Biology subjects

Modis, Y.

Publications and source records attributed to Modis, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

Cryo-EM structures and structure-guided design of Atlas virus capsids

Endogenous viruses and retroelements occupy a large fraction of genomes, but the properties of proteins they encode remain largely uncharted. Here, we define Atlas viruses, a new clade of endogenous viruses in nematodes with a retrovirus-like genome organization, phlebovirus-like envelope glycoproteins, and highly divergent Gag proteins. Cryo-EM structures of particles from three Atlas viruses reveal thin-shelled icosahedral assemblies containing 60, 240, 420 or 720 capsid proteins and ranging from 20 to 60 nm in diameter. The capsid fold and assembly interactions are distinct from those of orthoretroviruses but reminiscent of spumaretrovirus and dArc capsids. Atlas capsids package nucleic acids, enter cell by endocytosis, and disassemble at endolysosomal pH. Through structure-guided engineering, we generated recombinant Atlas particles with increased pH resilience and particles displaying a CTLA-4 receptor-targeting tag. This work identifies Atlas capsids as attractive candidates for development as engineerable scaffolds for tissue-specific nucleic acid delivery.

biochemistry↗

bioRxiv2025

Crystal structure of HERV-K envelope glycoprotein surface subunit

The most recently acquired and transcriptionally active family of human endogenous retroviruses (HERVs) is HERV-K. Of the approximately 100 copies of HERV-K in our genome, many retain the potential to proliferate by retrotransposition, express viral proteins, and form functional virus particles. Aberrant expression of the HERV-K envelope glycoprotein (Env) has been associated with cancer and neurodegeneration. Autoantibodies against HERV-K Env have been found in patients with various autoimmune diseases. Here, we report the crystal structures of the Env surface subunit (SU) from HERV-K HML-2, determined at 2.25 [A] resolution. The overall fold is somewhat similar to Syncycin-2 SU and distantly related to HIV-1 gp120. The structure contains five disulfide bonds and four N-linked glycans, including one glycan that appears important for structural stability. Two extended loops form a surface for potential interactions with cell-surface receptors or other cellular factors. The structure also contains three detergent molecules, similar in structure to cholesterol, bound to hydrophobic surface patches. This crystal structure provides a platform for future studies to map autoantigenic epitopes, identify small-molecules that interfere with HERV-K activity, and extend our mechanistic understanding of retroviruses. IMPORTANCE15% of the human genome consists of endogenous retroviruses and other virus-derived elements inherited from ancestral viral infections. Many endogenous retroviruses from the HERV-K family retain the ability to proliferate across the genome and produce virus-like particles. Aberrant expression of the HERV-K envelope glycoprotein is associated with cancer, neurodegeneration, and autoimmune disease. Here, we report the crystal structure of the HERV-K envelope glycoprotein surface subunit. The structure provides a view in atomic-level detail of the molecular components in HERV-K most likely to trigger autoimmune responses. The structure also identifies binding sites for drug-like molecules, paving the way for future studies to develop new therapeutics.

biochemistry↗

bioRxiv2025

ZNFX1, an immunoregulatory RNA helicase and E3 ubiquitin ligase, assembles into pleiomorphic polymers

ZNFX1 is an SF1-family RNA helicase essential for innate immunity. Patients with ZNFX1 mutations experience recurrent infections, yet the underlying mechanism remains unclear. We determined cryo-EM structures of ZNFX1 in apo and RNA-bound forms, revealing auto-inhibition of the helicase through a regulatory insertion occluding the RNA-binding groove. ZNFX1 also functions as a bi-catalytic E3 ubiquitin ligase, containing an RZ-finger homologous to RNF213 and a previously unidentified Miz-like domain that catalyze ubiquitylation independently or cooperatively, with activity enhanced by ubiquitin chains. Patient mutations demonstrate that the helicase, E3 ligase, and rigid zinc-finger stalk connecting them are required for function. ZNFX1 can assemble into structured, pleiomorphic polymers via multivalent protein-protein interactions, revealing a mechanism that may facilitate RNA sequestration in stress granules and antiviral activity. These findings establish ZNFX1 as a multifunctional enzyme in innate immunity that couples RNA sensing to ubiquitin signaling and assembles into higher-order structures for signal amplification.

biochemistry↗

bioRxiv2025

MORC2 directs transcription-dependent CpG methylation of human LINE-1 transposons in early neurodevelopment

