De novo identification of mammalian ciliary motility proteins using cryo-EM.

Dynein-decorated doublet microtubules (DMTs) are critical components of the oscillatory molecular machine of cilia, the axoneme, and have luminal surfaces patterned periodically by microtubule inner proteins (MIPs). Here we present an atomic model of the 48-nm repeat of a mammalian DMT, derived from a cryoelectron microscopy (cryo-EM) map of the complex isolated from bovine respiratory cilia. The structure uncovers principles of doublet microtubule organization and features specific to vertebrate cilia, including previously unknown MIPs, a luminal bundle of tektin filaments, and a pentameric dynein-docking complex. We identify a mechanism for bridging 48- to 24-nm periodicity across the microtubule wall and show that loss of the proteins involved causes defective ciliary motility and laterality abnormalities in zebrafish and mice. Our structure identifies candidate genes for diagnosis of ciliopathies and provides a framework to understand their functions in driving ciliary motility.

Infection with wild-type SARS-CoV-2 elicits broadly neutralizing and protective antibodies against omicron subvariants.

The omicron variants of SARS-CoV-2 have substantial ability to escape infection- and vaccine-elicited antibody immunity. Here, we investigated the extent of such escape in nine convalescent patients infected with the wild-type SARS-CoV-2 during the first wave of the pandemic. Among the total of 476 monoclonal antibodies (mAbs) isolated from peripheral memory B cells, we identified seven mAbs with broad neutralizing activity to all variants tested, including various omicron subvariants. Biochemical and structural analysis indicated the majority of these mAbs bound to the receptor-binding domain, mimicked the receptor ACE2 and were able to accommodate or inadvertently improve recognition of omicron substitutions. Passive delivery of representative antibodies protected K18-hACE2 mice from infection with omicron and beta SARS-CoV-2. A deeper understanding of how the memory B cells that produce these antibodies could be selectively boosted or recalled can augment antibody immunity against SARS-CoV-2 variants.

The Impact of Mutations in SARS-CoV-2 Spike on Viral Infectivity and Antigenicity.

The spike protein of SARS-CoV-2 has been undergoing mutations and is highly glycosylated. It is critically important to investigate the biological significance of these mutations. Here, we investigated 80 variants and 26 glycosylation site modifications for the infectivity and reactivity to a panel of neutralizing antibodies and sera from convalescent patients. D614G, along with several variants containing both D614G and another amino acid change, were significantly more infectious. Most variants with amino acid change at receptor binding domain were less infectious, but variants including A475V, L452R, V483A, and F490L became resistant to some neutralizing antibodies. Moreover, the majority of glycosylation deletions were less infectious, whereas deletion of both N331 and N343 glycosylation drastically reduced infectivity, revealing the importance of glycosylation for viral infectivity. Interestingly, N234Q was markedly resistant to neutralizing antibodies, whereas N165Q became more sensitive. These findings could be of value in the development of vaccine and therapeutic antibodies.

Dissecting the intricacies of human antibody responses to SARS-CoV-1 and SARS-CoV-2 infection.

The 2003 severe acute respiratory syndrome coronavirus (SARS-CoV-1) causes more severe disease than SARS-CoV-2, which is responsible for COVID-19. However, our understanding of antibody response to SARS-CoV-1 infection remains incomplete. Herein, we studied the antibody responses in 25 SARS-CoV-1 convalescent patients. Plasma neutralization was higher and lasted longer in SARS-CoV-1 patients than in severe SARS-CoV-2 patients. Among 77 monoclonal antibodies (mAbs) isolated, 60 targeted the receptor-binding domain (RBD) and formed 7 groups (RBD-1 to RBD-7) based on their distinct binding and structural profiles. Notably, RBD-7 antibodies bound to a unique RBD region interfaced with the N-terminal domain of the neighboring protomer (NTD proximal) and were more prevalent in SARS-CoV-1 patients. Broadly neutralizing antibodies for SARS-CoV-1, SARS-CoV-2, and bat and pangolin coronaviruses were also identified. These results provide further insights into the antibody response to SARS-CoV-1 and inform the design of more effective strategies against diverse human and animal coronaviruses.

