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At least 19 recordsLinked to original sources
PubMed2026

Alternative bipartite arrangements of VP1 BR1-3 drive efficient nuclear import of AAV2 capsids.

Adeno-associated viruses (AAVs) are among the most extensively studied viral gene-therapy vectors, yet the mechanisms governing their nuclear entry remain incompletely understood. Efficient transduction requires that the AAV capsid, or its structural subunit VP1, traverse the nuclear envelope to deliver the therapeutic genome. The N-terminal region of VP1 contains three clustered basic regions (BR1-3) proposed to function as nuclear localization signals (NLSs). Here, we combine cellular, biophysical, structural, and computational modelling approaches to define the nuclear import mechanism of AAV2 VP1 at molecular resolution. We show that VP1 engages the classical importin-α/β1 (IMPα/β1) pathway and binds multiple IMPα paralogs with distinct affinities. Crystallographic and mutational analyses reveal that two intact BRs are required to simultaneously occupy the major and minor binding pockets of IMPα in a bipartite configuration. Structural data indicate that mouse IMPα2 (mIMPα2) preferentially accommodates BR1 and BR3 at these sites, however, functional studies demonstrate that mutation of individual BRs does not abolish IMP binding or nuclear accumulation. This robustness arises from the ability of BR2 to flexibly engage both binding pockets, enabling the formation of alternative bipartite arrangements (BR1-BR2, BR2-BR3, or BR1-BR3). Together, these findings reveal an unexpected versatility in how AAV2 VP1 exploits the IMPα binding sites, providing a structural basis for efficient capsid nuclear import. The flexibility of BR1-3 expands the current paradigm of viral NLS organization and suggests new strategies to fine-tune nuclear targeting AAV-based gene-therapy vectors.

Adeno-associated virus↗
PubMed2025

Temporal gating of nuclear import: How Merkel cell polyomavirus exploits the cell cycle for nuclear entry.

Merkel cell polyomavirus (MCPyV) is a small, DNA tumor virus that is causally linked to an aggressive form of human skin cancer called Merkel cell carcinoma. MCPyV is the only polyomavirus definitively shown to cause cancer in humans, yet little is known about how it establishes infection in target cells. In this study, we report an unconventional mechanism by which MCPyV enters the host cell nucleus, where viral genome replication occurs. We demonstrate that, unlike other known polyomaviruses, MCPyV does not require the nuclear pore complex during entry. Instead, it takes advantage of cell cycle-dependent nuclear envelope breakdown to deliver its genetic material into the nucleus. We further show that the VP1 major capsid protein is sufficient to facilitate this process. Overall, our findings reveal a novel mechanism of polyomavirus nuclear entry and provide insight into the diverse mechanisms that these viruses use to cause infection.

Merkel cell polyomavirus↗
PubMed2026

Monitoring kinetic changes and restriction of influenza A virus RNA species during infection using a Flu-Stranded CRISPR platform.

UNLABELLED: Influenza A virus (IAV) generates three closely related RNA species: viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA), whose strand-specific quantification remains limited by sensitivity and quantitative dynamic range, particularly at low RNA abundance. Here, we developed Flu-Stranded CRISPR-Cas12a, a strand-specific detection platform integrating tagged reverse transcription, segment-specific PCR, and Cas12a collateral cleavage to support quantitative analysis of all three RNA species across a broad dynamic range. The assay enables reliable detection down to 102 copies per reaction, extending the lower quantitative boundary relative to both SYBR Green and TaqMan reverse transcription quantitative PCR (RT-qPCR) under matched conditions. Validated in infected cell lines, murine lung tissues, and clinical nasopharyngeal specimens, the platform enabled subtype-discriminating, strand-resolved detection, including samples near or below the quantitative range of SYBR Green RT-qPCR. Using finely resolved infection time-course analyses in NP and NA segments, we identified a reproducible early vRNA decline within the early post-infection phase. This decline was partially attenuated in RIG-I knockout A549 cells, while subsequent vRNA accumulation was enhanced, consistent with a modulatory rather than essential role for RIG-I in early viral RNA dynamics. Subcellular fractionation localized this decline to cytoplasmic incoming genomes. In contrast, importazole-mediated inhibition of nuclear import abolished vRNA recovery without affecting the early decline, indicating that nuclear entry functionally separates early genome reduction from subsequent productive replication. These findings establish Flu-Stranded CRISPR-Cas12a as a strand-resolved framework for monitoring IAV RNA dynamics and reveal an early window of genome vulnerability during cytoplasmic transit that shapes infection outcome. IMPORTANCE: The early fate of incoming influenza virus genomes remains unclear, limiting our understanding of how infection is established or aborted in host cells. We developed Flu-Stranded CRISPR-Cas12a, a strand-specific platform for sensitive and quantitative analysis of influenza viral RNA (vRNA), complementary RNA (cRNA), and messenger RNA (mRNA) across experimental and clinical samples. Using high-resolution time-course analysis, we identified a reproducible early decline in vRNA during the post-entry phase. Our data suggest that this early genome loss arises from multiple processes, with RIG-I acting as a modulatory factor rather than a primary driver. Subcellular fractionation localized this effect to cytoplasmic incoming genomes, whereas importin-β-mediated nuclear entry was required for subsequent vRNA recovery. These findings support a model of an early cytoplasmic phase of genome attrition that is distinct from replication and provide a framework for understanding early influenza RNA kinetics and for guiding strand-resolved diagnostics and antiviral evaluation.

