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HIV-1 integration favors nuclear speckle (NS)-proximal chromatin and viral infection induces the formation of capsid-dependent CPSF6 condensates that colocalize with nuclear speckles (NSs). Although CPSF6 displays liquid-liquid phase separation (LLPS) activity in vitro, the contributions of its different intrinsically disordered regions, which includes a central prion-like domain (PrLD) with capsid binding FG motif and C-terminal mixed-charge domain (MCD), to LLPS activity and to HIV-1 infection remain unclear. Herein, we determined that the PrLD and MCD both contribute to CPSF6 LLPS activity in vitro. Akin to FG mutant CPSF6, infection of cells expressing MCD-deleted CPSF6 uncharacteristically arrested at the nuclear rim. While heterologous MCDs effectively substituted for CPSF6 MCD function during HIV-1 infection, Arg-Ser domains from related SR proteins were largely ineffective. While MCD-deleted and wildtype CPSF6 proteins displayed similar capsid binding affinities, the MCD imparted LLPS-dependent higher-order binding and co-aggregation with capsids in vitro and in cellulo. NS depletion reduced CPSF6 puncta formation without significantly affecting integration into NS-proximal chromatin, and appending the MCD onto a heterologous capsid binding protein partially restored virus nuclear penetration and integration targeting in CPSF6 knockout cells. We conclude that MCD-dependent CPSF6 condensation with capsids underlies post-nuclear incursion for viral DNA integration and HIV-1 pathogenesis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

HIV-1 infection requires nuclear entry of the viral genome. Previous evidence suggests that this entry proceeds through nuclear pore complexes (NPCs), with the 120 × 60 nm capsid squeezing through an approximately 60-nm-wide central channel 1 and crossing the permeability barrier of the NPC. This barrier can be described as an FG phase 2 that is assembled from cohesively interacting phenylalanine-glycine (FG) repeats 3 and is selectively permeable to cargo captured by nuclear transport receptors (NTRs). Here we show that HIV-1 capsid assemblies can target NPCs efficiently in an NTR-independent manner and bind directly to several types of FG repeats, including barrier-forming cohesive repeats. Like NTRs, the capsid readily partitions into an in vitro assembled cohesive FG phase that can serve as an NPC mimic and excludes much smaller inert probes such as mCherry. Indeed, entry of the capsid protein into such an FG phase is greatly enhanced by capsid assembly, which also allows the encapsulated clients to enter. Thus, our data indicate that the HIV-1 capsid behaves like an NTR, with its interior serving as a cargo container. Because capsid-coating with trans-acting NTRs would increase the diameter by 10 nm or more, we suggest that such a 'self-translocating' capsid undermines the size restrictions imposed by the NPC scaffold, thereby bypassing an otherwise effective barrier to viral infection., (© 2024. The Author(s).)

Chemotherapy-induced peripheral neuropathy (CIPN) is a major unmet medical need with limited treatment options. Despite different mechanisms of action, diverse chemotherapeutics can cause CIPN through a converged pathway─an active axon degeneration program that engages the dual leucine zipper kinase (DLK). DLK is a neuronally enriched kinase upstream in the MAPK-JNK cascade, and while it is dormant under physiological conditions, DLK mediates a core mechanism for neuronal injury response under stress conditions, making it an attractive target for treatment of neuronal injury and neurodegenerative diseases. We have developed potent, selective, brain penetrant DLK inhibitors with excellent PK and activity in mouse models of CIPN. Lead compound IACS-52825 ( 22 ) showed strongly effective reversal of mechanical allodynia in a mouse model of CIPN and was advanced into preclinical development.

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

This paper introduces a novel bacterial identification assay based on thermal wave analysis through surface-imprinted polymers (SIPs). Aluminum chips are coated with SIPs, serving as synthetic cell receptors that have been combined previously with the heat-transfer method (HTM) for the selective detection of bacteria. In this work, the concept of bacterial identification is extended toward the detection of nine different bacterial species. In addition, a novel sensing approach, thermal wave transport analysis (TWTA), is introduced, which analyzes the propagation of a thermal wave through a functional interface. The results presented here demonstrate that bacterial rebinding to the SIP layer resulted in a measurable phase shift in the propagated wave, which is most pronounced at a frequency of 0.03 Hz. In this way, the sensor is able to selectively distinguish between the different bacterial species used in this study. Furthermore, a dose-response curve was constructed to determine a limit of detection of 1 × 10 4 CFU mL -1 , indicating that TWTA is advantageous over HTM in terms of sensitivity and response time. Additionally, the limit of selectivity of the sensor was tested in a mixed bacterial solution, containing the target species in the presence of a 99-fold excess of competitor species. Finally, a first application for the sensor in terms of infection diagnosis is presented, revealing that the platform is able to detect bacteria in clinically relevant concentrations as low as 3 × 10 4 CFU mL -1 in spiked urine samples.