Methylation of CpG dinucleotides is essential for silencing genomic repeats such as LINE-1 retrotransposons (L1s) in the germline and soma. Evolutionarily-young L1s are transcribed in human pluripotent stem cells, but how CpG methylation is patterned to these L1s upon exit of pluripotency is unknown. Here we investigate the critical functions of chromatin regulator MORC2 in epigenome reprogramming of the repetitive genome in early human neurodevelopment. We find that reversible ATP-dependent dimerization is required for MORC2 accumulation over L1s but not gene promoters. Engineered mutations of the MORC2 ATPase module severely disrupts the distribution of MORC2 chromatin binding, leading to simultaneous loss of L1 transcriptional control and hyper-repression of clustered ZNF genes in human pluripotent stem cells. Upon neural differentiation these phenotypes persist due to striking, targeted defects in CpG methylation patterning. Together our results define the vital role of MORC2 in safeguarding the somatic human genome upon exit of pluripotency by directing CpG methylation patterning over transcriptionally-active retrotransposons in a manner analogous to the piRNA pathway in the germline.

molecular biology↗

bioRxiv2024

Molecular basis of autoimmune disease protection by MDA5 variants

MDA5 recognizes dsRNA from viruses and retroelements. Cooperative filament formation and ATP-dependent proofreading confer MDA5 with the necessary sensitivity and specificity for dsRNA. The gene encoding MDA5 is a hotspot for disease-associated variants. Many MDA5 variants are associated with protection from autoimmune disease, while increasing the risk of infection and chronic inflammation, but how these variants affect MDA5-dependent RNA sensing remains unclear. Here, we determine the consequences of autoimmune-protective MDA5 variants on the molecular structure and activities of MDA5. The rare variants E627* and I923V reduce the cellular interferon response to picornavirus infection and are deficient in filament formation. The I923V variant is ATPase hyperactive, causing premature dissociation from dsRNA. Cryo-EM structures of MDA5 I923V bound to dsRNA at different stages of ATP hydrolysis reveal smaller RNA binding interfaces, leading to excessive proofreading activity. Variants R843H and T946A, which are genetically linked and cause mild phenotypes, have no effect on dsRNA recognition, suggesting an indirect disease mechanism. We conclude that the autoimmune-protective MDA5 variants lead to a loss of MDA5-dependent signaling via multiple distinct mechanisms.

biochemistry↗

bioRxiv2024

An alternative mechanism for activation of innate immune signaling by MDA5

Long double-stranded (ds) RNA in the cytosol acts as a potent inflammatory molecule recognized by the receptor MDA5, triggering the innate immune response. Mutations affecting MDA5 ATPase activity lead to severe pathological conditions. MDA5 nucleoprotein filament assembly-disassembly dynamic is proposed to regulate dsRNA recognition, though the exact mechanism remains unclear. Here, we employed magnetic tweezers to monitor the assembly and manipulate MDA5 filaments at the single dsRNA level. Following a slow nucleation event, MDA5 assembles cooperatively and directionally into (partial) filaments and utilizes ATP hydrolysis to compact dsRNA via single-stranded (ss) RNA loop extrusion, even against a significant opposing force. This compacted state is further stabilized by oligomerization of MDA5s caspase recruitment domain (CARD) and requires high force to be disrupted. ssRNA gaps impaired compaction, suggesting a new mechanism for dsRNA recognition. We propose that MDA5 mediated dsRNA compaction captures viral dsRNA, preventing further usage for viral replication.

biophysics↗

bioRxiv2023

Structure and methyl-lysine binding selectivity of the HUSH complex subunit MPP8

The Human Silencing Hub (HUSH) guards the genome from the pathogenic effects of retroelement expression. Composed of MPP8, TASOR, and Periphilin-1, HUSH recognizes actively transcribed retrotransposed sequences by the presence of long (>1.5-kb) nascent transcripts without introns. HUSH recruits effectors that alter chromatin structure, degrade transcripts, and deposit transcriptionally repressive epigenetic marks. Here, we report the crystal structure of the C-terminal domain (CTD) of MPP8 necessary for HUSH activity. The MPP8 CTD consists of five ankyrin repeats followed by a domain with structural homology to the PINIT domains of Siz/PIAS-family SUMO E3 ligases. AlphaFold-Multimer modeling predicts that the MPP8 CTD forms extended interaction interfaces with a SPOC domain and a domain with a novel fold in TASOR. The MPP8 chromodomain, known to bind the repressive mark H3K9me3, binds with similar or higher affinity to sequences in the H3K9 methyltransferase subunits SETDB1, ATF7IP, G9a, and GLP. Hence, MPP8 promotes heterochromatinization by recruiting H3K9 methyltransferases. Our work identifies novel structural elements in MPP8 required for HUSH complex assembly and silencing, thereby fulfilling vital functions in controlling retrotransposons.