CRISPR-Cas12a↗
PubMed2025

Proper 5'-3' cotranslational mRNA decay in yeast requires import of Xrn1 to the nucleus.

The budding yeast Xrn1 protein shuttles between the nucleus, where it stimulates transcription, and the cytoplasm, where it executes the major cytoplasmic mRNA decay. In the cytoplasm, apart from catalyzing 5'→3' decay onto non translated mRNAs, Xrn1 can follow the last translating ribosome to degrade the decapped mRNA template, a process known as "cotranslational mRNA decay". We have previously observed that the import of Xrn1 to the nucleus is required for efficient cytoplasmic mRNA decay. Here by using an Xrn1 mutant that cannot enter the nucleus, but is otherwise functional in ribonuclease activity, we show that nuclear import is necessary for proper global cotranslational decay of mRNAs along coding regions and also affects degradation in the of 5' region of a large group of mRNAs, which comprise about 20% of the transcriptome. Furthermore, a principal component analysis of the genomic datasets of this mutant and other Xrn1 mutants also shows that lack of a cytoplasmic 5'→3' exoribonuclease is the primary cause of the physiological defects seen in a xrn1Δ mutant, but also suggests that Xrn1 import into the nucleus is necessary for its full in vivo functions.

Exoribonucleases↗
PubMed2026

Molecular mechanism of HUWE1-HAPSTR1-USP7-mediated ubiquitin chain amplification on nuclear proteins.

Rapid protein turnover is essential for cellular stress adaptation. HUWE1 (HECT, UBA, and WWE domain containing 1), a large HECT-type E3 ligase, regulates many short-lived stress-responsive proteins, yet the mechanisms underlying its substrate selectivity remain unclear. Here, we reveal that HUWE1 functions as a ubiquitin chain amplifier that captures pre-ubiquitinated substrates and amplifies the degradation signal by assembling long ubiquitin chains containing K11-K48 branch points, a process regulated by its partners HUWE1-associated protein stress response 1 (HAPSTR1) and USP7 (ubiquitin-specific-processing protease 7). Structural and biochemical analyses show that HAPSTR1 engages HUWE1's ubiquitin-binding motifs to drive nuclear import and modulate substrate recruitment. A cryo-EM structure of the HUWE1-USP7 complex reveals a bidirectional regulatory mechanism: HUWE1 activates USP7's catalytic activity, while USP7 modulates HUWE1 conformational states. Global proteomic analyses demonstrate that this axis drives extensive remodeling of the short-lived nuclear proteome. These findings establish the HUWE1-HAPSTR1-USP7 complex as a key ubiquitin code modifier, providing a molecular rationale for HUWE1 dysregulation in neurodevelopmental disorders and cancer.

Ubiquitin-Specific Peptidase 7↗
PubMed2025

Fluorescence Loss After Photoactivation (FLAPh): A Pulse-Chase Cellular Assay for Understanding Kinetics and Dynamics of Viral Inclusions.

