Mechanism of phage sensing and restriction by toxin-antitoxin-chaperone systems.

Toxin-antitoxins (TAs) are prokaryotic two-gene systems composed of a toxin neutralized by an antitoxin. Toxin-antitoxin-chaperone (TAC) systems additionally include a SecB-like chaperone that stabilizes the antitoxin by recognizing its chaperone addiction (ChAD) element. TACs mediate antiphage defense, but the mechanisms of viral sensing and restriction are unexplored. We identify two Escherichia coli antiphage TAC systems containing host inhibition of growth (HigBA) and CmdTA TA modules, HigBAC and CmdTAC. HigBAC is triggered through recognition of the gpV major tail protein of phage λ. Chaperone HigC recognizes gpV and ChAD via analogous aromatic molecular patterns, with gpV outcompeting ChAD to trigger toxicity. For CmdTAC, the CmdT ADP-ribosyltransferase toxin modifies mRNA to halt protein synthesis and limit phage propagation. Finally, we establish the modularity of TACs by creating a hybrid broad-spectrum antiphage system combining the CmdTA TA warhead with a HigC chaperone phage sensor. Collectively, these findings reveal the potential of TAC systems in broad-spectrum antiphage defense.

Viral proteins activate PARIS-mediated tRNA degradation and viral tRNAs rescue infection.

Viruses compete with each other for limited cellular resources, and some viruses deliver defense mechanisms that protect the host from competing genetic parasites. PARIS is a defense system, often encoded in viral genomes, that is composed of a 53 kDa ABC ATPase (AriA) and a 35 kDa TOPRIM nuclease (AriB). Here we show that AriA and AriB assemble into a 425 kDa supramolecular immune complex. We use cryo-EM to determine the structure of this complex which explains how six molecules of AriA assemble into a propeller-shaped scaffold that coordinates three subunits of AriB. ATP-dependent detection of foreign proteins triggers the release of AriB, which assembles into a homodimeric nuclease that blocks infection by cleaving the host tRNALys. Phage T5 subverts PARIS immunity through expression of a tRNALys variant that prevents PARIS-mediated cleavage, and thereby restores viral infection. Collectively, these data explain how AriA functions as an ATP-dependent sensor that detects viral proteins and activates the AriB toxin. PARIS is one of an emerging set of immune systems that form macromolecular complexes for the recognition of foreign proteins, rather than foreign nucleic acids.

Nucleoside Analog 2',3'-Isopropylidene-5-Iodouridine as Novel Efficient Inhibitor of HIV-1.

Nucleoside reverse transcriptase inhibitors are the first class of drugs to be approved by the FDA for the suppression of HIV-1 and are widely used for this purpose in combination with drugs of other classes. Despite the progress in HIV-1 treatment, there is still the need to develop novel efficient antivirals. Here the efficiency of HIV-1 inhibition by a set of original 5-substituted uridine nucleosides was studied. We used the replication deficient human immunodeficiency virus (HIV-1)-based lentiviral particles and identified that among the studied compounds, 2',3'-isopropylidene-5-iodouridine was shown to cause anti-HIV-1 activity. Importantly, no toxic action of this compound against the cells of T-cell origin was found. We determined that this compound is significantly more efficient at suppressing HIV-1 compared to Azidothymidine (AZT) when taken at the high non-toxic concentrations. We did not find any profit when using AZT in combination with 2',3'-isopropylidene-5-iodouridine. 2',3'-Isopropylidene-5-iodouridine acts synergistically to repress HIV-1 when combined with the CDK4/6 inhibitor Palbociclib in low non-toxic concentration. No synergistic antiviral action was detected when AZT was combined with Palbociclib. We suggest 2',3'-isopropylidene-5-iodouridine as a novel perspective non-toxic compound that may be used for HIV-l suppression.

The structural basis of hyperpromiscuity in a core combinatorial network of type II toxin-antitoxin and related phage defense systems.

Toxin-antitoxin (TA) systems are a large group of small genetic modules found in prokaryotes and their mobile genetic elements. Type II TAs are encoded as bicistronic (two-gene) operons that encode two proteins: a toxin and a neutralizing antitoxin. Using our tool NetFlax (standing for Network-FlaGs for toxins and antitoxins), we have performed a large-scale bioinformatic analysis of proteinaceous TAs, revealing interconnected clusters constituting a core network of TA-like gene pairs. To understand the structural basis of toxin neutralization by antitoxins, we have predicted the structures of 3,419 complexes with AlphaFold2. Together with mutagenesis and functional assays, our structural predictions provide insights into the neutralizing mechanism of the hyperpromiscuous Panacea antitoxin domain. In antitoxins composed of standalone Panacea, the domain mediates direct toxin neutralization, while in multidomain antitoxins the neutralization is mediated by other domains, such as PAD1, Phd-C, and ZFD. We hypothesize that Panacea acts as a sensor that regulates TA activation. We have experimentally validated 16 NetFlax TA systems and used domain annotations and metabolic labeling assays to predict their potential mechanisms of toxicity (such as membrane disruption, and inhibition of cell division or protein synthesis) as well as biological functions (such as antiphage defense). We have validated the antiphage activity of a RosmerTA system encoded by Gordonia phage Kita, and used fluorescence microscopy to confirm its predicted membrane-depolarizing activity. The interactive version of the NetFlax TA network that includes structural predictions can be accessed at http://netflax.webflags.se/.