biochemistry↗

bioRxiv2023

Crystal structure and biochemical activity of the macro domain from rubella virus p150

Rubella virus remains a global health threat. Rubella infections during pregnancy can cause serious congenital pathology and no antiviral treatments are available. Rubella virus encodes a nonstructural polyprotein with RNA polymerase, methyltransferase, and papain-like cysteine protease activities, along with a putative macro domain of unknown function. Macro domains bind ADP-ribose adducts, a post-translational modification that plays a key role in host-virus conflicts. Some macro domains can also remove the mono-ADP-ribose adduct or degrade poly-ADP-ribose chains. Here, we report high-resolution crystal structures of the macro domain from rubella virus nonstructural protein p150, with and without ADP-ribose bound. The overall fold is most similar to macroD-type macro domains from various nonviral species. The specific composition and structure of the residues that poised for catalysis or coordinate ADP-ribose in the rubella virus macro domain are most similar to those of macro domains from alphaviruses. Isothermal calorimetry and enzymatic assays show that the rubella virus macro domain binds ADP-ribose in solution and has mono-ADP-ribosylhydrolase (de-MARylation) activity. IMPORTANCEOur work demonstrates that, like alpha- and coronaviruses, rubiviruses encode a mono-ADP-ribosylhydrolase with a structurally conserved macro domain fold to counteract MARylation by PARPs in the host innate immune response. Our structural data will guide future efforts to develop novel antiviral therapeutics against rubella or infections with related viruses.

biochemistry↗

bioRxiv2023

Contrasting functions of ATP hydrolysis by MDA5 and LGP2 in viral RNA sensing

Cytosolic long double-stranded RNA (dsRNA), among the most potent proinflammatory signals, is recognized by MDA5. MDA5 binds dsRNA cooperatively, forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNAs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-{beta} responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. This amplifies the RNA footprint expansion that accompanies each round of ATP hydrolysis and allows MDA5 to displace tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling of the MDA5-LGP2 interface suggests that MDA5 interacts with dsRNA stem-bound rather than end-bound LGP2. We conclude that NTPase-dependent binding of LGP2 to internal sites on dsRNA increases the number and signaling output of MDA5-dsRNA complexes. Our work identifies novel molecular mechanisms contributing the selectivity and sensitivity of cytosolic dsRNA sensing. KEY POINTSO_LICooperative ATP hydrolysis in MDA5 filaments confers selectivity for dsRNA and displaces other proteins from RNA C_LIO_LINoncooperative NTP hydrolysis by LGP2 induces binding to internal RNA sites with low selectivity C_LIO_LIRNA stem-bound LGP2 nucleates assembly of MDA5 signaling complexes on a broader set of RNA ligands C_LI

biochemistry↗

bioRxiv2022

Structure and functional mapping of the KRAB-KAP1 repressor complex

Transposable elements (TEs) are a genetic reservoir from which new genes and regulatory elements can emerge. Expression of TEs can be pathogenic, however, and is tightly regulated. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) recruit co-repressor KRAB-associated protein 1 (KAP1/TRIM28) to regulate many TEs but how KRAB-ZFPs and KAP1 interact remains unclear. Here, we report the crystal structure of the KAP1 tripartite motif (TRIM) in complex with the KRAB domain from a human KRAB-ZFP, ZNF93. Structure-guided mutations in the KAP1-KRAB binding interface abolished repressive activity in an epigenetic TE silencing assay. Deposition of H3K9me3 over thousands of loci is lost genome-wide in cells expressing a KAP1 variant with mutations that abolish KRAB binding. Our work identifies and functionally validates the KRAB-KAP1 molecular interface, which forms the nexus of a transcriptional control axis vital to vertebrates and underpins programmable gene repression by CRISPRi.

