms a stable hexameric complex with its upstream ncRNA and confers strong resistance against a broad range of phages, particularly Tevenvirinae, by inducing host growth arrest through an abortive infection mechanism. The cryo–electron microscopy structure of the DRT9-ncRNA complex revealed that DRT9 adopts a classical RT fold, whereas its associated ncRNA forms a distinctive "tree-like" structure with six stem-loops. Mutational analysis demonstrated that the ncRNA's secondary structure, rather than its primary sequence, is essential for antiphage defense. As a reverse transcriptase, DRT9 synthesizes long, poly(A)-rich single-stranded cDNA—up to ~5000 nucleotides, independently of any externally provided primer, with high deoxyadenosine triphosphate (dATP) concentrations substantially enhancing cDNA production in vitro. To understand how the DRT9 system senses phage infection, we isolated phage escaper mutants carrying mutations in nrdA and nrdB, which encode ribonucleotide reductase (RNR) subunits. Overexpression of nrdAB increased cellular dATP concentrations, triggering DRT9 activation and in vivo synthesis of long poly(A)-rich cDNA. These results indicate that DRT9 senses phage infection through an increase in RNR-mediated dATP levels. RNA-sequencing analysis revealed strong up-regulation of the gene encoding single-stranded DNA binding (SSB) protein during DRT9 activation. Notably, overexpression of the phage T4 SSB protein suppressed DRT9-mediated defense and restored phage propagation. We further showed that DRT9-produced poly(A)-rich cDNA directly binds T4 SSB in vitro and in vivo. Collectively, these results suggest that this poly(A)–rich single-stranded cDNA, lacking secondary structure, acts as an optimal substrate to bind and sequester the essential phage SSB, thereby blocking phage propagation. CONCLUSION: Our study reveals an unexpected antiviral strategy used by the DRT9 system, in which infection-induced increase in dATP concentration triggers the synthesis of long, poly(A)-rich single-stranded cDNA. This cDNA, which lacks secondary structure, likely acts as a molecular decoy that binds and sequesters essential phage replication associated protein SSB, thereby disrupting phage propagation. These findings highlight the mechanistic diversity of prokaryotic RTs in antiviral defense and expand our understanding of the biological roles of RT. Furthermore, the DRT9 system presents a potential platform for engineering new RT-based biotechnological tools. Antiviral immunity mediated by long poly(A)-rich cDNA synthesis.: The bacterial DRT9 system comprises a reverse transcriptase DRT9 and ncRNA. Phage infection increases intracellular dATP concentrations through phage-encoded ribonucleotide reductase NrdAB complex, activating DRT9 to produce long poly(A)-rich single-stranded cDNA that likely sequesters the essential phage SSB, disrupting phage propagation. [ABSTRACT FROM AUTHOR]