Analysis of SARS-CoV-2 variant mutations reveals neutralization escape mechanisms and the ability to use ACE2 receptors from additional species.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants continue to emerge during the global pandemic and may facilitate escape from current antibody therapies and vaccine protection. Here we showed that the South African variant B.1.351 was the most resistant to current monoclonal antibodies and convalescent plasma from coronavirus disease 2019 (COVID-19)-infected individuals, followed by the Brazilian variant P.1 and the United Kingdom variant B.1.1.7. This resistance hierarchy corresponded with Y144del and 242-244del mutations in the N-terminal domain and K417N/T, E484K, and N501Y mutations in the receptor-binding domain (RBD) of SARS-CoV-2. Crystal structure analysis of the B.1.351 triple mutant (417N-484K-501Y) RBD complexed with the monoclonal antibody P2C-1F11 revealed the molecular basis for antibody neutralization and escape. B.1.351 and P.1 also acquired the ability to use mouse and mink ACE2 receptors for entry. Our results demonstrate major antigenic shifts and potential broadening of the host range for B.1.351 and P.1 variants, which poses serious challenges to current antibody therapies and vaccine protection.

Mars's induced magnetosphere can degenerate.

The interaction between planets and stellar winds can lead to atmospheric loss and is, thus, important for the evolution of planetary atmospheres1. The planets in our Solar System typically interact with the solar wind, whose velocity is at a large angle to the embedded stellar magnetic field. For planets without an intrinsic magnetic field, this interaction creates an induced magnetosphere and a bow shock in front of the planet2. However, when the angle between the solar wind velocity and the solar wind magnetic field (cone angle) is small, the interaction is very different3. Here we show that when the cone angle is small at Mars, the induced magnetosphere degenerates. There is no shock on the dayside, only weak flank shocks. A cross-flow plume appears and the ambipolar field drives planetary ions upstream. Hybrid simulations with a 4° cone angle show agreement with observations by the Mars Atmosphere and Volatile Evolution mission4 and Mars Express5. Degenerate, induced magnetospheres are complex and not yet explored objects. It remains to be studied what the secondary effects are on processes like atmospheric loss through ion escape.

A hybrid topological quantum state in an elemental solid.

Topology1-3 and interactions are foundational concepts in the modern understanding of quantum matter. Their nexus yields three important research directions: (1) the competition between distinct interactions, as in several intertwined phases, (2) the interplay between interactions and topology that drives the phenomena in twisted layered materials and topological magnets, and (3) the coalescence of several topological orders to generate distinct novel phases. The first two examples have grown into major areas of research, although the last example remains mostly unexplored, mainly because of the lack of a material platform for experimental studies. Here, using tunnelling microscopy, photoemission spectroscopy and a theoretical analysis, we unveil a 'hybrid' topological phase of matter in the simple elemental-solid arsenic. Through a unique bulk-surface-edge correspondence, we uncover that arsenic features a conjoined strong and higher-order topology that stabilizes a hybrid topological phase. Although momentum-space spectroscopy measurements show signs of topological surface states, real-space microscopy measurements unravel a unique geometry of topologically induced step-edge conduction channels revealed on various natural nanostructures on the surface. Using theoretical models, we show that the existence of gapless step-edge states in arsenic relies on the simultaneous presence of both a non-trivial strong Z2 invariant and a non-trivial higher-order topological invariant, which provide experimental evidence for hybrid topology. Our study highlights pathways for exploring the interplay of different band topologies and harnessing the associated topological conduction channels in engineered quantum or nano-devices.

MRE11 liberates cGAS from nucleosome sequestration during tumorigenesis.

Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression1-3. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA)4-10. Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.

Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

Organoid cultures derived from patients with advanced prostate cancer.

The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D organoid system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression, and CHD1 loss. Whole-exome sequencing shows a low mutational burden, consistent with genomics studies, but with mutations in FOXA1 and PIK3R1, as well as in DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies.

Spin-mediated shear oscillators in a van der Waals antiferromagnet.

Understanding how microscopic spin configuration gives rise to exotic properties at the macroscopic length scale has long been pursued in magnetic materials1-5. One seminal example is the Einstein-de Haas effect in ferromagnets1,6,7, in which angular momentum of spins can be converted into mechanical rotation of an entire object. However, for antiferromagnets without net magnetic moment, how spin ordering couples to macroscopic movement remains elusive. Here we observed a seesaw-like rotation of reciprocal lattice peaks of an antiferromagnetic nanolayer film, whose gigahertz structural resonance exhibits more than an order-of-magnitude amplification after cooling below the Néel temperature. Using a suite of ultrafast diffraction and microscopy techniques, we directly visualize this spin-driven rotation in reciprocal space at the nanoscale. This motion corresponds to interlayer shear in real space, in which individual micro-patches of the film behave as coherent oscillators that are phase-locked and shear along the same in-plane axis. Using time-resolved optical polarimetry, we further show that the enhanced mechanical response strongly correlates with ultrafast demagnetization, which releases elastic energy stored in local strain gradients to drive the oscillators. Our work not only offers the first microscopic view of spin-mediated mechanical motion of an antiferromagnet but it also identifies a new route towards realizing high-frequency resonators8,9 up to the millimetre band, so the capability of controlling magnetic states on the ultrafast timescale10-13 can be readily transferred to engineering the mechanical properties of nanodevices.