biochemistry↗

bioRxiv2022

The Vaccinia virus chondroitin sulfate binding protein drives host membrane curvature to facilitate fusion

Virus binding serves to define virus tropism and species specificity1. Virus binding proteins are classically considered as facilitators of cell surface attachment prior to receptor engagement and virus internalization. For efficient entry vaccinia virus (VACV) - the prototypic poxvirus - relies on four binding proteins and an eleven-protein entry fusion complex (EFC)2. We recently demonstrated that VACV binding and fusion proteins are organized into distinct functional domains, with localization of EFC proteins to virion tips directly influencing membrane fusion activity3. However, the relationship between virus binding protein distribution, virion binding orientation and subsequent membrane fusion remain unexplored. Here, we show that virus binding proteins guide side-on virion binding and promote curvature of the host membrane towards EFC-containing virion tips to facilitate virus fusion. Using a cell-derived membrane-bleb model system together with super-resolution and electron microscopy we found that side-bound VACV virions induce membrane invagination in the presence of low pH. Repression or deletion of individual binding proteins revealed that three of four contribute to binding orientation, amongst which the chondroitin sulphate binding protein, D8, is required for host membrane bending. Consistent with low-pH dependent macropinocytic entry of vaccinia virus4,5, loss of D8 prevents virion-associated macropinosome membrane bending, disrupts fusion pore formation and infection kinetics. Our results extend the role of viral binding proteins from mere attachment factors to active participants in successful viral membrane fusion and further illustrate the influence of virus protein architecture on successful infection.

microbiology↗

bioRxiv2021

A bioactive phlebovirus-like envelope protein in a hookworm endogenous virus

Endogenous retroviruses (ERVs), accounting for 14% of our genome, serve as a genetic reservoir from which new genes can emerge. Nematode ERVs are particularly diverse and informative of retrovirus evolution. We identify Atlas virus - a novel, intact ERV in the human hookworm Ancylostoma ceylanicum with an envelope protein genetically related to GN-GC glycoproteins from phleboviruses. A cryo-EM structure of Atlas GC reveals a class II viral membrane fusion protein fold not previously seen in retroviruses. Atlas GC has the structural hallmarks of an active fusogen. Atlas GC trimers insert into membranes with endosomal lipid compositions and low pH. When expressed on the plasma membrane, Atlas GC has cell-cell fusion activity. Atlas virus is transcriptionally active at specific stages of hookworm development. The preserved activities and expression pattern of Atlas GC suggest it has acquired a cellular function. Our work reveals an unexpected degree of structural and genetic plasticity in retroviruses.

molecular biology↗

bioRxiv2021

Cryo-electron tomography of ASC signalling sites in pyroptotic cells

NLRP3 induces caspase-1-dependent pyroptotic cell death to drive inflammation. Aberrant activity of NLRP3 occurs in many human diseases. NLRP3 activation induces ASC polymerization into a single, micron-scale perinuclear punctum. Higher resolution imaging of this signaling platform is needed to understand how it induces pyroptosis. Here, we apply correlative cryo-light microscopy and cryo-electron tomography to visualize ASC/caspase-1 in NLRP3-activated cells. The puncta are composed of branched ASC filaments, with a tubular core formed by the pyrin domain. Ribosomes and Golgi-like vesicles permeate the filament network, consistent with roles for these organelles in NLRP3 activation. Mitochondria are not associated with ASC but have outer-membrane discontinuities the same size as gasdermin D pores, consistent with our data showing gasdermin D associates with mitochondria and contributes to mitochondrial depolarization. One-Sentence SummaryElectron tomography of frozen cells reveals the ultrastructure of ASC specks and gasdermin D pores in adjacent mitochondria.

immunology↗

bioRxiv2021

MDA5 autoimmune disease variant M854K prevents ATP-dependent structural discrimination of viral and cellular RNA

Our innate immune responses to viral RNA are vital defenses. Long cytosolic double-stranded RNA (dsRNA) is recognized by MDA5. The ATPase activity of MDA5 contributes to its dsRNA binding selectivity. Mutations that reduce RNA selectivity can cause autoimmune disease. Here, we show how the disease-associated MDA5 variant M854K perturbs MDA5-dsRNA recognition. M854K MDA5 constitutively activates interferon signaling in the absence of exogenous RNA. M854K MDA5 lacks ATPase activity and binds more tightly to synthetic Alu:Alu dsRNA. CryoEM structures MDA5-dsRNA filaments at different stages of ATP hydrolysis show that the K854 side-chain forms polar bonds that constrain the conformation of MDA5 subdomains, disrupting key steps in the ATPase cycle-RNA footprint expansion and helical twist modulation. The M854K mutation inhibits ATP-dependent RNA proofreading via a novel allosteric mechanism, allowing MDA5 to form signaling complexes on endogenous RNAs. This work provides new insights on how MDA5 recognizes dsRNA in health and disease.