Influenza A virus (IAV) relies on host cellular machinery for replication. Upon infection, the eight genomic segments, independently packed as viral ribonucleoproteins (vRNPs), are released into the cytosol before nuclear import for viral replication. After nucleocytoplasmic transport, the resulting progeny vRNPs reach the cytosol, accumulating in highly mobile and dynamic viral inclusions that display liquid properties. Being sites postulated to support IAV genome assembly, the biophysical properties of IAV inclusions may be critical for function. In agreement, imposing liquid-to-solid transitions was demonstrated to impact viral replication negatively. Therefore, screening for host factors or compounds able to alter the material properties may provide the molecular basis for how influenza genomic complex forms as well as identify novel antivirals. Conventional techniques employed to investigate biomolecular condensates' material properties include fluorescence correlation spectroscopy, raster image correlation spectroscopy, single molecule or microrheology particle tracking, and Fluorescence Recovery After Photobleaching (FRAP). These approaches allow measuring molecular dynamics in systems that do not move very much. However, the analysis of highly mobile intracellular condensates, such as IAV inclusions, poses significant challenges as these structures not only constantly move within the cell but also exchange material, fusing, and dividing, rendering the quantitation of internal rearrangements and diffusion coefficients of molecules within condensates inaccurate. As an alternative, we opted for measuring the kinetics and the exchange of material between IAV inclusions using the Fluorescence Loss After Photoactivation (FLAPh) technique. It involves pulse photoactivation of individual or pools of viral inclusions in the cell, and chasing over time in photoactivated and non-photoactivated regions. This approach is suitable for quantifying the movement and spatial distribution of components within inclusions over time, enabling the determination of both the distance and speed from a specific cellular location. As a result, this method allows the quantification of decay profiles, half-lives, decay constant rate, and mobile and immobile fractions in viral inclusions. It, therefore, enables high throughput screenings for compounds or host factors that affect this dynamism and indirectly allows assessing the material properties of IAV inclusions.

Humans↗
PubMed2026

Live dynamics of induced cell-cell fusion between mitotic and interphasic cells.

The cell cycle is tightly regulated by checkpoint mechanisms that ensure faithful duplication and segregation of the genome. Here, we induced cell-cell fusion between mitotic and interphase cells to study how nuclei from different cell cycle stages behave in a shared cytoplasm. We found that mitosis is a dominant cell cycle state: the mitotic cytoplasm can drive interphase nuclei into mitosis, whereas, in high ratios of interphase versus mitotic nuclei, fusion forced mitotic nuclei to exit mitosis. Both outcomes represent checkpoint override events with impactful consequences. Interphase nuclei forced into mitosis form aberrant mitotic spindles, show partially condensed DNA and ultimately undergo mitotic catastrophe. Conversely, forced mitotic exit resulted in reformation of nuclear envelope membranes around condensed chromosomes, forming nuclei with a defective nuclear import machinery. Altogether, cell-cell fusion revealed the consequences of checkpoint override, forcing nuclei through untimely cell cycle transitions, and highlight how cell-cell fusion experiments can be a powerful system to study how competing cytoplasmic states are integrated in a shared cytoplasm, such as in muscle, placenta formation and in cancer.

Cell cycle↗
PubMed2025

Reverse transcription progression and genome length regulate HIV-1 core elasticity and disassembly.

The structural and mechanical properties of the HIV-1 core are critical for successful infection, balancing stability for early replication and controlled disassembly for genome release. Recent studies have highlighted the role of core elasticity in nuclear entry, yet the molecular determinants regulating this property remain poorly understood. Here, atomic force microscopy (AFM) was used to investigate the relationship between reverse transcription progression, genome length, core elasticity, and disassembly. The results demonstrate that reverse transcription induces a gradual loss of elasticity, rendering the core increasingly brittle as DNA synthesis progresses. Cores containing shorter genomes remained highly elastic, whereas those with longer genomes exhibited increased brittleness, structural damage, and a higher degree of disassembly, after 4 hours of reverse transcription. Additionally, cores from an RNase H-deficient HIV-1 mutant retained high elasticity. These findings provide insight into the interplay between genome synthesis, core integrity, and nuclear entry, supporting a model in which reverse transcription-generated mechanical stress facilitates uncoating. Furthermore, early-stage reverse transcription preserved core elasticity, suggesting a temporal window for successful nuclear import before structural destabilization compromises infectivity.

HIV-1↗
PubMed1975

Content of nuclear estradiol receptor complex in rat corpora lutea during pregnancy: relationship to estrogen concentrations and cytosol receptor availability.

