RIPK3 restricts viral pathogenesis via cell death-independent neuroinflammation

Receptor-interacting protein kinase-3 (RIPK3) is an activator of necroptotic cell death, but recent work has implicated additional roles for RIPK3 in inflammatory signaling independent of cell death. However, while necroptosis has been shown to contribute to antiviral immunity, death-independent roles for RIPK3 in host defense have not been demonstrated. Using a mouse model of West Nile virus (WNV) encephalitis, we show that RIPK3 restricts WNV pathogenesis independently of cell death. Ripk3(-/-) mice exhibited enhanced mortality compared to wild-type (WT) controls, while mice lacking the necroptotic effector MLKL, or both MLKL and caspase-8, were unaffected. The enhanced susceptibility of Ripk3(-/-) mice arose from suppressed neuronal chemokine expression and decreased central nervous system (CNS) recruitment of T lymphocytes and inflammatory myeloid cells, while peripheral immunity remained intact. These data identify pleiotropic functions for RIPK3 in the restriction of viral pathogenesis and implicate RIPK3 as a key coordinator of immune responses within the CNS

MLKL activation triggers NLRP3-mediated processing and release of IL-1β independently of gasdermin-D

Necroptosis is a form of programmed cell death defined by activation of the kinase receptor interacting protein kinase 3 and its downstream effector, the pseudokinase mixed lineage kinase domain-like (MLKL). Activated MLKL translocates to the cell membrane and disrupts it, leading to loss of cellular ion homeostasis. In this study, we use a system in which this event can be specifically triggered by a small-molecule ligand to show that MLKL activation is sufficient to induce the processing and release of bioactive IL-1β. MLKL activation triggers potassium efflux and assembly of the NLRP3 inflammasome, which is required for the processing and activity of IL-1β released during necroptosis. Notably, MLKL activation also causes cell membrane disruption, which allows efficient release of IL-1β independently of the recently described pyroptotic effector gasdermin-D. Taken together, our findings indicate that MLKL is an endogenous activator of the NLRP3 inflammasome, and that MLKL activation provides a mechanism for concurrent processing and release of IL-1β independently of gasdermin-D

A RecA protein surface required for activation of DNA polymerase V.

DNA polymerase V (pol V) of Escherichia coli is a translesion DNA polymerase responsible for most of the mutagenesis observed during the SOS response. Pol V is activated by transfer of a RecA subunit from the 3'-proximal end of a RecA nucleoprotein filament to form a functional complex called DNA polymerase V Mutasome (pol V Mut). We identify a RecA surface, defined by residues 112-117, that either directly interacts with or is in very close proximity to amino acid residues on two distinct surfaces of the UmuC subunit of pol V. One of these surfaces is uniquely prominent in the active pol V Mut. Several conformational states are populated in the inactive and active complexes of RecA with pol V. The RecA D112R and RecA D112R N113R double mutant proteins exhibit successively reduced capacity for pol V activation. The double mutant RecA is specifically defective in the ATP binding step of the activation pathway. Unlike the classic non-mutable RecA S117F (recA1730), the RecA D112R N113R variant exhibits no defect in filament formation on DNA and promotes all other RecA activities efficiently. An important pol V activation surface of RecA protein is thus centered in a region encompassing amino acid residues 112, 113, and 117, a surface exposed at the 3'-proximal end of a RecA filament. The same RecA surface is not utilized in the RecA activation of the homologous and highly mutagenic RumA'2B polymerase encoded by the integrating-conjugative element (ICE) R391, indicating a lack of structural conservation between the two systems. The RecA D112R N113R protein represents a new separation of function mutant, proficient in all RecA functions except SOS mutagenesis

Plasmodium 18S rRNA of intravenously administered sporozoites does not persist in peripheral blood

Abstract Background Plasmodium 18S rRNA is a biomarker used to monitor blood-stage infections in malaria clinical trials. Plasmodium sporozoites also express this biomarker, and there is conflicting evidence about how long sporozoite-derived 18S rRNA persists in peripheral blood. If present in blood for an extended timeframe, sporozoite-derived 18S rRNA could complicate use as a blood-stage biomarker. Methods Blood samples from Plasmodium yoelii infected mice were tested for Plasmodium 18S rRNA and their coding genes (rDNA) using sensitive quantitative reverse transcription PCR and quantitative PCR assays, respectively. Blood and tissues from Plasmodium falciparum sporozoite (PfSPZ)-infected rhesus macaques were similarly tested. Results In mice, when P. yoelii sporozoite inoculation and blood collection were performed at the same site (tail vein), low level rDNA positivity persisted for 2 days post-infection. Compared to intact parasites with high rRNA-to-rDNA ratios, this low level positivity was accompanied by no increase in rRNA-to-rDNA, indicating detection of residual, non-viable parasite rDNA. When P. yoelii sporozoites were administered via the retro-orbital vein and blood sampled by cardiac puncture, neither P. yoelii 18S rRNA nor rDNA were detected 24 h post-infection. Similarly, there was no P. falciparum 18S rRNA detected in blood of rhesus macaques 3 days after intravenous injection with extremely high doses of PfSPZ. Plasmodium 18S rRNA in the rhesus livers increased by approximately 101-fold from 3 to 6 days post infection, indicating liver-stage proliferation. Conclusions Beyond the first few hours after injection, sporozoite-derived Plasmodium 18S rRNA was not detected in peripheral blood. Diagnostics based on 18S rRNA are unlikely to be confounded by sporozoite inocula in human clinical trials

Effects of ATP and primer-template binding on RecA-UmuC cross-linking efficiency in pol V Mut.

<p>Western blot using an antibody to UmuC demonstrating increased UmuC-RecA cross-linking with increasing ATPγS concentration and decreasing UmuC-RecA cross-linking with increasing primer-template concentration in the presence of 500 μM ATPγS. pol V Mut was generated using RecA N113Bpa as described in Methods, with ATPγS and primer-template hairpin DNA added where indicated. ATPγS concentration ranges from 0.8 to 500 μM and primer-template DNA concentration ranges from 0.01–5 μM.</p

RecA D112R and D112R N113R exhibit wild-type RecA binding affinities for pol V.

<p>(A) Location of investigated residues on the RecA protein surface. The D112 and N113 residues compose an acidic knob on the RecA surface. The RecA monomer represented in electrostatic coloring scheme (red = negative charged residues, blue = positive charged residues) was generated in Pymol (PDB 3CMU [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.ref067" target="_blank">67</a>]). In this illustration, the monomer shown is located at the 3' end of the ssDNA (the 3'-proximal RecA monomer), which is the monomer removed by pol V during the activation cycle. (B) Altering this acidic surface to basic residues does not affect the binding affinity for pol V. Equilibrium binding isotherms of wild-type RecA, RecA D112R, and RecA D112R titrated with pol V as monitored by fluorescence depolarization. All data are the average of at least three experiments. Error bars are one standard deviation from the mean.</p

Analysis of cross-linked RecA-UmuC product #1.

<p>The first of the two identified cross-linked products appeared in samples generated via both active and inactive pol V protocols, although it seemed to be somewhat more prominent in the inactive samples. The UmuC peptide involved in the cross-linking encompasses residues 361–376. MS/MS spectra for each of the three peaks seen in the extracted ion chromatogram (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005066#pgen.1005066.g012" target="_blank">Fig. 12</a>) for the RecA-UmuC cross-linked product are shown at the top. The predicted crosslinking locations for peaks A,B, and C are shown at the bottom. The lettering corresponds to the unique elution profile for the same precursor ion.</p