TBK1-Zyxin signaling controls tumor-associated macrophage recruitment to mitigate antitumor immunity.

Mechanical control is fundamental for cellular localization within a tissue, including for tumor-associated macrophages (TAMs). While the innate immune sensing pathways cGAS-STING and RLR-MAVS impact the pathogenesis and therapeutics of malignant diseases, their effects on cell residency and motility remain incompletely understood. Here, we uncovered that TBK1 kinase, activated by cGAS-STING or RLR-MAVS signaling in macrophages, directly phosphorylates and mobilizes Zyxin, a key regulator of actin dynamics. Under pathological conditions and in STING or MAVS signalosomes, TBK1-mediated Zyxin phosphorylation at S143 facilitates rapid recruitment of phospho-Zyxin to focal adhesions, leading to subsequent F-actin reorganization and reduced macrophage migration. Intratumoral STING-TBK1-Zyxin signaling was evident in TAMs and critical in antitumor immunity. Furthermore, myeloid-specific or global disruption of this signaling decreased the population of CD11b+ F4/80+ TAMs and promoted PD-1-mediated antitumor immunotherapy. Thus, our findings identify a new biological function of innate immune sensing pathways by regulating macrophage tissue localization, thus providing insights into context-dependent mitigation of antitumor immunity.

Inhibition of colorectal cancer in Alzheimer's disease is mediated by gut microbiota via induction of inflammatory tolerance.

Epidemiological studies have revealed an inverse relationship between the incidence of Alzheimer's disease (AD) and various cancers, including colorectal cancer (CRC). We aimed to determine whether the incidence of CRC is reduced in AD-like mice and whether gut microbiota confers resistance to tumorigenesis through inducing inflammatory tolerance using 16S ribosomal RNA gene sequencing and fecal microbiota transplantation (FMT). AD-like mice experienced a significantly decreased incidence of CRC tumorigenesis induced by azoxymethane-dextran sodium sulfate as evidenced by suppressed intestinal inflammation compared with control mice. However, FMT from age-matched control mice reversed the inhibitory effects on the tumorigenesis of CRC and inflammatory response in AD-like mice. The key bacterial genera in gut microbiota, including Prevotella, were increased in both the AD-like mice and in patients with amnestic mild cognitive impairment (aMCI) but were decreased in patients with CRC. Pretreatment with low-dose Prevotella-derived lipopolysaccharides (LPS) induced inflammatory tolerance both in vivo and in vitro and inhibited CRC tumorigenesis in mice. Imbalanced gut microbiota increased intestinal barrier permeability, which facilitated LPS absorption from the gut into the blood, causing cognitive decline in AD-like mice and patients with aMCI. These data reveal that intestinal Prevotella-derived LPS exerts a resistant effect to CRC tumorigenesis via inducing inflammatory tolerance in the presence of AD. These findings provide biological evidence demonstrating the inverse relationship between the incidence of AD and CRC.

Improving normothermic machine perfusion and blood transfusion through biocompatible blood silicification.

The growing world population and increasing life expectancy are driving the need to improve the quality of blood transfusion, organ transplantation, and preservation. Here, to improve the ability of red blood cells (RBCs) for normothermic machine perfusion, a biocompatible blood silicification approach termed "shielding-augmenting RBC-in-nanoscale amorphous silica (SARNAS)" has been developed. The key to RBC surface engineering and structure augmentation is the precise control of the hydrolysis form of silicic acid to realize stabilization of RBC within conformal nanoscale silica-based exoskeletons. The formed silicified RBCs (Si-RBCs) maintain membrane/structural integrity, normal cellular functions (e.g., metabolism, oxygen-carrying capability), and enhance resistance to external stressors as well as tunable mechanical properties, resulting in nearly 100% RBC cryoprotection. In vivo experiments confirm their excellent biocompatibility. By shielding RBC surface antigens, the Si-RBCs provide universal blood compatibility, the ability for allogeneic mechanical perfusion, and more importantly, the possibility for cross-species transfusion. Being simple, reliable, and easily scalable, the SARNAS strategy holds great promise to revolutionize the use of engineered blood for future clinical applications.