The content of estradiol receptor in cytosol and nuclear cell fractions of rat corpora lutea changed during pregnancy. The binding of (3-H)) estradiol to luteal cell cytosol was high early in pregnancy between days 3-11, decreased on days 12and15 and was low throughout of the remainder of pregnancy. In contrast, the binding of (3-H) estradiol to nuclear receptor, as measured by nuclear exchange assay, was low early in pregnancy, increased between days 10-15, remained high through day 18 and decreased on days 20 and 22. The administration in vivo of estradiol-17beta (40 mug) 1 hr prior to sacrifice stimulated an increase in nuclear receptor content early in pregnancy (days 3,6,8,10-12) but not later in pregnancy (15,18,20, and 22,). These results suggested that the estradiol binding component(s) present in rat luteal cell cytosol early in pregnancy represented available estradiol receptor capable of being translocated to the nucleus in the presence of sufficient estradiol. However, once available receptor has been saturated by endogenous hormone at midgestation, exogenous hormones hasno further effect on no nuclear receptor content. Importantly,loss of nuclear receptor content late in pregnancy appears to reflect decreased levels of total cellular receptor indicating that corpora lutea at the end of pregnancy have lost the primary mecchanism for responding to estrogens Thus, various stages of luteal cell differentiation are associated with changes in the intra-cellular distribution and total cellular content of estradiol receptor suggesting that luteal cell function may be regulated selectively and separately by hormone concentrations and hormone receptor availability.

Animals↗
PubMed1979

Importance of mammalian nuclear-envelope nucleoside triphosphatase in nucleo-cytoplasmic transport of ribonucleoproteins.

The nucleoside triphosphate-stimulated efflux of RNA from isolated nuclei was studied under a range of conditions, and the effects of these conditions on the process were compared with the properties of the nucleoside triphosphatase located in the pore complex. A marked similarity between the rate of efflux and the rate of nucleoside triphosphate hydrolysis was apparent, in terms of substrate specificity, sensitivity to treatment with insolubilized trypsin, kinetics and the effects of increased ionic strength and of many inhibitors. These results are taken, in view of earlier evidence, to suggest that the activity of the nucleoside triphosphatase is a prerequisite for nucleo-cytoplasmic RNA transport in vivo. There are some indications that the nuclear-envelope lipid is also involved in regulating the efflux process.

Animals↗
PubMed2025

Anthracyclines attenuate Nrf1-dependent proteolytic pathways and potentiate proteasome inhibitor cytotoxicity.

Proteasome inhibitors such as bortezomib, carfilzomib, and ixazomib are FDA-approved treatments for multiple myeloma, but resistance frequently limits their effectiveness. The transcription factor Nrf1 (NFE2L1) upregulates proteasome and autophagy genes upon proteasome inhibition, contributing to adaptive resistance. In this study, we identified anthracyclines, including doxorubicin, as suppressors of the Nrf1-driven transcriptional response. Mechanistically, doxorubicin impaired Nrf1 binding to antioxidant response elements (AREs) within promoter regions of target genes without affecting Nrf1 processing or nuclear localization. Importantly, aclarubicin, a non-DNA-damaging anthracycline, also attenuated Nrf1 transcriptional activity, indicating that DNA damage is not required for this inhibition. Doxorubicin cotreatment delayed proteasome recovery after pulse inhibition and partially restored sensitivity to carfilzomib in bortezomib-resistant U266 myeloma cells, consistent with genetic knockout of Nrf1. These findings identify a DNA-damage-independent mechanism by which anthracyclines directly obstruct Nrf1-mediated transcriptional induction. Thus, anthracyclines serve as chemical tools to probe the molecular control of proteostasis and suggest a strategy to mitigate Nrf1-driven adaptive response to proteasome inhibition.

Humans↗
PubMedDate not supplied

The effect of certain variables on the tumor and tissue distribution of tracers. Part 1; Carrier.

Buffalo rats bearing thigh-implanted strain-7777 Morris hepatomas were used as a model for studying the effect of carrier material on the body distribution, tumor uptake, excretion, and tumor-to-background ratios of 67Ga and 54Mn. An effort was also made to observe the changes in 67Ga and 54Mn concentrations induced by carrier in viable tumor and skeletal muscle, relative to their interstitial fluid space. This value is referred to as the Tissue Distribution Index. Carrier manipulation resulted in striking changes in the distribution of the two ions from the carrier-free state. The data also indicated a difference in the pharmacodynamics of 67Ga and 54Mn in malignant and healthy tissues which could be of importance to nuclear medicine and oncology.

Animals↗
PubMed1975

DNA synthesis in membrane-denuded nuclei and nuclear fractions from host liver and Morris hepatomas.

Incorporation of [3H]TTP into membrane-denuded nuclei and fractions of these nuclei from host liver and Morris hepatomas has been compared. Treatment of sucrose nuclei with Triton X-100 removed 95% of the phospholipids and 15 to 20% of the protein. These membrane-denuded nuclei remained physically stable. The Triton X-100-extracted nuclei incorporated label into their DNA in nuclear-incorporating system similar to sucrose nuclei with their membranes intact. Triton X-100-treated nuclei from hepatoma 7777 incorporated six times more label and those from hepatoma 7800 incorporated three times more label than Triton X-100-treated host liver nuclei. Nuclei from the three sources incorporated more label when exogenous DNA was added to the incubation system, but the difference in incorporation between the hepatoma nuclei and the host liver nuclei disappeared. When Triton X-100-treated nuclei, prepared from a tumor-bearing animal given injections of [3H]thymidine for 10 min were fractionated on sucrose gradients after disruption by high Mg2+ concentration, the fractions from hepatoma 7777 nuclei contained six times as much label as the host liver nuclear fractions. Nuclear fractions prepared from unabeled hepatomas or host livers had DNA polymerase activity. The activity, however, is the same in fractions prepared from hepatoma 7777 or host liver nuclei. It is suggested that the nuclear membrane does not play an important role in nuclear DNA synthesis. It is further suggested that the increased incorporation found with hepatoma nuclei is dependent on a physical or chemical arrangement of components within the nucleus and not solely on different enzyme levels.

Animals↗
PubMed1977

Renal serendipity in whole body scintiscan.

The importance in nuclear medicine procedures of critical examination of all of the data, beyond the problem in question, is emphasized. These case reports exemplify such situations. Renal pathology was diagnosed serendipitously while performing skeletal screening in patients in whom renal diseases had not been thought of.

Bone Neoplasms↗
PubMed2026

Signaling into the nucleus through the importin 7 pathway.

Importin 7 (IPO7) is a nuclear transport receptor of the β-karyopherin family that mediates the translocation of a broad spectrum of macromolecules, commonly referred to as cargoes. Discovered nearly three decades ago, IPO7 was initially identified as an import receptor for constitutive cellular cargoes, including histone H1 and ribosomal proteins, and was shown to function synergistically and partially redundantly with canonical receptors such as importin β1 and karyopherin β2. Over the past 15 years, however, accumulating evidence has established IPO7 as an important mediator of signal-dependent nuclear trafficking in response to extracellular stimuli, including cytokines, growth factors, and cellular stress. Thus, IPO7 has emerged as a versatile nuclear transport receptor that couples extracellular signaling to dynamic changes in nuclear composition and gene expression. Mechanistically, many IPO7 cargoes lack classical nuclear localization signals and instead contain noncanonical targeting motifs that directly engage IPO7. In several cases, phosphorylation-dependent conformational changes expose these motifs, promoting IPO7 binding and translocation through the nuclear pore complex. The expanding repertoire of IPO7 cargoes, including ERK, SMAD3, EGR1, GLI1, the glucocorticoid receptor, HIF-1α, YAP1, and RUNX2, highlights its prominent role at the interface of signaling and transcriptional control. Consistent with these functions, dysregulation of IPO7-mediated transport has been implicated in cancer, hypoxia, and other pathological states. Beyond cellular signaling, IPO7 also contributes to the nuclear trafficking of viral genomes and proteins. Here, we review the molecular mechanisms of IPO7-dependent nuclear import, emerging principles of cargo recognition, and pathways that regulate IPO7 activity during cellular signaling.

NLS↗
PubMed1978

Electron microscopic morphometric analysis of human T and B peripheral blood lymphocytes.

Using the system of morphometric analysis described in this paper, human peripheral blood T and B lymphocytes, labeled with specific surface markers, can be compared on different analytical levels. They show differences in their surface and the eccentricity of cells, in the relative surfaces occupied by peripheral and central condensed chromatin, in the average surface of the central chromatin clumps and in the number of perichromatin granules per nuclear surface. The morphometric analysis reveals the importance of examining the nuclear and the surface parameters in the characterization of lymphocytes, confirming that a detailed analysis of the nuclear characteristics can contribute to the identification of T and B lymphocytes by transmission electron microscopy.

Animals↗