MDM2 Influences ACE2 Stability and SARS-CoV-2 Uptake.

Angiotensin-converting enzyme 2 (ACE2) is the central entry receptor for SARS-CoV-2. However, surprisingly little is known about the effects of host regulators on ACE2 localization, expression, and the associated influence on SARS-CoV-2 infection. Here we identify that ACE2 expression levels are regulated by the E3 ligase MDM2 and that MDM2 levels indirectly influence infection with SARS-CoV-2. Genetic depletion of MDM2 elevated ACE2 expression levels, which strongly promoted infection with all SARS-CoV-2 isolates tested. SARS-CoV-2 spike-pseudotyped viruses and the uptake of non-replication-competent virus-like particles showed that MDM2 affects the viral uptake process. MDM2 ubiquitinates Lysine 788 of ACE2 to induce proteasomal degradation, and degradation of this residue led to higher ACE2 expression levels and superior virus particle uptake. Our study illustrates that cellular regulators of ACE2 stability, such as MDM2, play an important role in defining the infection capabilities of SARS-CoV-2.

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.

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.

Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics.

Over the past decades, starting from crude cell extracts, a variety of successful preparation protocols and optimized reaction conditions have been established for the production of cell-free gene expression systems. One of the crucial steps during the preparation of cell extract-based expression systems is the cell lysis procedure itself, which largely determines the quality of the active components of the extract. Here we evaluate the utility of an E. coli cell extract, which was prepared using a combination of lysozyme incubation and a gentle sonication step. As quality measure, we demonstrate the cell-free expression of YFP at concentrations up to 0.6 mg/mL. In addition, we produced and assembled T7 bacteriophages up to a titer of 108  PFU/mL. State-of-the-art quantitative proteomics was used to compare the produced extracts with each other and with a commercial extract. The differences in protein composition were surprisingly small between lysozyme-assisted sonication (LAS) extracts, but we observed an increase in the release of DNA-binding proteins for increasing numbers of sonication cycles. Proteins taking part in carbohydrate metabolism, glycolysis, amino acid and nucleotide related pathways were found to be more abundant in the LAS extract, while proteins related to RNA modification and processing, DNA modification and replication, transcription regulation, initiation, termination and the TCA cycle were found enriched in the commercial extract.

SEQU-INTO: Early detection of impurities, contamination and off-targets (ICOs) in long read/MinION sequencing.

The MinION sequencer by Oxford Nanopore Technologies turns DNA and RNA sequencing into a routine task in biology laboratories or in field research. For downstream analysis it is required to have a sufficient amount of target reads. Especially prokaryotic or bacteriophagic sequencing samples can contain a significant amount of off-target sequences in the processed sample, stemming from human DNA/RNA contamination, insufficient rRNA depletion, or remaining DNA/RNA from other organisms (e.g. host organism from bacteriophage cultivation). Such impurity, contamination and off-targets (ICOs) block read capacity, requiring to sequence deeper. In comparison to second-generation sequencing, MinION sequencing allows to reuse its chip after a (partial) run. This allows further usage of the same chip with more sample, even after adjusting the library preparation to reduce ICOs. The earlier a sample's ICOs are detected, the better the sequencing chip can be conserved for future use. Here we present sequ-into, a low-resource and user-friendly cross-platform tool to detect ICO sequences from a predefined ICO database in samples early during a MinION sequencing run. The data provided by sequ-into empowers the user to quickly take action to preserve sample material and chip capacity. sequ-into is available from https://github.com/mjoppich/sequ-into.