Genetic improvement of phosphate-limited photosynthesis for high yield in rice.

Low phosphate (Pi) availability decreases photosynthesis, with phosphate limitation of photosynthesis occurring particularly during grain filling of cereal crops; however, effective genetic solutions remain to be established. We previously discovered that rice phosphate transporter OsPHO1;2 controls seed (sink) development through Pi reallocation during grain filling. Here, we find that OsPHO1;2 regulates Pi homeostasis and thus photosynthesis in leaves (source). Loss-of-function of OsPHO1;2 decreased Pi levels in leaves, leading to decreased photosynthetic electron transport activity, CO2 assimilation rate, and early occurrence of phosphate-limited photosynthesis. Interestingly, ectopic expression of OsPHO1;2 greatly increased Pi availability, and thereby, increased photosynthetic rate in leaves during grain filling, contributing to increased yield. This was supported by the effect of foliar Pi application. Moreover, analysis of core rice germplasm resources revealed that higher OsPHO1;2 expression was associated with enhanced photosynthesis and yield potential compared to those with lower expression. These findings reveal that phosphate-limitation of photosynthesis can be relieved via a genetic approach, and the OsPHO1;2 gene can be employed to reinforce crop breeding strategies for achieving higher photosynthetic efficiency.

Searching across-cohort relatives in 54,092 GWAS samples via encrypted genotype regression.

Explicitly sharing individual level data in genomics studies has many merits comparing to sharing summary statistics, including more strict QCs, common statistical analyses, relative identification and improved statistical power in GWAS, but it is hampered by privacy or ethical constraints. In this study, we developed encG-reg, a regression approach that can detect relatives of various degrees based on encrypted genomic data, which is immune of ethical constraints. The encryption properties of encG-reg are based on the random matrix theory by masking the original genotypic matrix without sacrificing precision of individual-level genotype data. We established a connection between the dimension of a random matrix, which masked genotype matrices, and the required precision of a study for encrypted genotype data. encG-reg has false positive and false negative rates equivalent to sharing original individual level data, and is computationally efficient when searching relatives. We split the UK Biobank into their respective centers, and then encrypted the genotype data. We observed that the relatives estimated using encG-reg was equivalently accurate with the estimation by KING, which is a widely used software but requires original genotype data. In a more complex application, we launched a finely devised multi-center collaboration across 5 research institutes in China, covering 9 cohorts of 54,092 GWAS samples. encG-reg again identified true relatives existing across the cohorts with even different ethnic backgrounds and genotypic qualities. Our study clearly demonstrates that encrypted genomic data can be used for data sharing without loss of information or data sharing barrier.

Construction of a prognostic model for lung adenocarcinoma based on m6A/m5C/m1A genes.

BACKGROUND: Developing a prognostic model for lung adenocarcinoma (LUAD) that utilizes m6A/m5C/m1A genes holds immense importance in providing precise prognosis predictions for individuals. METHODS: This study mined m6A/m5C/m1A-related differential genes in LUAD based on public databases, identified LUAD tumor subtypes based on these genes, and further built a risk prognostic model grounded in differential genes between subtypes. The immune status between high- and low-risk groups was investigated, and the distribution of feature genes in tumor immune cells was analyzed using single-cell analysis. Based on the expression levels of feature genes, a projection of chemotherapeutic and targeted drugs was made for individuals identified as high-risk. Ultimately, cell experiments were further verified. RESULTS: The 6-gene risk prognosis model based on differential genes between tumor subtypes had good predictive performance. Individuals classified as low-risk exhibited a higher (P < 0.05) abundance of infiltrating immune cells. Feature genes were mainly distributed in tumor immune cells like CD4+T cells, CD8+T cells, and regulatory T cells. Four drugs with relatively low IC50 values were found in the high-risk group: Elesclomol, Pyrimethamine, Saracatinib, and Temsirolimus. In addition, four drugs with significant positive correlation (P < 0.001) between IC50 values and feature gene expression were found, including Alectinib, Estramustine, Brigatinib, and Elesclomol. The low expression of key gene NTSR1 reduced the IC50 value of irinotecan. CONCLUSION: Based on the m6A/m5C/m1A-related genes in LUAD, LUAD patients were divided into 2 subtypes, and a m6A/m5C/m1A-related LUAD prognostic model was constructed to provide a reference for the prognosis prediction of LUAD.

Functional conservation and divergence of the helix-turn-helix motif of E2 ubiquitin-conjugating enzymes.

Polyubiquitination by E2 and E3 enzymes is crucial to cell cycle control, epigenetic regulation, and development. The hallmark of the E2 family is the ubiquitin (Ub)-conjugating (UBC) domain that forms a dynamic thioester conjugate with ubiquitin (E2~Ub). Numerous studies have focused on E2 surfaces, such as the N-terminal and crossover helices, that directly interact with an E3 or the conjugated ubiquitin to stabilize the active, "closed" state of the E2~Ub. However, it remains unclear how other E2 surfaces regulate ubiquitin transfer. Here, we demonstrate the helix-turn-helix (HTH) motif of the UBC tunes the intrinsic polyubiquitination activity through distinct functions in different E2s. Interestingly, the E2HTH motif is repurposed in UBE2S and UBE2R2 to interact with the conjugated or acceptor ubiquitin, respectively, modulating ubiquitin transfer. Furthermore, we propose that Anaphase-Promoting Complex/Cyclosome binding to the UBE2SHTH reduces the conformational space of the flexible E2~Ub, demonstrating an atypical E3-dependent activation mechanism. Altogether, we postulate the E2HTH motif evolved to provide new functionalities that can be harnessed by E3s and permits additional regulation to facilitate specific E2-E3-mediated polyubiquitination.

Graphormer supervised de novo protein design method and function validation.

Protein design is central to nearly all protein engineering problems, as it can enable the creation of proteins with new biological functions, such as improving the catalytic efficiency of enzymes. One key facet of protein design, fixed-backbone protein sequence design, seeks to design new sequences that will conform to a prescribed protein backbone structure. Nonetheless, existing sequence design methods present limitations, such as low sequence diversity and shortcomings in experimental validation of the designed functional proteins. These inadequacies obstruct the goal of functional protein design. To improve these limitations, we initially developed the Graphormer-based Protein Design (GPD) model. This model utilizes the Transformer on a graph-based representation of three-dimensional protein structures and incorporates Gaussian noise and a sequence random masks to node features, thereby enhancing sequence recovery and diversity. The performance of the GPD model was significantly better than that of the state-of-the-art ProteinMPNN model on multiple independent tests, especially for sequence diversity. We employed GPD to design CalB hydrolase and generated nine artificially designed CalB proteins. The results show a 1.7-fold increase in catalytic activity compared to that of the wild-type CalB and strong substrate selectivity on p-nitrophenyl acetate with different carbon chain lengths (C2-C16). Thus, the GPD method could be used for the de novo design of industrial enzymes and protein drugs. The code was released at https://github.com/decodermu/GPD.

Porcine circovirus type 2 infection promotes the SUMOylation of nucleophosmin-1 to facilitate the viral circular single-stranded DNA replication.

The mechanism of genome DNA replication in circular single-stranded DNA viruses is currently a mystery, except for the fact that it undergoes rolling-circle replication. Herein, we identified SUMOylated porcine nucleophosmin-1 (pNPM1), which is previously reported to be an interacting protein of the viral capsid protein, as a key regulator that promotes the genome DNA replication of porcine single-stranded DNA circovirus. Upon porcine circovirus type 2 (PCV2) infection, SUMO2/3 were recruited and conjugated with the K263 site of pNPM1's C-terminal domain to SUMOylate pNPM1, subsequently, the SUMOylated pNPM1 were translocated in nucleoli to promote the replication of PCV2 genome DNA. The mutation of the K263 site reduced the SUMOylation levels of pNPM1 and the nucleolar localization of pNPM1, resulting in a decrease in the level of PCV2 DNA replication. Meanwhile, the mutation of the K263 site prevented the interaction of pNPM1 with PCV2 DNA, but not the interaction of pNPM1 with PCV2 Cap. Mechanistically, PCV2 infection increased the expression levels of Ubc9, the only E2 enzyme involved in SUMOylation, through the Cap-mediated activation of ERK signaling. The upregulation of Ubc9 promoted the interaction between pNPM1 and TRIM24, a potential E3 ligase for SUMOylation, thereby facilitating the SUMOylation of pNPM1. The inhibition of ERK activation could significantly reduce the SUMOylation levels and the nucleolar localization of pNPM1, as well as the PCV2 DNA replication levels. These results provide new insights into the mechanism of circular single-stranded DNA virus replication and highlight NPM1 as a potential target for inhibiting PCV2 replication.