Biology subjects

Sundquist, W. I.

Publications and source records attributed to Sundquist, W. I..

14 recordsLinked to original sources

bioRxiv2026

Lenacapavir allosterically remodels the HIV-1 capsid

Lenacapavir (LEN) is a highly potent, long-acting HIV-1 capsid inhibitor that holds exceptional promise for pre-exposure prophylaxis. LEN causes the mature viral capsid to rupture and lose integrity, but the underlying mechanism has been unclear. Here, we show that LEN is an allosteric modulator of HIV-1 capsid structure that breaks the capsids fullerene cone architecture in two steps: loss of high-curvature declinations occurs early, followed by failure of the capsid body. At the molecular level, LEN alters the non-covalent bonding interactions between capsid subunits and reduces local lattice curvature. LEN also alters the material properties of the capsid, by increasing brittleness. These results provide molecular rationales for how LEN remodels HIV-1 capsid structure and impairs the replication capacity of the virus.

microbiology↗

bioRxiv2025

RNA splicing is required for timely completion of abscission and is modulated by the abscission checkpoint

The abscission checkpoint pauses the progression of cytokinetic abscission at the intercellular bridge when various checkpoint-sustaining errors are sensed. A unique cytoplasmic organelle, the abscission checkpoint body (ACB), forms when the abscission checkpoint is active. This organelle houses proteins involved in abscission and RNA splicing factors. Here, we investigate the role of RNA splicing factors in abscission checkpoint maintenance. A specific subset of RNA splicing factors localizes to ACBs, while others move back into the nucleus. The key RNA splicing factor SF3b1 is aberrantly localized in the cytoplasm and its catalytic activity limited when ACBs are present. Efficient abscission was found to rely on RNA splicing, and we have identified patterns in gene expression and RNA splicing that occur at this time in cell division. Finally, we found a unique RNA landscape associated with the formation of ACBs due to a specific set of changes in RNA expression and alternative splicing, revealing a previously unrecognized level of regulation imparted by abscission checkpoint activity.

cell biology↗

bioRxiv2025

T cell ectosomes promote antibody responses through cognate TCR-pMHC interactions

Protective antibody-mediated immunity requires effective T cell-mediated help. Recognition of peptide antigens presented by major histocompatibility complex class II molecules (pMHCII), via cognate T cell antigen receptors (TCR), activates CD4+ T helper cells to upregulate expression of CD40L and helper cytokines. CD40L-CD40 interactions at T-B cell synapses and secreted cytokines are well established mediators of T cell help. Whether engaged pMHCII also transmits signals that help B cells remains unresolved. Here, we show that TCR-enriched nanoscale vesicles shed by activated T cells (ectosomes) are transferred to antigen-primed B cells, where they engage and cluster cognate pMHCII, triggering signaling and specific IgG antibody production. Disruption of ectosome release attenuates B cell antibody production, while native and synthetic ectosomes boost antibody responses. We conclude that T cell ectosomes constitute a new modality of help for B cells, delivered through engagement of pMHCII by cognate ectosomal TCR.

immunology↗

bioRxiv2024

Cell-free reconstitution of HIV-1 innate immune sensing reveals the capsid is a molecular cloak that protects reverse transcripts from cGAS

Retroviruses can be detected by the innate immune sensor cyclic GMP-AMP synthase (cGAS), which recognizes reverse-transcribed DNA and activates an antiviral response. However, the extent to which HIV-1 shields its genome from cGAS recognition remains unclear. To study this process in mechanistic detail, we reconstituted reverse transcription, genome release, and innate immune sensing of HIV-1 in a cell-free system. We found that wild-type HIV-1 capsids protect viral genomes from cGAS even after completing reverse transcription. Viral DNA could be "deprotected" by thermal stress, capsid mutations, or reduced concentrations of inositol hexakisphosphate (IP6) that destabilize the capsid. Strikingly, the capsid inhibitor lenacapavir also disrupted viral cores and dramatically potentiated cGAS activity, both in vitro and in cellular infections. Our results provide biochemical evidence that the HIV-1 capsid lattice conceals the genome from cGAS and that chemical or physical disruption of the viral core can expose HIV-1 DNA and activate innate immune signaling.

microbiology↗

bioRxiv2022

The Calpain-7 protease functions together with the ESCRT-III protein IST1 within the midbody to regulate the timing and completion of abscission

The Endosomal Sorting Complexes Required for Transport (ESCRT) machinery mediates the membrane fission step that completes cytokinetic abscission and separates dividing cells. Filaments composed of ESCRT-III subunits constrict membranes of the intercellular bridge midbody to the abscission point. These filaments also bind and recruit cofactors whose activities help execute abscission and/or delay abscission timing in response to mitotic errors via the NoCut/Abscission checkpoint. We previously showed that the ESCRT-III subunit IST1 binds the cysteine protease CAPN7 (Calpain-7) and that CAPN7 is required for both efficient abscission and NoCut checkpoint maintenance (Wenzel et al., 2022). Here, we report biochemical and crystallographic studies showing that the tandem MIT domains of CAPN7 bind simultaneously to two distinct IST1 MIT interaction motifs. Structure-guided point mutations in either CAPN7 MIT domain disrupted IST1 binding in vitro and in cells, and depletion/rescue experiments showed that the CAPN7-IST1 interaction is required for: 1) CAPN7 recruitment to midbodies, 2) efficient abscission, and 3) NoCut checkpoint arrest. CAPN7 proteolytic activity is also required for abscission and checkpoint maintenance. Hence, IST1 recruits CAPN7 to midbodies, where its proteolytic activity is required to regulate and complete abscission.

biochemistry↗

bioRxiv2022

HIV-1 preintegration complex preferentially integrates the viral DNA into nucleosomes containing trimethylated histone 3-lysine 36 modification

HIV-1 DNA integration into the host chromosomes is carried out by the preintegration complex (PIC). The PIC contains the viral DNA, virally encoded integrase enzyme and other critical viral/host factors. The PIC-associated viral DNA is preferentially integrated into gene bodies of actively transcribing genes. Here, we identify a biochemical mechanism underlying the preference of PIC-mediated viral DNA integration (PIC-VDI). Specifically, we observed that the PIC-VDI into human chromatin is preferred over the genomic DNA. Surprisingly, nucleosome core particles without any histone modifications were not preferred for PIC-VDI when compared to the analogous naked DNA. However, PIC-VDI was markedly enhanced with nucleosomes containing the trimethylated histone 3 lysine 36 (H3K36me3), an epigenetic mark linked to HIV-1 DNA integration preference. Interestingly, we observed that nucleosomes with flanking linker DNA promoted PIC-VDI in the presence of LEDGF/p75. We also discovered that nucleosomes with linker DNA and H3K36me3 served as the optimal substrate for PIC-VDI. Mapping of the integration sites within these substrates identified preference of specific regions of the nucleosome core DNA for integration. Finally, we provide biochemical and genetic evidence that histone H1 protein, that condenses the chromatin, negatively regulates HIV-1 DNA integration, consistent with the integration preference for open chromatin structure. Collectively, these results identify the role of specific chromatin marks that drive HIV-1 integration preference and define the optimal substrate requirement for efficient DNA integration by the PIC.

microbiology↗

bioRxiv2022

Comprehensive analysis of the human ESCRT-III-MIT domain interactome reveals new cofactors for cytokinetic abscission

The 12 related human ESCRT-III proteins form filaments that constrict membranes and mediate fission, including during cytokinetic abscission. The C-terminal tails of polymerized ESCRT-III subunits also bind proteins that contain Microtubule-Interacting and Trafficking (MIT) domains. MIT domains can interact with ESCRT-III tails in many different ways to create a complex binding code that recruits essential cofactors to sites of ESCRT activity. Here, we have comprehensively and quantitatively mapped the interactions between all known ESCRT-III tails and 19 pure recombinant human MIT domains. We measured 228 pairwise interactions, quantified 58 positive interactions, and discovered 16 previously unreported interactions. We also report the crystal structure of the SPASTIN MIT domain in complex with the IST1 C-terminal tail. Three MIT enzymes were studied in detail and shown to: 1) localize to cytokinetic midbody membrane bridges through interactions with their specific ESCRT-III binding partners (SPASTIN-IST1, KATNA1-CHMP3, and CAPN7-IST1), 2) function in abscission (SPASTIN, KATNA1, and CAPN7), and 3) function in the "NoCut" abscission checkpoint (SPASTIN and CAPN7). Our studies define the human MIT-ESCRT-III interactome, identify new factors and activities required for cytokinetic abscission and its regulation, and provide a platform for analyzing ESCRT-III and MIT cofactor interactions in all ESCRT-mediated processes.

biochemistry↗

bioRxiv2021

Recurrent Emergence of an Antiviral Defense through Repeated Retrotransposition and Truncation of CHMP3

Most restriction factors recognize virus features to execute antiviral functions. In contrast, we discovered retroCHMP3, which instead impairs the host endosomal complexes required for transport (ESCRT) pathway to inhibit budding of enveloped viruses, including HIV-1. The ESCRT pathway is essential, so ESCRT inhibition creates the potential for cytotoxicity. We chart independent courses of retroCHMP3 emergence and reduction of cytotoxicity in New World monkeys and mice using ancestral reconstructions. Overexpression of full-length CHMP3 results in modest antiviral activity, which is enhanced by truncating mutations but causes increased cytotoxicity. We show that retroCHMP3 from squirrel monkeys acquired ancient mutations mitigating cytotoxicity before gaining the activating truncation. In contrast, a truncating mutation arose soon after the independent appearance of murine retroCHMP3, but the variant exhibits regulated expression by interferon signaling, illustrating distinct paths in the emergence of antiviral functions. RetroCHMP3 genes can repeatedly emerge in different species to independently create new immune functions.

evolutionary biology↗

bioRxiv2020

Abscission Checkpoint Bodies Reveal a New Facet of Abscission Checkpoint Control

The abscission checkpoint regulates the ESCRT membrane fission machinery and thereby delays cytokinetic abscission to protect genomic integrity in response to residual mitotic errors. The checkpoint is maintained by Aurora B kinase, which phosphorylates multiple targets, including CHMP4C, a regulatory ESCRT-III subunit necessary for this checkpoint. We now report the discovery that cytoplasmic abscission checkpoint bodies (ACBs) containing phospho-Aurora B and tri-phospho-CHMP4C develop in telophase under an active checkpoint. ACBs are derived from Mitotic Interchromatin Granules (MIGs), transient mitotic structures whose components are housed in splicing-related nuclear speckles during interphase. ACB formation requires CHMP4C, and the ESCRT factor ALIX also contributes. ACB formation is conserved across cell types and under multiple circumstances that activate the checkpoint. Finally, ACBs retain a population of ALIX, and their presence correlates with delayed recruitment of ALIX to the midbody where it would normally promote abscission. Thus, a cytoplasmic mechanism helps regulate midbody machinery to delay abscission.

cell biology↗

bioRxiv2020

A Duplicated ESCRT-III Factor Blocks Enveloped Virus Budding Without Inhibiting Cellular ESCRT Functions

Many enveloped viruses require the endosomal sorting complexes required for transport (ESCRT) pathway to exit infected cells. This highly conserved pathway mediates essential cellular membrane fission events, which restricts the acquisition of adaptive mutations to counteract viral co-option. Here, we describe duplicated and truncated copies of the ESCRT-III factor CHMP3 that block ESCRT-dependent virus budding and that arose independently in New World monkeys and mice. When expressed in human cells, these retroCHMP3 proteins potently inhibit release of retroviruses, paramyxoviruses, and filoviruses. Remarkably, retroCHMP3 proteins have evolved to reduce interactions with other ESCRT-III factors and to have little effect on cellular ESCRT processes, revealing routes for decoupling cellular ESCRT functions from viral exploitation. The repurposing of duplicated ESCRT-III proteins thus provides a mechanism to generate broad-spectrum viral budding inhibitors without blocking highly conserved essential cellular ESCRT functions.

immunology↗

bioRxiv2020

Vaccinia virus hijacks ESCRT-mediated multivesicular body formation for virus egress

Unlike most enveloped viruses, poxvirus egress is a complex process whereby cytoplasmic single membrane-bound virions are wrapped in a cell-derived double membrane. These triple membrane-bound particles, termed intracellular enveloped virions (IEVs), are then released from infected cells by fusion. While the wrapping double membrane is thought to be derived from virus-modified trans-Golgi or early endosomal cisternae, the cellular factors that regulate virus wrapping remain largely undefined. To identify novel cell factors required for this process the prototypic poxvirus, vaccinia virus (VACV), was subjected to a high-throughput RNAi screen directed against cellular membrane trafficking proteins. Focusing on the endosomal sorting complexes required for transport (ESCRT), we demonstrate that ESCRT-III and VPS4 are required for packaging of virus into multivesicular bodies (MVBs). EM-based characterization of these MVB-IEVs showed that they account for half of IEV production indicating that MVBs serve as a second major source of VACV wrapping membrane. These data support a model whereby, in addition to cisternae-based wrapping, VACV hijacks ESCRT-mediated MVB formation to facilitate virus egress and spread.

cell biology↗

bioRxiv2019

Membrane constriction and thinning by sequential ESCRT-III polymerization

The Endosomal Sorting Complexes Required for Transport (ESCRTs) mediate diverse membrane remodeling events. These activities typically require ESCRT-III proteins to stabilize negatively-curved membranes, although recent work has indicated that certain ESCRT-IIIs also participate in positive-curvature membrane shaping reactions. ESCRT-IIIs polymerize into membrane-binding filaments, but the structural basis for negative versus positive membrane curvature shaping by these proteins remains poorly understood. To learn how ESCRT-IIIs shape membranes, we determined structures of human membrane-bound CHMP1B-only, membrane-bound CHMP1B+IST1, and IST1-only filaments by electron cryomicroscopy. Our structures show how CHMP1B first polymerizes into a single-stranded helical filament, shaping membranes into moderate-curvature tubules. Subsequently, IST1 assembles a second strand upon the CHMP1B filament, further constricting the membrane tube and reducing its diameter nearly to the fission point. Each step of constriction, moreover, thins the underlying bilayer and lowers the barrier to membrane fission. Together, our structures reveal how a two-component, sequential polymerization mechanism drives membrane tubulation, tube constriction, and bilayer thinning.

biophysics↗

bioRxiv2018

The ESCRT-III proteins IST1 and CHMP1B assemble around nucleic acids

ESCRT-III proteins can promote inside-out or outside-in membrane tubulation and fission. In addition, several observations suggest that ESCRT factors may also associate with nucleic acids during development, different stages of the cell cycle, and during retro-transposition of parasitic nucleic acids like LINE1 elements. Two ESCRT-III subunits, IST1 (aka CHMP8) and CHMP1B, can coassemble as an external protein coat around liposomes in vitro and around recycling endosomal tubules in living cells. Here we show that recombinant IST1 and CHMP1B can also copolymerize into double stranded filaments that surround nucleic acids. Electron cryo-microscopy reconstructions of nucleic acid-bound IST1-CHMP1B copolymers revealed that the polynucleotides track along a binding groove formed between filaments of the inner CHMP1B strand. The well-ordered structures also reveal that the C-terminal tails of CHMP1B subunits extrude through the outer IST1 layer to the tube exterior. As a result, the MIT domain binding motifs of both CHMP1B and IST1 are arrayed on the outer surface of the copolymer, where they could bind and recruit MIT domain-containing co-factors, such as the SPASTIN ATPase or the USP8 ubiquitin protease. Our structure raises the possibility that ESCRT-III proteins may form nucleic acid complexes in mammalian cells.

biochemistry↗

bioRxiv2018

A cancer-associated polymorphism in ESCRT-III disrupts the abscission checkpoint and promotes genome instability

Cytokinetic abscission facilitates the irreversible separation of daughter cells. This process requires the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery and is tightly regulated by Charged Multivesicular body Protein 4C (CHMP4C), an ESCRT-III subunit that engages the abscission checkpoint (NoCut) in response to mitotic problems such as persisting chromatin bridges within the midbody. Importantly, a human polymorphism in CHMP4CT232 (rs35094336), increases cancer susceptibility. Here, we explain the structural and functional basis for this cancer association: the CHMP4CT232 allele unwinds the C-terminal helix of CHMP4C, impairs binding to the early-acting ESCRT factor ALIX, and disrupts the abscission checkpoint. Cells expressing CHMP4CT232 exhibit increased levels of DNA damage and are sensitized to several conditions that increase chromosome mis-segregation, including DNA replication stress, inhibition of the mitotic checkpoint, and loss of p53. Our data demonstrate the biological importance of the abscission checkpoint, and suggest that dysregulation of abscission by CHMP4CT232 may synergize with oncogene-induced mitotic stress to promote genomic instability and tumorigenesis.\n\nSignificance StatementThe final step of cell division, abscission, is temporally regulated by the Aurora B kinase and CHMP4C in a conserved pathway called the abscission checkpoint which arrests abscission in the presence of lingering mitotic problems. Despite extensive study, the physiological importance of this pathway to human health has remained elusive. We now demonstrate that a cancer predisposing polymorphism in CHMP4C disrupts the abscission checkpoint and results in DNA damage accumulation. Moreover, deficits in this checkpoint synergize with p53 loss and generate aneuploidy under stress conditions that increase the frequency of chromosome missegregation. Therefore, cells expressing the cancer-associated polymorphism in CHMP4C are genetically unstable, thus suggesting a novel oncogenic mechanism that may involve the dysregulation of abscission.

cell biology↗