biochemistry↗

bioRxiv2020

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control

SummaryThe Human Silencing Hub (HUSH) complex epigenetically represses retroviruses, transposons and genes in vertebrates. HUSH therefore maintains genome integrity and is central in the interplay between intrinsic immunity, transposable elements and transcriptional regulation. Comprising three subunits - TASOR, MPP8 and Periphilin - HUSH regulates SETDB1-dependent deposition of the transcriptionally repressive epigenetic mark H3K9me3 and recruits MORC2 to modify local chromatin structure. However the mechanistic roles of each HUSH subunit remain undetermined. Here we show that TASOR lies at the heart of HUSH, providing a platform for assembling the other subunits. Targeted epigenomic profiling supports the model that TASOR binds and regulates H3K9me3 specifically over LINE-1 repeats and other repetitive exons in transcribed genes. We find TASOR associates with several components of the nuclear RNA processing machinery and its modular domain architecture bears striking similarities to that of Chp1, the central component of the yeast RNA-induced transcriptional silencing (RITS) complex. Together these observations suggest that an RNA intermediate may be important for HUSH activity. We identify the TASOR domains necessary for HUSH assembly and transgene repression. Structural and genomic analyses reveal that TASOR contains a poly-ADP ribose polymerase (PARP) domain dispensable for assembly and chromatin localization, but critical for epigenetic regulation of target elements. This domain contains a degenerated and obstructed active site and has hence lost catalytic activity. Together our data demonstrate that TASOR is a pseudo-PARP critical for HUSH complex assembly and H3K9me3 deposition over its genomic targets.

molecular biology↗

bioRxiv2019

Periphilin self-association underpins epigenetic silencing by the HUSH complex

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR core complex shows Periphilin forms -helical homodimers, which each bind a single TASOR molecule. The NTD binds RNA non-specifically and forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation on nascent transcripts.

biochemistry↗

bioRxiv2019

An equilibrium binding model for CpG DNA-dependent dimerization of Toll-like receptor 9

Microbial nucleic acids in the extracellular milieu are recognized in vertebrates by Toll-like receptors (TLRs), one of the most important families of innate immune receptors. TLR9 recognizes single-stranded unmethylated CpG DNA in endosomes. DNA binding induces dimerization of TLR9 and activation of a potent inflammatory response. To provide insights on how DNA ligands induce TLR9 dimerization, we developed a detailed theoretical equilibrium ligand binding model. Light scattering and fluorescence polarization assays performed with a recombinant TLR9 ectodomain fragment and a panel of agonistic and antagonistic DNA ligands provide data that restrain the binding parameters in our binding model. This work brings us one step closer to establishing a rigorous biochemical understanding of how TLRs are activated by their ligands.

biochemistry↗

bioRxiv2018

MAVS polymers smaller than 80 nm induce mitochondrial membrane remodeling and interferon signaling

Double-stranded RNA (dsRNA) is a potent proinflammatory signature of viral infection. Oligomerization of RIG-I-like receptors on cytosolic dsRNA nucleates self-assembly of the mitochondrial antiviral signaling protein (MAVS). In the current signaling model, the caspase recruitment domains of MAVS form helical fibrils that self-propagate like prions to promote signaling complex assembly. However, there is no conclusive evidence that MAVS forms fibrils in cells or with the transmembrane anchor present. We show here with super-resolution light microscopy that MAVS activation by dsRNA induces mitochondrial membrane remodeling. Quantitative image analysis at imaging resolutions as high as 32 nm shows that in the cellular context MAVS signaling complexes and the fibrils within them are smaller than 80 nm. The transmembrane domain of MAVS is required for its membrane remodeling, interferon signaling and proapoptotic activities. We conclude that membrane tethering of MAVS restrains its polymerization and contributes to mitochondrial remodeling and apoptosis upon dsRNA sensing.

molecular biology↗