NUDT2 initiates viral RNA degradation by removal of 5′-phosphates.

While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5 '-triphosphate (PPP-) group that impairs degradation by the canonical 5 '-3 ' degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5 '-3 ' exonuclease XRN1. NUDT2 removes 5 '-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5 '-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals. RNA of some viruses is protected from degradation by a 5 ' triphosphate group. Here the authors identify nudix hydrolase 2 (NUDT2) as novel antiviral defense protein that dephosphorylates viral RNA and thereby enables its degradation.We thank the core facility of the MPI of biochemistry for support

Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages